Study of Gene Trafficking between Acanthamoeba and Giant Viruses Suggests an Undiscovered Family of Amoeba-Infecting Viruses.

PubMed

Maumus, Florian; Blanc, Guillaume

2016-12-14

The nucleocytoplasmic large DNA viruses (NCLDV) are a group of extremely complex double-stranded DNA viruses, which are major parasites of a variety of eukaryotes. Recent studies showed that certain unicellular eukaryotes contain fragments of NCLDV DNA integrated in their genome, when surprisingly many of these organisms were not previously shown to be infected by NCLDVs. These findings prompted us to search the genome of Acanthamoeba castellanii strain Neff (Neff), one of the most prolific hosts in the discovery of giant NCLDVs, for possible DNA inserts of viral origin. We report the identification of 267 markers of lateral gene transfer with viruses, approximately half of which are clustered in Neff genome regions of viral origins, transcriptionally inactive or exhibit nucleotide-composition signatures suggestive of a foreign origin. The integrated viral genes had diverse origin among relatives of viruses that infect Neff, including Mollivirus, Pandoravirus, Marseillevirus, Pithovirus, and Mimivirus However, phylogenetic analysis suggests the existence of a yet-undiscovered family of amoeba-infecting NCLDV in addition to the five already characterized. The active transcription of some apparently anciently integrated virus-like genes suggests that some viral genes might have been domesticated during the amoeba evolution. These insights confirm that genomic insertion of NCLDV DNA is a common theme in eukaryotes. This gene flow contributed fertilizing the eukaryotic gene repertoire and participated in the occurrence of orphan genes, a long standing issue in genomics. Search for viral inserts in eukaryotic genomes followed by environmental screening of the original viruses should be used to isolate radically new NCLDVs. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Evolutionary Dynamics of Influenza A Viruses in US Exhibition Swine

PubMed Central

Nelson, Martha I.; Wentworth, David E.; Das, Suman R.; Sreevatsan, Srinand; Killian, Mary L.; Nolting, Jacqueline M.; Slemons, Richard D.; Bowman, Andrew S.

2016-01-01

The role of exhibition swine in influenza A virus transmission was recently demonstrated by >300 infections with influenza A(H3N2) variant viruses among individuals who attended agricultural fairs. Through active influenza A virus surveillance in US exhibition swine and whole-genome sequencing of 380 isolates, we demonstrate that exhibition swine are actively involved in the evolution of influenza A viruses, including zoonotic strains. First, frequent introduction of influenza A viruses from commercial swine populations provides new genetic diversity in exhibition pigs each year locally. Second, genomic reassortment between viruses cocirculating in exhibition swine increases viral diversity. Third, viral migration between exhibition swine in neighboring states demonstrates that movements of exhibition pigs contributes to the spread of genetic diversity. The unexpected frequency of viral exchange between commercial and exhibition swine raises questions about the understudied interface between these populations. Overall, the complexity of viral evolution in exhibition swine indicates that novel viruses are likely to continually reemerge, presenting threats to humans. PMID:26243317

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

PubMed

Stepanauskas, Ramunas; Fergusson, Elizabeth A; Brown, Joseph; Poulton, Nicole J; Tupper, Ben; Labonté, Jessica M; Becraft, Eric D; Brown, Julia M; Pachiadaki, Maria G; Povilaitis, Tadas; Thompson, Brian P; Mascena, Corianna J; Bellows, Wendy K; Lubys, Arvydas

2017-07-20

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.

Viral symbiosis and the holobiontic nature of the human genome.

PubMed

Ryan, Francis Patrick

2016-01-01

The human genome is a holobiontic union of the mammalian nuclear genome, the mitochondrial genome and large numbers of endogenized retroviral genomes. This article defines and explores this symbiogenetic pattern of evolution, looking at the implications for human genetics, epigenetics, embryogenesis, physiology and the pathogenesis of inborn errors of metabolism and many other diseases. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

PubMed

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

2017-04-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genomic fossils reveal adaptation of non-autonomous pararetroviruses driven by concerted evolution of noncoding regulatory sequences.

PubMed

Chen, Sunlu; Zheng, Huizhen; Kishima, Yuji

2017-06-01

The interplay of different virus species in a host cell after infection can affect the adaptation of each virus. Endogenous viral elements, such as endogenous pararetroviruses (PRVs), have arisen from vertical inheritance of viral sequences integrated into host germline genomes. As viral genomic fossils, these sequences can thus serve as valuable paleogenomic data to study the long-term evolutionary dynamics of virus-virus interactions, but they have rarely been applied for this purpose. All extant PRVs have been considered autonomous species in their parasitic life cycle in host cells. Here, we provide evidence for multiple non-autonomous PRV species with structural defects in viral activity that have frequently infected ancient grass hosts and adapted through interplay between viruses. Our paleogenomic analyses using endogenous PRVs in grass genomes revealed that these non-autonomous PRV species have participated in interplay with autonomous PRVs in a possible commensal partnership, or, alternatively, with one another in a possible mutualistic partnership. These partnerships, which have been established by the sharing of noncoding regulatory sequences (NRSs) in intergenic regions between two partner viruses, have been further maintained and altered by the sequence homogenization of NRSs between partners. Strikingly, we found that frequent region-specific recombination, rather than mutation selection, is the main causative mechanism of NRS homogenization. Our results, obtained from ancient DNA records of viruses, suggest that adaptation of PRVs has occurred by concerted evolution of NRSs between different virus species in the same host. Our findings further imply that evaluation of within-host NRS interactions within and between populations of viral pathogens may be important.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

PubMed

Cornelissen, Marion; Gall, Astrid; Vink, Monique; Zorgdrager, Fokla; Binter, Špela; Edwards, Stephanie; Jurriaans, Suzanne; Bakker, Margreet; Ong, Swee Hoe; Gras, Luuk; van Sighem, Ard; Bezemer, Daniela; de Wolf, Frank; Reiss, Peter; Kellam, Paul; Berkhout, Ben; Fraser, Christophe; van der Kuyl, Antoinette C

2017-07-15

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection.

PubMed

Weiss, Eric R; Lamers, Susanna L; Henderson, Jennifer L; Melnikov, Alexandre; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Nusbaum, Chad; Luzuriaga, Katherine

2018-01-15

Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time ( P < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence ( P < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection. IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection. Copyright © 2018 American Society for Microbiology.

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

PubMed Central

Krupovic, Mart; Prangishvili, David; Hendrix, Roger W.; Bamford, Dennis H.

2011-01-01

Summary: Prokaryotes, bacteria and archaea, are the most abundant cellular organisms among those sharing the planet Earth with human beings (among others). However, numerous ecological studies have revealed that it is actually prokaryotic viruses that predominate on our planet and outnumber their hosts by at least an order of magnitude. An understanding of how this viral domain is organized and what are the mechanisms governing its evolution is therefore of great interest and importance. The vast majority of characterized prokaryotic viruses belong to the order Caudovirales, double-stranded DNA (dsDNA) bacteriophages with tails. Consequently, these viruses have been studied (and reviewed) extensively from both genomic and functional perspectives. However, albeit numerous, tailed phages represent only a minor fraction of the prokaryotic virus diversity. Therefore, the knowledge which has been generated for this viral system does not offer a comprehensive view of the prokaryotic virosphere. In this review, we discuss all families of bacterial and archaeal viruses that contain more than one characterized member and for which evolutionary conclusions can be attempted by use of comparative genomic analysis. We focus on the molecular mechanisms of their genome evolution as well as on the relationships between different viral groups and plasmids. It becomes clear that evolutionary mechanisms shaping the genomes of prokaryotic viruses vary between different families and depend on the type of the nucleic acid, characteristics of the virion structure, as well as the mode of the life cycle. We also point out that horizontal gene transfer is not equally prevalent in different virus families and is not uniformly unrestricted for diverse viral functions. PMID:22126996

ACVP-05: Virus Genetic Analysis from Cell-Free Plasma, Virally Infected Cells or Tissues and Cultured Supernatant Via Single Genome Amplification and Direct Sequencing | Frederick National Laboratory for Cancer Research

Cancer.gov

The Viral Evolution Core within the AIDS and Cancer Virus Program will extract viral RNA/DNA from cell-free or cell-associated samples. Complementary (cDNA) will be generated as needed, and cDNA or DNA will be diluted to a single copy prior to nested

Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution.

PubMed

Renner, Daniel W; Szpara, Moriah L

2018-01-01

Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well. Copyright © 2017 Renner and Szpara.

Single-Genome Sequencing of Hepatitis C Virus in Donor-Recipient Pairs Distinguishes Modes and Models of Virus Transmission and Early Diversification.

PubMed

Li, Hui; Stoddard, Mark B; Wang, Shuyi; Giorgi, Elena E; Blair, Lily M; Learn, Gerald H; Hahn, Beatrice H; Alter, Harvey J; Busch, Michael P; Fierer, Daniel S; Ribeiro, Ruy M; Perelson, Alan S; Bhattacharya, Tanmoy; Shaw, George M

2016-01-01

Despite the recent development of highly effective anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and development of an effective vaccine. A precise molecular identification of transmitted/founder (T/F) HCV genomes that lead to productive clinical infection could play a critical role in vaccine research, as it has for HIV-1. However, the replication schema of these two RNA viruses differ substantially, as do viral responses to innate and adaptive host defenses. These differences raise questions as to the certainty of T/F HCV genome inferences, particularly in cases where multiple closely related sequence lineages have been observed. To clarify these issues and distinguish between competing models of early HCV diversification, we examined seven cases of acute HCV infection in humans and chimpanzees, including three examples of virus transmission between linked donors and recipients. Using single-genome sequencing (SGS) of plasma vRNA, we found that inferred T/F sequences in recipients were identical to viral sequences in their respective donors. Early in infection, HCV genomes generally evolved according to a simple model of random evolution where the coalescent corresponded to the T/F sequence. Closely related sequence lineages could be explained by high multiplicity infection from a donor whose viral sequences had undergone a pretransmission bottleneck due to treatment, immune selection, or recent infection. These findings validate SGS, together with mathematical modeling and phylogenetic analysis, as a novel strategy to infer T/F HCV genome sequences. Despite the recent development of highly effective, interferon-sparing anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and the development of an effective vaccine, which could be facilitated by a precise molecular identification of transmitted/founder (T/F) viral genomes and their progeny. We used single-genome sequencing to show that inferred HCV T/F sequences in recipients were identical to viral sequences in their respective donors and that viral genomes generally evolved early in infection according to a simple model of random sequence evolution. Altogether, the findings validate T/F genome inferences and illustrate how T/F sequence identification can illuminate studies of HCV transmission, immunopathogenesis, drug resistance development, and vaccine protection, including sieving effects on breakthrough virus strains. Copyright © 2015 Li et al.

Fluid spatial dynamics of West Nile virus in the USA: Rapid spread in a permissive host environment

USGS Publications Warehouse

Di Giallonardo , Francesca; Geoghegan, Jemma L.; Docherty, Douglas E.; McLean, Robert G.; Zody, Michael C.; Qu, James; Yang, Xiao; Birren, Bruce W.; Malboeuf, Christine M.; Newman, R.; Ip, Hon S.; Holmes, Edward C.

2016-01-01

The introduction of West Nile virus (WNV) into North America in 1999 is a classical example of viral emergence in a new environment, with its subsequent dispersion across the continent having a major impact on local bird populations. Despite the importance of this epizootic, the pattern, dynamics and determinants of WNV spread in its natural hosts remain uncertain. In particular, it is unclear whether the virus encountered major barriers to transmission, or spread in an unconstrained manner, and if specific viral lineages were favored over others indicative of intrinsic differences in fitness. To address these key questions in WNV evolution and ecology we sequenced the complete genomes of approximately 300 avian isolates sampled across the USA between 2001-2012. Phylogenetic analysis revealed a relatively ‘star-like' tree structure, indicative of explosive viral spread in US, although with some replacement of viral genotypes through time. These data are striking in that viral sequences exhibit relatively limited clustering according to geographic region, particularly for those viruses sampled from birds, and no strong phylogenetic association with well sampled avian species. The genome sequence data analysed here also contain relatively little evidence for adaptive evolution, particularly on structural proteins, suggesting that most viral lineages are of similar fitness, and that WNV is well adapted to the ecology of mosquito vectors and diverse avian hosts in the USA. In sum, the molecular evolution of WNV in North America depicts a largely unfettered expansion within a permissive host and geographic population with little evidence of major adaptive barriers.

15year fulminant hepatitis B follow-up in Belgium: Viral evolution and signature of demographic change.

PubMed

Mina, Thomas; Amini-Bavil-Olyaee, Samad; Shirvani-Dastgerdi, Elham; Trovão, Nídia Sequeira; Van Ranst, Marc; Pourkarim, Mahmoud Reza

2017-04-01

Fulminant hepatitis among different clinical outcomes of hepatitis B virus infection is very rare and manifests high mortality rate, however it has not been investigated in Belgian inhabitants yet. In the frame of a retrospective study between 1995 and 2010, 80 serum samples (in some cases serial samples) archived in Biobank, were collected from 24 patients who had clinically developed fulminant infection of hepatitis B virus. In total, 33 hepatitis B virus (HBV) strains (31 full-length genome and 2 partial viral genes) of different HBV genotypes and subgenotypes including A2, B2, D1, D2, D3 and E, were amplified, sequenced and phylogenetically analyzed. HBV isolated strains from native and exotic patients were characterized by genome variations associated with viral invasiveness. Although several mutations at nucleotide and protein levels were detected, evolutionary analyses revealed a negative selective pressure over the viral genomes. This study revealed influence of immigration through a steady change in the viral epidemiological profile of the Belgian population. Copyright © 2017 Elsevier B.V. All rights reserved.

The rediscovery of smallpox.

PubMed

Thèves, C; Biagini, P; Crubézy, E

2014-03-01

Smallpox is an infectious disease that is unique to humans, caused by a poxvirus. It is one of the most lethal of diseases; the virus variant Variola major has a mortality rate of 30%. People surviving this disease have life-long consequences, but also assured immunity. Historically, smallpox was recognized early in human populations. This led to prevention attempts--variolation, quarantine, and the isolation of infected subjects--until Jenner's discovery of the first steps of vaccination in the 18th century. After vaccination campaigns throughout the 19th and 20th centuries, the WHO declared the eradication of smallpox in 1980. With the development of microscopy techniques, the structural characterization of the virus began in the early 20th century. In 1990, the genomes of different smallpox viruses were determined; viruses could be classified in order to investigate their origin, diffusion, and evolution. To study the evolution and possible re-emergence of this viral pathogen, however, researchers can only use viral genomes collected during the 20th century. Cases of smallpox in ancient periods are sometimes well documented, so palaeomicrobiology and, more precisely, the study of ancient smallpox viral strains could be an exceptional opportunity. The analysis of poxvirus fragmented genomes could give new insights into the genetic evolution of the poxvirus. Recently, small fragments of the poxvirus genome were detected. With the genetic information obtained, a new phylogeny of smallpox virus was described. The interest in conducting studies on ancient strains is discussed, in order to explore the natural history of this disease. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

An experimental and computational evolution-based method to study a mode of co-evolution of overlapping open reading frames in the AAV2 viral genome.

PubMed

Kawano, Yasuhiro; Neeley, Shane; Adachi, Kei; Nakai, Hiroyuki

2013-01-01

Overlapping open reading frames (ORFs) in viral genomes undergo co-evolution; however, how individual amino acids coded by overlapping ORFs are structurally, functionally, and co-evolutionarily constrained remains difficult to address by conventional homologous sequence alignment approaches. We report here a new experimental and computational evolution-based methodology to address this question and report its preliminary application to elucidating a mode of co-evolution of the frame-shifted overlapping ORFs in the adeno-associated virus (AAV) serotype 2 viral genome. These ORFs encode both capsid VP protein and non-structural assembly-activating protein (AAP). To show proof of principle of the new method, we focused on the evolutionarily conserved QVKEVTQ and KSKRSRR motifs, a pair of overlapping heptapeptides in VP and AAP, respectively. In the new method, we first identified a large number of capsid-forming VP3 mutants and functionally competent AAP mutants of these motifs from mutant libraries by experimental directed evolution under no co-evolutionary constraints. We used Illumina sequencing to obtain a large dataset and then statistically assessed the viability of VP and AAP heptapeptide mutants. The obtained heptapeptide information was then integrated into an evolutionary algorithm, with which VP and AAP were co-evolved from random or native nucleotide sequences in silico. As a result, we demonstrate that these two heptapeptide motifs could exhibit high degeneracy if coded by separate nucleotide sequences, and elucidate how overlap-evoked co-evolutionary constraints play a role in making the VP and AAP heptapeptide sequences into the present shape. Specifically, we demonstrate that two valine (V) residues and β-strand propensity in QVKEVTQ are structurally important, the strongly negative and hydrophilic nature of KSKRSRR is functionally important, and overlap-evoked co-evolution imposes strong constraints on serine (S) residues in KSKRSRR, despite high degeneracy of the motifs in the absence of co-evolutionary constraints.

Genome-Wide Networks of Amino Acid Covariances Are Common among Viruses

PubMed Central

Donlin, Maureen J.; Szeto, Brandon; Gohara, David W.; Aurora, Rajeev

2012-01-01

Coordinated variation among positions in amino acid sequence alignments can reveal genetic dependencies at noncontiguous positions, but methods to assess these interactions are incompletely developed. Previously, we found genome-wide networks of covarying residue positions in the hepatitis C virus genome (R. Aurora, M. J. Donlin, N. A. Cannon, and J. E. Tavis, J. Clin. Invest. 119:225–236, 2009). Here, we asked whether such networks are present in a diverse set of viruses and, if so, what they may imply about viral biology. Viral sequences were obtained for 16 viruses in 13 species from 9 families. The entire viral coding potential for each virus was aligned, all possible amino acid covariances were identified using the observed-minus-expected-squared algorithm at a false-discovery rate of ≤1%, and networks of covariances were assessed using standard methods. Covariances that spanned the viral coding potential were common in all viruses. In all cases, the covariances formed a single network that contained essentially all of the covariances. The hepatitis C virus networks had hub-and-spoke topologies, but all other networks had random topologies with an unusually large number of highly connected nodes. These results indicate that genome-wide networks of genetic associations and the coordinated evolution they imply are very common in viral genomes, that the networks rarely have the hub-and-spoke topology that dominates other biological networks, and that network topologies can vary substantially even within a given viral group. Five examples with hepatitis B virus and poliovirus are presented to illustrate how covariance network analysis can lead to inferences about viral biology. PMID:22238298

Genome Sequencing and Analysis of Geographically Diverse Clinical Isolates of Herpes Simplex Virus 2

PubMed Central

Lamers, Susanna L.; Weiner, Brian; Ray, Stuart C.; Colgrove, Robert C.; Diaz, Fernando; Jing, Lichen; Wang, Kening; Saif, Sakina; Young, Sarah; Henn, Matthew; Laeyendecker, Oliver; Tobian, Aaron A. R.; Cohen, Jeffrey I.; Koelle, David M.; Quinn, Thomas C.; Knipe, David M.

2015-01-01

ABSTRACT Herpes simplex virus 2 (HSV-2), the principal causative agent of recurrent genital herpes, is a highly prevalent viral infection worldwide. Limited information is available on the amount of genomic DNA variation between HSV-2 strains because only two genomes have been determined, the HG52 laboratory strain and the newly sequenced SD90e low-passage-number clinical isolate strain, each from a different geographical area. In this study, we report the nearly complete genome sequences of 34 HSV-2 low-passage-number and laboratory strains, 14 of which were collected in Uganda, 1 in South Africa, 11 in the United States, and 8 in Japan. Our analyses of these genomes demonstrated remarkable sequence conservation, regardless of geographic origin, with the maximum nucleotide divergence between strains being 0.4% across the genome. In contrast, prior studies indicated that HSV-1 genomes exhibit more sequence diversity, as well as geographical clustering. Additionally, unlike HSV-1, little viral recombination between HSV-2 strains could be substantiated. These results are interpreted in light of HSV-2 evolution, epidemiology, and pathogenesis. Finally, the newly generated sequences more closely resemble the low-passage-number SD90e than HG52, supporting the use of the former as the new reference genome of HSV-2. IMPORTANCE Herpes simplex virus 2 (HSV-2) is a causative agent of genital and neonatal herpes. Therefore, knowledge of its DNA genome and genetic variability is central to preventing and treating genital herpes. However, only two full-length HSV-2 genomes have been reported. In this study, we sequenced 34 additional HSV-2 low-passage-number and laboratory viral genomes and initiated analysis of the genetic diversity of HSV-2 strains from around the world. The analysis of these genomes will facilitate research aimed at vaccine development, diagnosis, and the evaluation of clinical manifestations and transmission of HSV-2. This information will also contribute to our understanding of HSV evolution. PMID:26018166

Identification of full-length transmitted/founder viruses and their progeny in primary HIV-1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korber, Bette; Hraber, Peter; Giorgi, Elena

2009-01-01

Identification of transmitted/founder virus genomes and their progeny by is a novel strategy for probing the molecular basis of HIV-1 transmission and for evaluating the genetic imprint of viral and host factors that act to constrain or facilitate virus replication. Here, we show in a cohort of twelve acutely infected subjects (9 clade B; 3 clade C), that complete genomic sequences of transmitted/founder viruses could be inferred using single genome amplification of plasma viral RNA, direct amplicon sequencing, and a model of random virus evolution. This allowed for the precise identification, chemical synthesis, molecular cloning, and biological analysis of thosemore » viruses actually responsible for productive clinical infection and for a comprehensive mapping of sequential viral genomes and proteomes for mutations that are necessary or incidental to the establishment of HIV-1 persistence. Transmitted/founder viruses were CD4 and CCR5 tropic, replicated preferentially in activated primary T-Iymphocytes but not monocyte-derived macrophages, and were effectively shielded from most heterologous or broadly neutralizing antibodies. By 3 months of infection, the evolving viral quasispecies in three subjects showed mutational fixation at only 2-5 discreet genomic loci. By 6-12 months, mutational fixation was evident at 18-27 genomic loci. Some, but not all, of these mutations were attributable to virus escape from cytotoxic Tlymphocytes or neutralizing antibodies, suggesting that other viral or host factors may influence early HIV -1 fitness.« less

Genomic reassortment of influenza A virus in North American swine, 1998–2011

PubMed Central

Detmer, Susan E.; Wentworth, David E.; Tan, Yi; Schwartzbard, Aaron; Halpin, Rebecca A.; Stockwell, Timothy B.; Lin, Xudong; Vincent, Amy L.; Gramer, Marie R.; Holmes, Edward C.

2012-01-01

Revealing the frequency and determinants of reassortment among RNA genome segments is fundamental to understanding basic aspects of the biology and evolution of the influenza virus. To estimate the extent of genomic reassortment in influenza viruses circulating in North American swine, we performed a phylogenetic analysis of 139 whole-genome viral sequences sampled during 1998–2011 and representing seven antigenically distinct viral lineages. The highest amounts of reassortment were detected between the H3 and the internal gene segments (PB2, PB1, PA, NP, M and NS), while the lowest reassortment frequencies were observed among the H1γ, H1pdm and neuraminidase segments, particularly N1. Less reassortment was observed among specific haemagglutinin–neuraminidase combinations that were more prevalent in swine, suggesting that some genome constellations may be evolutionarily more stable. PMID:22993190

Going, going, gone: predicting the fate of genomic insertions in plant RNA viruses.

PubMed

Willemsen, Anouk; Carrasco, José L; Elena, Santiago F; Zwart, Mark P

2018-05-10

Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate-the rate at which deletions removing the insertion occur-and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome

PubMed Central

Hartley, Carol A.; Vaz, Paola K.; Diaz-Méndez, Andrés; García, Maricarmen; Spatz, Stephen; Devlin, Joanne M.

2017-01-01

ABSTRACT Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome. IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease. PMID:28939604

Viral genome structures, charge, and sequences are optimal for capsid assembly

NASA Astrophysics Data System (ADS)

Hagan, Michael

2014-03-01

For many viruses, the spontaneous assembly of a capsid shell around the nu-cleic acid (NA) genome is an essential step in the viral life cycle. Capsid formation is a multicomponent, out-of-equilibrium assembly process for which kinetic effects and thermodynamic constraints compete to determine the outcome. Understand-ing how viral components drive highly efficient assembly under these constraints could promote biomedical efforts to block viral propagation, and would elucidate the factors controlling assembly in a wide range of systems containing proteins and polyelectrolytes. This talk will describe coarse-grained models of capsid proteins and NAs with which we investigate the dynamics and thermodynamics of virus assembly. In con-trast to recent theoretical models, we find that capsids spontaneously `overcharge' that is, the NA length which is kinetically and thermodynamically optimal possess-es a negative charge greater than the positive charge of the capsid. When applied to specific virus capsids, the calculated optimal NA lengths closely correspond to the natural viral genome lengths. These results suggest that the features included in this model (i.e. electrostatics, excluded volume, and NA tertiary structure) play key roles in determining assembly thermodynamics and consequently exert selec-tive pressure on viral evolution. I will then discuss mechanisms by which se-quence-specific interactions between NAs and capsid proteins promote selective encapsidation of the viral genome. This work was supported by NIH R01GM108021 and the Brandeis MRSEC NSF-MRSEC-0820492.

What can we learn about lyssavirus genomes using 454 sequencing?

PubMed

Höper, Dirk; Finke, Stefan; Freuling, Conrad M; Hoffmann, Bernd; Beer, Martin

2012-01-01

The main task of the individual project number four"Whole genome sequencing, virus-host adaptation, and molecular epidemiological analyses of lyssaviruses "within the network" Lyssaviruses--a potential re-emerging public health threat" is to provide high quality complete genome sequences from lyssaviruses. These sequences are analysed in-depth with regard to the diversity of the viral populations as to both quasi-species and so-called defective interfering RNAs. Moreover, the sequence data will facilitate further epidemiological analyses, will provide insight into the evolution of lyssaviruses and will be the basis for the design of novel nucleic acid based diagnostics. The first results presented here indicate that not only high quality full-length lyssavirus genome sequences can be generated, but indeed efficient analysis of the viral population gets feasible.

Evolution of puma lentivirus in bobcats (Lynx rufus) and mountain lions (Puma concolor) in North America.

PubMed

Lee, Justin S; Bevins, Sarah N; Serieys, Laurel E K; Vickers, Winston; Logan, Ken A; Aldredge, Mat; Boydston, Erin E; Lyren, Lisa M; McBride, Roy; Roelke-Parker, Melody; Pecon-Slattery, Jill; Troyer, Jennifer L; Riley, Seth P; Boyce, Walter M; Crooks, Kevin R; VandeWoude, Sue

2014-07-01

Mountain lions (Puma concolor) throughout North and South America are infected with puma lentivirus clade B (PLVB). A second, highly divergent lentiviral clade, PLVA, infects mountain lions in southern California and Florida. Bobcats (Lynx rufus) in these two geographic regions are also infected with PLVA, and to date, this is the only strain of lentivirus identified in bobcats. We sequenced full-length PLV genomes in order to characterize the molecular evolution of PLV in bobcats and mountain lions. Low sequence homology (88% average pairwise identity) and frequent recombination (1 recombination breakpoint per 3 isolates analyzed) were observed in both clades. Viral proteins have markedly different patterns of evolution; sequence homology and negative selection were highest in Gag and Pol and lowest in Vif and Env. A total of 1.7% of sites across the PLV genome evolve under positive selection, indicating that host-imposed selection pressure is an important force shaping PLV evolution. PLVA strains are highly spatially structured, reflecting the population dynamics of their primary host, the bobcat. In contrast, the phylogeography of PLVB reflects the highly mobile mountain lion, with diverse PLVB isolates cocirculating in some areas and genetically related viruses being present in populations separated by thousands of kilometers. We conclude that PLVA and PLVB are two different viral species with distinct feline hosts and evolutionary histories. Importance: An understanding of viral evolution in natural host populations is a fundamental goal of virology, molecular biology, and disease ecology. Here we provide a detailed analysis of puma lentivirus (PLV) evolution in two natural carnivore hosts, the bobcat and mountain lion. Our results illustrate that PLV evolution is a dynamic process that results from high rates of viral mutation/recombination and host-imposed selection pressure. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Structural and Functional Basis of the Fidelity of Nucleotide Selection by Flavivirus RNA-Dependent RNA Polymerases

PubMed Central

Canard, Bruno

2018-01-01

Viral RNA-dependent RNA polymerases (RdRps) play a central role not only in viral replication, but also in the genetic evolution of viral RNAs. After binding to an RNA template and selecting 5′-triphosphate ribonucleosides, viral RdRps synthesize an RNA copy according to Watson-Crick base-pairing rules. The copy process sometimes deviates from both the base-pairing rules specified by the template and the natural ribose selectivity and, thus, the process is error-prone due to the intrinsic (in)fidelity of viral RdRps. These enzymes share a number of conserved amino-acid sequence strings, called motifs A–G, which can be defined from a structural and functional point-of-view. A co-relation is gradually emerging between mutations in these motifs and viral genome evolution or observed mutation rates. Here, we review our current knowledge on these motifs and their role on the structural and mechanistic basis of the fidelity of nucleotide selection and RNA synthesis by Flavivirus RdRps. PMID:29385764

Within-Host Evolution of Simian Arteriviruses in Crab-Eating Macaques

PubMed Central

Moncla, Louise H.; Weiler, Andrea M.; Barry, Gabrielle; Weinfurter, Jason T.; Dinis, Jorge M.; Charlier, Olivia; Lauck, Michael; Bailey, Adam L.; Wahl-Jensen, Victoria; Nelson, Chase W.; Johnson, Joshua C.; Caì, Yíngyún; Goldberg, Tony L.; O'Connor, David H.; Jahrling, Peter B.

2016-01-01

ABSTRACT Simian arteriviruses are a diverse clade of viruses infecting captive and wild nonhuman primates. We recently reported that Kibale red colobus virus 1 (KRCV-1) causes a mild and self-limiting disease in experimentally infected crab-eating macaques, while simian hemorrhagic fever virus (SHFV) causes lethal viral hemorrhagic fever. Here we characterize how these viruses evolved during replication in cell culture and in experimentally infected macaques. During passage in cell culture, 68 substitutions that were localized in open reading frames (ORFs) likely associated with host cell entry and exit became fixed in the KRCV-1 genome. However, we did not detect any strong signatures of selection during replication in macaques. We uncovered patterns of evolution that were distinct from those observed in surveys of wild red colobus monkeys, suggesting that these species may exert different adaptive challenges for KRCV-1. During SHFV infection, we detected signatures of selection on ORF 5a and on a small subset of sites in the genome. Overall, our data suggest that patterns of evolution differ markedly among simian arteriviruses and among host species. IMPORTANCE Certain RNA viruses can cross species barriers and cause disease in new hosts. Simian arteriviruses are a diverse group of related viruses that infect captive and wild nonhuman primates, with associated disease severity ranging from apparently asymptomatic infections to severe, viral hemorrhagic fevers. We infected nonhuman primate cell cultures and then crab-eating macaques with either simian hemorrhagic fever virus (SHFV) or Kibale red colobus virus 1 (KRCV-1) and assessed within-host viral evolution. We found that KRCV-1 quickly acquired a large number of substitutions in its genome during replication in cell culture but that evolution in macaques was limited. In contrast, we detected selection focused on SHFV ORFs 5a and 5, which encode putative membrane proteins. These patterns suggest that in addition to diverse pathogenic phenotypes, these viruses may also exhibit distinct patterns of within-host evolution both in vitro and in vivo. PMID:27974564

Genome sequences of seven foot-and-mouth disease virus isolates collected from serial samples from one persistently infected carrier cow in Vietnam

USDA-ARS?s Scientific Manuscript database

Several FMDV carrier cattle were identified in Vietnam by recovery of infectious virus from oropharyngeal fluid. This report contains the first near-complete genome sequences of seven viruses isolated from a single carrier animal over the course of one year. Understanding within-host viral evolution...

Probing the Structures of Viral RNA Regulatory Elements with SHAPE and Related Methodologies

PubMed Central

Rausch, Jason W.; Sztuba-Solinska, Joanna; Le Grice, Stuart F. J.

2018-01-01

Viral RNAs were selected by evolution to possess maximum functionality in a minimal sequence. Depending on the classification of the virus and the type of RNA in question, viral RNAs must alternately be replicated, spliced, transcribed, transported from the nucleus into the cytoplasm, translated and/or packaged into nascent virions, and in most cases, provide the sequence and structural determinants to facilitate these processes. One consequence of this compact multifunctionality is that viral RNA structures can be exquisitely complex, often involving intermolecular interactions with RNA or protein, intramolecular interactions between sequence segments separated by several thousands of nucleotides, or specialized motifs such as pseudoknots or kissing loops. The fluidity of viral RNA structure can also present a challenge when attempting to characterize it, as genomic RNAs especially are likely to sample numerous conformations at various stages of the virus life cycle. Here we review advances in chemoenzymatic structure probing that have made it possible to address such challenges with respect to cis-acting elements, full-length viral genomes and long non-coding RNAs that play a major role in regulating viral gene expression. PMID:29375504

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

PubMed Central

Quick, Josh; Grubaugh, Nathan D; Pullan, Steven T; Claro, Ingra M; Smith, Andrew D; Gangavarapu, Karthik; Oliveira, Glenn; Robles-Sikisaka, Refugio; Rogers, Thomas F; Beutler, Nathan A; Burton, Dennis R; Lewis-Ximenez, Lia Laura; de Jesus, Jaqueline Goes; Giovanetti, Marta; Hill, Sarah; Black, Allison; Bedford, Trevor; Carroll, Miles W; Nunes, Marcio; Alcantara, Luiz Carlos; Sabino, Ester C; Baylis, Sally A; Faria, Nuno; Loose, Matthew; Simpson, Jared T; Pybus, Oliver G; Andersen, Kristian G; Loman, Nicholas J

2018-01-01

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. PMID:28538739

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.

PubMed

Quick, Joshua; Grubaugh, Nathan D; Pullan, Steven T; Claro, Ingra M; Smith, Andrew D; Gangavarapu, Karthik; Oliveira, Glenn; Robles-Sikisaka, Refugio; Rogers, Thomas F; Beutler, Nathan A; Burton, Dennis R; Lewis-Ximenez, Lia Laura; de Jesus, Jaqueline Goes; Giovanetti, Marta; Hill, Sarah C; Black, Allison; Bedford, Trevor; Carroll, Miles W; Nunes, Marcio; Alcantara, Luiz Carlos; Sabino, Ester C; Baylis, Sally A; Faria, Nuno R; Loose, Matthew; Simpson, Jared T; Pybus, Oliver G; Andersen, Kristian G; Loman, Nicholas J

2017-06-01

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.

[The great virus comeback].

PubMed

Forterre, Patrick

2013-01-01

Viruses have been considered for a long time as by-products of biological evolution. This view is changing now as a result of several recent discoveries. Viral ecologists have shown that viral particles are the most abundant biological entities on our planet, whereas metagenomic analyses have revealed an unexpected abundance and diversity of viral genes in the biosphere. Comparative genomics have highlighted the uniqueness of viral sequences, in contradiction with the traditional view of viruses as pickpockets of cellular genes. On the contrary, cellular genomes, especially eukaryotic ones, turned out to be full of genes derived from viruses or related elements (plasmids, transposons, retroelements and so on). The discovery of unusual viruses infecting archaea has shown that the viral world is much more diverse than previously thought, ruining the traditional dichotomy between bacteriophages and viruses. Finally, the discovery of giant viruses has blurred the traditional image of viruses as small entities. Furthermore, essential clues on virus history have been obtained in the last ten years. In particular, structural analyses of capsid proteins have uncovered deeply rooted homologies between viruses infecting different cellular domains, suggesting that viruses originated before the last universal common ancestor (LUCA). These studies have shown that several lineages of viruses originated independently, i.e., viruses are polyphyletic. From the time of LUCA, viruses have coevolved with their hosts, and viral lineages can be viewed as lianas wrapping around the trunk, branches and leaves of the tree of life. Although viruses are very diverse, with genomes encoding from one to more than one thousand proteins, they can all be simply defined as organisms producing virions. Virions themselves can be defined as infectious particles made of at least one protein associated with the viral nucleic acid, endowed with the capability to protect the viral genome and ensure its delivery to the infected cell. These definitions, which clearly distinguish viruses from plasmids, suggest that infectious RNA molecules that only encode an RNA replicase presently classified among viruses by the ICTV (International Committee for the Taxonomy of Viruses) into families of Endornaviridae and Hypoviridae are in fact RNA plasmids. Since a viral genome should encode for at least one structural protein, these definitions also imply that viruses originated after the emergence of the ribosome in an RNA-protein cellular world. Although virions are the hallmarks of viruses, viruses and virions should not be confused. The infection transforms the ribocell (cell encoding ribosomes and dividing by binary fission) into a virocell (cell producing virions) or ribovirocell (cell that produces virions but can still divide by binary fission). In the ribovirocell, two different organisms, defined by their distinct evolutionary histories, coexist in symbiosis in the same cell. The virocells or ribovirocells are the living forms of the virus, which can be in fine considered to be a living organism. In the virocell, the metabolism is reorganized for the production of virions, while the ability to capture and store free energy is retained, as in other cellular organisms. In the virocell, viral genomes replicate, recombine and evolve, leading to the emergence of new viral proteins and potentially novel functions. Some of these new functions can be later on transferred to the cell, explaining how viruses can play a major (often underestimated) role in the evolution of cellular organisms. The virocell concept thus helps to understand recent hypotheses suggesting that viruses played a critical role in major evolutionary transitions, such as the origin of DNA genomes or else the origin of the eukaryotic nucleus. Finally, it is more and more recognized that viruses are the major source of variation and selection in living organisms (both viruses and cells), the two pillars of darwinism. One can thus conclude that the continuous interaction between viruses and cells, all along the history of life, has been, and still is, a major engine of biological evolution. © Société de Biologie, 2013.

Tracking interspecies transmission and long-term evolution of an ancient retrovirus using the genomes of modern mammals

PubMed Central

Diehl, William E; Patel, Nirali; Halm, Kate; Johnson, Welkin E

2016-01-01

Mammalian genomes typically contain hundreds of thousands of endogenous retroviruses (ERVs), derived from ancient retroviral infections. Using this molecular 'fossil' record, we reconstructed the natural history of a specific retrovirus lineage (ERV-Fc) that disseminated widely between ~33 and ~15 million years ago, corresponding to the Oligocene and early Miocene epochs. Intercontinental viral spread, numerous instances of interspecies transmission and emergence in hosts representing at least 11 mammalian orders, and a significant role for recombination in diversification of this viral lineage were also revealed. By reconstructing the canonical retroviral genes, we identified patterns of adaptation consistent with selection to maintain essential viral protein functions. Our results demonstrate the unique potential of the ERV fossil record for studying the processes of viral spread and emergence as they play out across macro-evolutionary timescales, such that looking back in time may prove insightful for predicting the long-term consequences of newly emerging viral infections. DOI: http://dx.doi.org/10.7554/eLife.12704.001 PMID:26952212

Tracking interspecies transmission and long-term evolution of an ancient retrovirus using the genomes of modern mammals.

PubMed

Diehl, William E; Patel, Nirali; Halm, Kate; Johnson, Welkin E

2016-03-08

Mammalian genomes typically contain hundreds of thousands of endogenous retroviruses (ERVs), derived from ancient retroviral infections. Using this molecular 'fossil' record, we reconstructed the natural history of a specific retrovirus lineage (ERV-Fc) that disseminated widely between ~33 and ~15 million years ago, corresponding to the Oligocene and early Miocene epochs. Intercontinental viral spread, numerous instances of interspecies transmission and emergence in hosts representing at least 11 mammalian orders, and a significant role for recombination in diversification of this viral lineage were also revealed. By reconstructing the canonical retroviral genes, we identified patterns of adaptation consistent with selection to maintain essential viral protein functions. Our results demonstrate the unique potential of the ERV fossil record for studying the processes of viral spread and emergence as they play out across macro-evolutionary timescales, such that looking back in time may prove insightful for predicting the long-term consequences of newly emerging viral infections.

A traditional evolutionary history of foot-and-mouth disease viruses in Southeast Asia challenged by analyses of non-structural protein coding sequences

USDA-ARS?s Scientific Manuscript database

Molecular epidemiology and evolution of foot-and-mouth disease virus (FMDV) are widely studied using genomic sequences encoding VP1, the capsid protein containing the most relevant antigenic domains. Although sequencing of the full viral genome is not used as a routine diagnostic or surveillance too...

Using hidden Markov models and observed evolution to annotate viral genomes.

PubMed

McCauley, Stephen; Hein, Jotun

2006-06-01

ssRNA (single stranded) viral genomes are generally constrained in length and utilize overlapping reading frames to maximally exploit the coding potential within the genome length restrictions. This overlapping coding phenomenon leads to complex evolutionary constraints operating on the genome. In regions which code for more than one protein, silent mutations in one reading frame generally have a protein coding effect in another. To maximize coding flexibility in all reading frames, overlapping regions are often compositionally biased towards amino acids which are 6-fold degenerate with respect to the 64 codon alphabet. Previous methodologies have used this fact in an ad hoc manner to look for overlapping genes by motif matching. In this paper differentiated nucleotide compositional patterns in overlapping regions are incorporated into a probabilistic hidden Markov model (HMM) framework which is used to annotate ssRNA viral genomes. This work focuses on single sequence annotation and applies an HMM framework to ssRNA viral annotation. A description of how the HMM is parameterized, whilst annotating within a missing data framework is given. A Phylogenetic HMM (Phylo-HMM) extension, as applied to 14 aligned HIV2 sequences is also presented. This evolutionary extension serves as an illustration of the potential of the Phylo-HMM framework for ssRNA viral genomic annotation. The single sequence annotation procedure (SSA) is applied to 14 different strains of the HIV2 virus. Further results on alternative ssRNA viral genomes are presented to illustrate more generally the performance of the method. The results of the SSA method are encouraging however there is still room for improvement, and since there is overwhelming evidence to indicate that comparative methods can improve coding sequence (CDS) annotation, the SSA method is extended to a Phylo-HMM to incorporate evolutionary information. The Phylo-HMM extension is applied to the same set of 14 HIV2 sequences which are pre-aligned. The performance improvement that results from including the evolutionary information in the analysis is illustrated.

Evolution of bird genomes-a transposon's-eye view.

PubMed

Kapusta, Aurélie; Suh, Alexander

2017-02-01

Birds, the most species-rich monophyletic group of land vertebrates, have been subject to some of the most intense sequencing efforts to date, making them an ideal case study for recent developments in genomics research. Here, we review how our understanding of bird genomes has changed with the recent sequencing of more than 75 species from all major avian taxa. We illuminate avian genome evolution from a previously neglected perspective: their repetitive genomic parasites, transposable elements (TEs) and endogenous viral elements (EVEs). We show that (1) birds are unique among vertebrates in terms of their genome organization; (2) information about the diversity of avian TEs and EVEs is changing rapidly; (3) flying birds have smaller genomes yet more TEs than flightless birds; (4) current second-generation genome assemblies fail to capture the variation in avian chromosome number and genome size determined with cytogenetics; (5) the genomic microcosm of bird-TE "arms races" has yet to be explored; and (6) upcoming third-generation genome assemblies suggest that birds exhibit stability in gene-rich regions and instability in TE-rich regions. We emphasize that integration of cytogenetics and single-molecule technologies with repeat-resolved genome assemblies is essential for understanding the evolution of (bird) genomes. © 2016 New York Academy of Sciences.

Genomic characterization of H14 subtype influenza A viruses in New World waterfowl and experimental infectivity in mallards Anas platyrhynchos

USGS Publications Warehouse

Ramey, Andy M.; Poulson, Rebecca L.; Gonzalez-Reiche, Ana S.; Perez, Daniel R.; Stalknecht, David E.; Brown, Justin D.

2014-01-01

Recent repeated isolation of H14 hemagglutinin subtype influenza A viruses (IAVs) in the New World waterfowl provides evidence to suggest that host and/or geographic ranges for viruses of this subtype may be expanding. In this study, we used genomic analyses to gain inference on the origin and evolution of H14 viruses in New World waterfowl and conducted an experimental challenge study in mallards (Anas platyrhynchos) to evaluate pathogenicity, viral replication, and transmissibility of a representative viral strain in a natural host species. Genomic characterization of H14 subtype IAVs isolated from New World waterfowl, including three isolates sequenced specifically for this study, revealed high nucleotide identity among individual gene segments (e.g. ≥95% shared identity among H14 HA gene segments). In contrast, lower shared identity was observed among internal gene segments. Furthermore, multiple neuraminidase subtypes were observed for H14 IAVs isolated in the New World. Gene segments of H14 viruses isolated after 2010 shared ancestral genetic lineages with IAVs isolated from wild birds throughout North America. Thus, genomic characterization provided evidence for viral evolution in New World waterfowl through genetic drift and genetic shift since purported introduction from Eurasia. In the challenge study, no clinical disease or lesions were observed among mallards experimentally inoculated with A/blue-winged teal/Texas/AI13-1028/2013(H14N5) or exposed via contact with infected birds. Titers of viral shedding for mallards challenged with the H14N5 IAV were highest at two days post-inoculation (DPI); however shedding was detected up to nine DPI using cloacal swabs. The distribution of viral antigen among mallards infected with H14N5 IAV was largely restricted to enterocytes lining the villi in the lower intestinal tract and in the epithelium of the bursa of Fabricius. Characterization of the infectivity of A/blue-winged teal/Texas/AI13-1028/2013(H14N5) in mallards provides support for similarities in viral replication and shedding as compared to previously described waterfowl-adapted, low pathogenic IAV strains in ducks.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome.

PubMed

Loncoman, Carlos A; Hartley, Carol A; Coppo, Mauricio J C; Vaz, Paola K; Diaz-Méndez, Andrés; Browning, Glenn F; García, Maricarmen; Spatz, Stephen; Devlin, Joanne M

2017-12-01

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1 ) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome. IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease. Copyright © 2017 American Society for Microbiology.

Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

PubMed

Luteijn, Rutger David; Drexler, Ingo; Smith, Geoffrey L; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

2018-06-01

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

A virocentric perspective on the evolution of life

PubMed Central

Koonin, Eugene V.; Dolja, Valerian V.

2015-01-01

Viruses and/or virus-like selfish elements are associated with all cellular life forms and are the most abundant biological entities on Earth, with the number of virus particles in many environments exceeding the number of cells by one to two orders of magnitude. The genetic diversity of viruses is commensurately enormous and might substantially exceed the diversity of cellular organisms. Unlike cellular organisms with their uniform replication-expression scheme, viruses possess either RNA or DNA genomes and exploit all conceivable replication-expression strategies. Although viruses extensively exchange genes with their hosts, there exists a set of viral hallmark genes that are shared by extremely diverse groups of viruses to the exclusion of cellular life forms. Coevolution of viruses and host defense systems is a key aspect in the evolution of both viruses and cells, and viral genes are often recruited for cellular functions. Together with the fundamental inevitability of the emergence of genomic parasites in any evolving replicator system, these multiple lines of evidence reveal the central role of viruses in the entire evolution of life. PMID:23850169

2D-dynamic representation of DNA sequences as a graphical tool in bioinformatics

NASA Astrophysics Data System (ADS)

Bielińska-Wa̧Ż, D.; Wa̧Ż, P.

2016-10-01

2D-dynamic representation of DNA sequences is briefly reviewed. Some new examples of 2D-dynamic graphs which are the graphical tool of the method are shown. Using the examples of the complete genome sequences of the Zika virus it is shown that the present method can be applied for the study of the evolution of viral genomes.

Genome sequence of a novel H14N7 subtype influenza A virus isolated from a blue-winged teal (Anas discors) harvested in Texas, USA

USGS Publications Warehouse

Ramey, Andy M.; Reeves, Andrew; Poulson, Rebecca L.; Carter, Deborah L.; Davis-Fields, Nicholas; Stallknecht, David E.

2016-01-01

We report here the complete genome sequence of a novel H14N7 subtype influenza A virus (IAV) isolated from a blue-winged teal (Anas discors) harvested in Texas, USA. The genomic characteristics of this IAV strain with a previously undetected subtype combination suggest recent viral evolution within the New World wild-bird IAV reservoir.                   

How endogenous plant pararetroviruses shed light on Musa evolution

PubMed Central

Duroy, Pierre-Olivier; Perrier, Xavier; Laboureau, Nathalie; Jacquemoud-Collet, Jean-Pierre; Iskra-Caruana, Marie-Line

2016-01-01

Background and Aims Banana genomes harbour numerous copies of viral sequences derived from banana streak viruses (BSVs) – dsDNA viruses belonging to the family Caulimoviridae. These viral integrants (eBSVs) are mostly defective, probably as a result of ‘pseudogenization’ driven by host genome evolution. However, some can give rise to infection by releasing a functional viral genome following abiotic stresses. These distinct infective eBSVs correspond to the three main widespread BSV species (BSOLV, BSGFV and BSIMV), fully described within the Musa balbisiana B genomes of the seedy diploid ‘Pisang Klutuk Wulung’ (PKW). Methods We characterize eBSV distribution among a Musa sampling including seedy BB diploids and interspecific hybrids with Musa acuminata exhibiting different levels of ploidy for the B genome (ABB, AAB, AB). We used representative samples of the two areas of sympatry between M. acuminata and M. balbisiana species representing the native area of the most widely cultivated AAB cultivars (in India and in East Asia, ranging from the Philippines to New Guinea). Seventy-seven accessions were characterized using eBSV-related PCR markers and Southern hybridization approaches. We coded both sets of results to create a common dissimilarity matrix with which to interpret eBSV distribution. Key Results We propose a Musa phylogeny driven by the M. balbisiana genome based on a dendrogram resulting from a joint neighbour-joining analysis of the three BSV species, showing for the first time lineages between BB and ABB/AAB hybrids. eBSVs appear to be relevant phylogenetic markers that can illustrate the M. balbisiana phylogeography story. Conclusion The theoretical implications of this study for further elucidation of the historical and geographical process of Musa domestication are numerous. Discovery of banana plants with B genome non-infective for eBSV opens the way to the introduction of new genitors in programmes of genetic banana improvement. PMID:26971286

How endogenous plant pararetroviruses shed light on Musa evolution.

PubMed

Duroy, Pierre-Olivier; Perrier, Xavier; Laboureau, Nathalie; Jacquemoud-Collet, Jean-Pierre; Iskra-Caruana, Marie-Line

2016-04-01

Banana genomes harbour numerous copies of viral sequences derived from banana streak viruses (BSVs) - dsDNA viruses belonging to the family Caulimoviridae.These viral integrants (eBSVs) are mostly defective, probably as a result of 'pseudogenization' driven by host genome evolution. However, some can give rise to infection by releasing a functional viral genome following abiotic stresses. These distinct infective eBSVs correspond to the three main widespread BSV species (BSOLV, BSGFV and BSIMV), fully described within the Musa balbisiana B genomes of the seedy diploid 'Pisang Klutuk Wulung' (PKW). We characterize eBSV distribution among a Musa sampling including seedy BB diploids and interspecific hybrids with Musa acuminate exhibiting different levels of ploidy for the B genome (ABB, AAB, AB). We used representative samples of the two areas of sympatry between M. acuminate and M. balbisiana species representing the native area of the most widely cultivated AAB cultivars (in India and in East Asia, ranging from the Philippines to New Guinea). Seventy-seven accessions were characterized using eBSV-related PCR markers and Southern hybridization approaches. We coded both sets of results to create a common dissimilarity matrix with which to interpret eBSV distribution. We propose a Musa phylogeny driven by the M. balbisiana genome based on a dendrogram resulting from a joint neighbour-joining analysis of the three BSV species, showing for the first time lineages between BB and ABB/AAB hybrids. eBSVs appear to be relevant phylogenetic markers that can illustrate theM. balbisiana phylogeography story. The theoretical implications of this study for further elucidation of the historical and geographical process of Musa domestication are numerous. Discovery of banana plants with B genome non-infective for eBSV opens the way to the introduction of new genitors in programmes of genetic banana improvement. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Molecular Evolution and Intraclade Recombination of Enterovirus D68 during the 2014 Outbreak in the United States

PubMed Central

Tan, Yi; Hassan, Ferdaus; Schuster, Jennifer E.; Simenauer, Ari; Selvarangan, Rangaraj; Halpin, Rebecca A.; Lin, Xudong; Fedorova, Nadia; Stockwell, Timothy B.; Lam, Tommy Tsan-Yuk; Chappell, James D.; Hartert, Tina V.; Holmes, Edward C.

2015-01-01

ABSTRACT In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies. IMPORTANCE Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68. PMID:26656685

The molecular biology and evolution of feline immunodeficiency viruses of cougars

PubMed Central

Poss, Mary; Ross, Howard; Rodrigo, Allen; Terwee, Julie; VandeWoude, Sue; Biek, Roman

2008-01-01

Feline immunodeficiency virus (FIV) is a lentivirus that has been identified in many members of the family Felidae but domestic cats are the only FIV host in which infection results in disease. We studied FIVpco infection of cougars (Puma concolor) as a model for asymptomatic lentivirus infections to understand the mechanisms of host-virus coexistence. Several natural cougar populations were evaluated to determine if there are any consequences of FIVpco infection on cougar fecundity, survival, or susceptibility to other infections. We have sequenced full length viral genomes and conducted a detailed analysis of viral molecular evolution on these sequences and on genome fragments of serially sampled animals to determine the evolutionary forces experienced by this virus in cougars. In addition, we have evaluated the molecular genetics of FIVpco in a new host, domestic cats, to determine the evolutionary consequences to a host-adapted virus associated with cross-species infection. Our results indicate that there are no significant differences in survival, fecundity or susceptibility to other infections between FIVpco-infected and uninfected cougars. The molecular evolution of FIVpco is characterized by a slower evolutionary rate and an absence of positive selection, but also by proviral and plasma viral loads comparable to those of epidemic lentiviruses such as HIV-1 or FIVfca. Evolutionary and recombination rates and selection profiles change significantly when FIVpco replicates in a new host. PMID:18295904

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation

PubMed Central

Nikolaitchik, Olga A.; Burdick, Ryan C.; Gorelick, Robert J.; Keele, Brandon F.; Hu, Wei-Shau; Pathak, Vinay K.

2016-01-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10−5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10−21 and1 × 10−11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication. PMID:27186986

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.

PubMed

Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K

2016-05-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication.

Exploration of Panviral Proteome: High-Throughput Cloning and Functional Implications in Virus-host Interactions

PubMed Central

Yu, Xiaobo; Bian, Xiaofang; Throop, Andrea; Song, Lusheng; Moral, Lerys Del; Park, Jin; Seiler, Catherine; Fiacco, Michael; Steel, Jason; Hunter, Preston; Saul, Justin; Wang, Jie; Qiu, Ji; Pipas, James M.; LaBaer, Joshua

2014-01-01

Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies. PMID:24955142

Exploration of panviral proteome: high-throughput cloning and functional implications in virus-host interactions.

PubMed

Yu, Xiaobo; Bian, Xiaofang; Throop, Andrea; Song, Lusheng; Moral, Lerys Del; Park, Jin; Seiler, Catherine; Fiacco, Michael; Steel, Jason; Hunter, Preston; Saul, Justin; Wang, Jie; Qiu, Ji; Pipas, James M; LaBaer, Joshua

2014-01-01

Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

PubMed Central

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

2017-01-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

Preliminary Evaluation of the Effect of Investigational Ebola Virus Disease Treatments on Viral Genome Sequences.

PubMed

Whitmer, Shannon L M; Albariño, César; Shepard, Samuel S; Dudas, Gytis; Sheth, Mili; Brown, Shelley C; Cannon, Deborah; Erickson, Bobbie R; Gibbons, Aridth; Schuh, Amy; Sealy, Tara; Ervin, Elizabeth; Frace, Mike; Uyeki, Timothy M; Nichol, Stuart T; Ströher, Ute

2016-10-15

 Several patients with Ebola virus disease (EVD) managed in the United States have received ZMapp monoclonal antibodies, TKM-Ebola small interfering RNA, brincidofovir, and/or convalescent plasma as investigational therapeutics.  To investigate whether treatment selected for Ebola virus (EBOV) mutations conferring resistance, viral sequencing was performed on RNA extracted from clinical blood specimens from patients with EVD following treatment, and putative viral targets were analyzed.  We observed no major or minor EBOV mutations within regions targeted by therapeutics.  This small subset of patients and clinical specimens suggests that evolution of resistance is not a direct consequence of antiviral treatment. As EVD antiviral treatments are introduced into wider use, it is essential that continuous viral full-genome surveillance is performed, to monitor for the emergence of escape mutations. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Molecular hyperdiversity and evolution in very large populations.

PubMed

Cutter, Asher D; Jovelin, Richard; Dey, Alivia

2013-04-01

The genomic density of sequence polymorphisms critically affects the sensitivity of inferences about ongoing sequence evolution, function and demographic history. Most animal and plant genomes have relatively low densities of polymorphisms, but some species are hyperdiverse with neutral nucleotide heterozygosity exceeding 5%. Eukaryotes with extremely large populations, mimicking bacterial and viral populations, present novel opportunities for studying molecular evolution in sexually reproducing taxa with complex development. In particular, hyperdiverse species can help answer controversial questions about the evolution of genome complexity, the limits of natural selection, modes of adaptation and subtleties of the mutation process. However, such systems have some inherent complications and here we identify topics in need of theoretical developments. Close relatives of the model organisms Caenorhabditis elegans and Drosophila melanogaster provide known examples of hyperdiverse eukaryotes, encouraging functional dissection of resulting molecular evolutionary patterns. We recommend how best to exploit hyperdiverse populations for analysis, for example, in quantifying the impact of noncrossover recombination in genomes and for determining the identity and micro-evolutionary selective pressures on noncoding regulatory elements. © 2013 Blackwell Publishing Ltd.

Whole-genome characterization of Uruguayan strains of avian infectious bronchitis virus reveals extensive recombination between the two major South American lineages.

PubMed

Marandino, Ana; Tomás, Gonzalo; Panzera, Yanina; Greif, Gonzalo; Parodi-Talice, Adriana; Hernández, Martín; Techera, Claudia; Hernández, Diego; Pérez, Ruben

2017-10-01

Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus that causes one of the most persistent respiratory diseases in poultry. The virus is classified in genotypes and lineages with different epidemiological relevance. Two lineages of the GI genotype (11 and 16) have been widely circulating for decades in South America. GI-11 is an exclusive South American lineage while the GI-16 lineage is distributed in Asia, Europe and South America. Here, we obtained the whole genome of two Uruguayan strains of the GI-11 and GI-16 lineages using Illumina high-throughput sequencing. The strains here sequenced are the first obtained in South America for the infectious bronchitis virus and provide new insights into the origin, spreading and evolution of viral variants. The complete genome of the GI-11 and GI-16 strains have 27,621 and 27,638 nucleotides, respectively, and possess the same genomic organization. Phylogenetic incongruence analysis reveals that both strains have a mosaic genome that arose by recombination between Euro Asiatic strains of the GI-16 lineage and ancestral South American GI-11 viruses. The recombination occurred in South America and produced two viral variants that have retained the full-length S1 sequences of the parental lineages but are extremely similar in the rest of their genomes. These recombinant virus have been extraordinary successful, persisting in the continent for several years with a notorious wide geographic distribution. Our findings reveal a singular viral dynamics and emphasize the importance of complete genomic characterization to understand the emergence and evolutionary history of viral variants. Copyright © 2017 Elsevier B.V. All rights reserved.

Hepatitis C virus quasispecies and pseudotype analysis from acute infection to chronicity in HIV-1 co-infected individuals.

PubMed

Ferns, R Bridget; Tarr, Alexander W; Hue, Stephane; Urbanowicz, Richard A; McClure, C Patrick; Gilson, Richard; Ball, Jonathan K; Nastouli, Eleni; Garson, Jeremy A; Pillay, Deenan

2016-05-01

HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days). We observed that one to three founder viruses were transmitted. Relatively low viral sequence diversity, possibly related to an impaired immune response, due to HIV infection was observed in three patients. However, the fourth patient, after an early purifying selection displayed increasing E2 sequence evolution, possibly related to being on suppressive antiretroviral therapy. Viral pseudotypes generated from HCV variants showed relative resistance to neutralization by autologous plasma but not to plasma collected from later time points, confirming ongoing virus escape from antibody neutralization. Copyright © 2016 Elsevier Inc. All rights reserved.

Influenza Virus Database (IVDB): an integrated information resource and analysis platform for influenza virus research.

PubMed

Chang, Suhua; Zhang, Jiajie; Liao, Xiaoyun; Zhu, Xinxing; Wang, Dahai; Zhu, Jiang; Feng, Tao; Zhu, Baoli; Gao, George F; Wang, Jian; Yang, Huanming; Yu, Jun; Wang, Jing

2007-01-01

Frequent outbreaks of highly pathogenic avian influenza and the increasing data available for comparative analysis require a central database specialized in influenza viruses (IVs). We have established the Influenza Virus Database (IVDB) to integrate information and create an analysis platform for genetic, genomic, and phylogenetic studies of the virus. IVDB hosts complete genome sequences of influenza A virus generated by Beijing Institute of Genomics (BIG) and curates all other published IV sequences after expert annotation. Our Q-Filter system classifies and ranks all nucleotide sequences into seven categories according to sequence content and integrity. IVDB provides a series of tools and viewers for comparative analysis of the viral genomes, genes, genetic polymorphisms and phylogenetic relationships. A search system has been developed for users to retrieve a combination of different data types by setting search options. To facilitate analysis of global viral transmission and evolution, the IV Sequence Distribution Tool (IVDT) has been developed to display the worldwide geographic distribution of chosen viral genotypes and to couple genomic data with epidemiological data. The BLAST, multiple sequence alignment and phylogenetic analysis tools were integrated for online data analysis. Furthermore, IVDB offers instant access to pre-computed alignments and polymorphisms of IV genes and proteins, and presents the results as SNP distribution plots and minor allele distributions. IVDB is publicly available at http://influenza.genomics.org.cn.

Zika virus evolution and spread in the Americas.

PubMed

Metsky, Hayden C; Matranga, Christian B; Wohl, Shirlee; Schaffner, Stephen F; Freije, Catherine A; Winnicki, Sarah M; West, Kendra; Qu, James; Baniecki, Mary Lynn; Gladden-Young, Adrianne; Lin, Aaron E; Tomkins-Tinch, Christopher H; Ye, Simon H; Park, Daniel J; Luo, Cynthia Y; Barnes, Kayla G; Shah, Rickey R; Chak, Bridget; Barbosa-Lima, Giselle; Delatorre, Edson; Vieira, Yasmine R; Paul, Lauren M; Tan, Amanda L; Barcellona, Carolyn M; Porcelli, Mario C; Vasquez, Chalmers; Cannons, Andrew C; Cone, Marshall R; Hogan, Kelly N; Kopp, Edgar W; Anzinger, Joshua J; Garcia, Kimberly F; Parham, Leda A; Ramírez, Rosa M Gélvez; Montoya, Maria C Miranda; Rojas, Diana P; Brown, Catherine M; Hennigan, Scott; Sabina, Brandon; Scotland, Sarah; Gangavarapu, Karthik; Grubaugh, Nathan D; Oliveira, Glenn; Robles-Sikisaka, Refugio; Rambaut, Andrew; Gehrke, Lee; Smole, Sandra; Halloran, M Elizabeth; Villar, Luis; Mattar, Salim; Lorenzana, Ivette; Cerbino-Neto, Jose; Valim, Clarissa; Degrave, Wim; Bozza, Patricia T; Gnirke, Andreas; Andersen, Kristian G; Isern, Sharon; Michael, Scott F; Bozza, Fernando A; Souza, Thiago M L; Bosch, Irene; Yozwiak, Nathan L; MacInnis, Bronwyn L; Sabeti, Pardis C

2017-06-15

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests.

Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance

PubMed Central

Fenton-May, Angharad E.; Dilernia, Dario A.; Kilembe, William; Allen, Susan A.; Borrow, Persephone; Hunter, Eric

2015-01-01

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential. PMID:26378795

Evolution of Influenza A Virus by Mutation and Re-Assortment

PubMed Central

Shao, Wenhan; Li, Xinxin; Goraya, Mohsan Ullah; Wang, Song; Chen, Ji-Long

2017-01-01

Influenza A virus (IAV), a highly infectious respiratory pathogen, has continued to be a significant threat to global public health. To complete their life cycle, influenza viruses have evolved multiple strategies to interact with a host. A large number of studies have revealed that the evolution of influenza A virus is mainly mediated through the mutation of the virus itself and the re-assortment of viral genomes derived from various strains. The evolution of influenza A virus through these mechanisms causes worldwide annual epidemics and occasional pandemics. Importantly, influenza A virus can evolve from an animal infected pathogen to a human infected pathogen. The highly pathogenic influenza virus has resulted in stupendous economic losses due to its morbidity and mortality both in human and animals. Influenza viruses fall into a category of viruses that can cause zoonotic infection with stable adaptation to human, leading to sustained horizontal transmission. The rapid mutations of influenza A virus result in the loss of vaccine optimal efficacy, and challenge the complete eradication of the virus. In this review, we highlight the current understanding of influenza A virus evolution caused by the mutation and re-assortment of viral genomes. In addition, we discuss the specific mechanisms by which the virus evolves. PMID:28783091

Extensive gene remodeling in the viral world: new evidence for nongradual evolution in the mobilome network.

PubMed

Jachiet, Pierre-Alain; Colson, Philippe; Lopez, Philippe; Bapteste, Eric

2014-08-07

Complex nongradual evolutionary processes such as gene remodeling are difficult to model, to visualize, and to investigate systematically. Despite these challenges, the creation of composite (or mosaic) genes by combination of genetic segments from unrelated gene families was established as an important adaptive phenomena in eukaryotic genomes. In contrast, almost no general studies have been conducted to quantify composite genes in viruses. Although viral genome mosaicism has been well-described, the extent of gene mosaicism and its rules of emergence remain largely unexplored. Applying methods from graph theory to inclusive similarity networks, and using data from more than 3,000 complete viral genomes, we provide the first demonstration that composite genes in viruses are 1) functionally biased, 2) involved in key aspects of the arm race between cells and viruses, and 3) can be classified into two distinct types of composite genes in all viral classes. Beyond the quantification of the widespread recombination of genes among different viruses of the same class, we also report a striking sharing of genetic information between viruses of different classes and with different nucleic acid types. This latter discovery provides novel evidence for the existence of a large and complex mobilome network, which appears partly bound by the sharing of genetic information and by the formation of composite genes between mobile entities with different genetic material. Considering that there are around 10E31 viruses on the planet, gene remodeling appears as a hugely significant way of generating and moving novel sequences between different kinds of organisms on Earth. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Viral Diversity Threshold for Adaptive Immunity in Prokaryotes

PubMed Central

Weinberger, Ariel D.; Wolf, Yuri I.; Lobkovsky, Alexander E.; Gilmore, Michael S.; Koonin, Eugene V.

2012-01-01

ABSTRACT Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological sensors (Lamarckian evolution) are predicted. PMID:23221803

Sequence variation of the feline immunodeficiency virus genome and its clinical relevance.

PubMed

Stickney, A L; Dunowska, M; Cave, N J

2013-06-08

The ongoing evolution of feline immunodeficiency virus (FIV) has resulted in the existence of a diverse continuum of viruses. FIV isolates differ with regards to their mutation and replication rates, plasma viral loads, cell tropism and the ability to induce apoptosis. Clinical disease in FIV-infected cats is also inconsistent. Genomic sequence variation of FIV is likely to be responsible for some of the variation in viral behaviour. The specific genetic sequences that influence these key viral properties remain to be determined. With knowledge of the specific key determinants of pathogenicity, there is the potential for veterinarians in the future to apply this information for prognostic purposes. Genomic sequence variation of FIV also presents an obstacle to effective vaccine development. Most challenge studies demonstrate acceptable efficacy of a dual-subtype FIV vaccine (Fel-O-Vax FIV) against FIV infection under experimental settings; however, vaccine efficacy in the field still remains to be proven. It is important that we discover the key determinants of immunity induced by this vaccine; such data would compliment vaccine field efficacy studies and provide the basis to make informed recommendations on its use.

Untangling the origin of viruses and their impact on cellular evolution.

PubMed

Nasir, Arshan; Sun, Feng-Jie; Kim, Kyung Mo; Caetano-Anollés, Gustavo

2015-04-01

The origin and evolution of viruses remain mysterious. Here, we focus on the distribution of viral replicons in host organisms, their morphological features, and the evolution of highly conserved protein and nucleic acid structures. The apparent inability of RNA viral replicons to infect contemporary akaryotic species suggests an early origin of RNA viruses and their subsequent loss in akaryotes. A census of virion morphotypes reveals that advanced forms were unique to viruses infecting a specific supergroup, while simpler forms were observed in viruses infecting organisms in all forms of cellular life. Results hint toward an ancient origin of viruses from an ancestral virus harboring either filamentous or spherical virions. Finally, phylogenetic trees built from protein domain and tRNA structures in thousands of genomes suggest that viruses evolved via reductive evolution from ancient cells. The analysis presents a complete account of the evolutionary history of cells and viruses and identifies viruses as crucial agents influencing cellular evolution. © 2015 New York Academy of Sciences.

The evolution of life history trade-offs in viruses.

PubMed

Goldhill, Daniel H; Turner, Paul E

2014-10-01

Viruses can suffer 'life-history' trade-offs that prevent simultaneous improvement in fitness traits, such as improved intrahost reproduction at the expense of reduced extrahost survival. Here we examine reproduction-survival trade-offs and other trait compromises, highlighting that experimental evolution can reveal trade-offs and their associated mechanisms. Whereas 'curse of the pharaoh' (high virulence with extreme stability) may generally apply for viruses of eukaryotes, we suggest phages are instead likely to suffer virulence/stability trade-offs. We examine how survival/reproduction trade-offs in viruses are affected by environmental stressors, proteins governing viral host range, and organization of the virus genome. Future studies incorporating comparative biology, experimental evolution, and structural biology, could thoroughly determine how viral trade-offs evolve, and whether they transiently or permanently constrain virus adaptation. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiple incursions and recurrent epidemic fade-out of H3N2 canine influenza A virus in the United States.

PubMed

Voorhees, Ian E H; Dalziel, Benjamin D; Glaser, Amy; Dubovi, Edward J; Murcia, Pablo R; Newbury, Sandra; Toohey-Kurth, Kathy; Su, Shuo; Kriti, Divya; Van Bakel, Harm; Goodman, Laura B; Leutenegger, Christian; Holmes, Edward C; Parrish, Colin R

2018-06-06

Avian-origin H3N2 canine influenza virus (CIV) transferred to dogs in Asia around 2005, becoming enzootic throughout China and Korea before reaching the USA in early 2015. To understand the post-transfer evolution and epidemiology of this virus, particularly the cause of recent and ongoing increases in incidence in the USA, we performed an integrated analysis of whole-genome sequence data from 64 newly sequenced viruses and comprehensive surveillance data. This reveals that the circulation of H3N2 CIV within the USA is typified by recurrent epidemic burst-fadeout dynamics driven by multiple introductions of virus from Asia. Although all major viral lineages displayed similar rates of genomic sequence evolution, H3N2 CIV consistently exhibited proportionally more non-synonymous substitutions per site compared to avian reservoir viruses, indicative of a large-scale change in selection pressures. Despite these genotypic differences, we found no evidence of adaptive evolution or increased viral transmission, with epidemiological models indicating a basic reproductive number, R 0 , of between 1 and 1.5 across nearly all USA outbreaks, consistent with maintained, but heterogeneous circulation. We propose that CIV's mode of viral circulation may have resulted in evolutionary cul-de-sacs, in which there is little opportunity for the selection of the more transmissible H3N2 CIV phenotypes necessary to enable circulation through a general dog population characterized by widespread contact heterogeneity. CIV must therefore rely on metapopulations of high host density (notably animal shelters) within the greater dog population and reintroduction from other populations or face complete epidemic extinction. IMPORTANCE The relatively recent appearance of influenza A virus (IAV) epidemics in dogs expands our understanding of IAV host-range and ecology, providing useful and relevant models for understanding critical factors involved in viral emergence. Here, we integrate viral whole-genome sequence analysis and comprehensive surveillance data to examine the evolution of the emerging avian-origin H3N2 canine influenza virus (CIV), particularly the factors driving ongoing circulation and recent increase in incidence of the virus within the USA. Our results provide a detailed understanding of how H3N2 CIV achieves sustained circulation within the USA, despite widespread host contact heterogeneity and recurrent epidemic fade-out. Moreover, our findings suggest that the types and intensity of selection pressures an emerging virus experiences are highly dependent on host population structure and ecology, and may inhibit an emerging virus from acquiring sustained epidemic or pandemic circulation. Copyright © 2018 American Society for Microbiology.

Demographic processes affect HIV-1 evolution in primary infection before the onset of selective processes.

PubMed

Herbeck, Joshua T; Rolland, Morgane; Liu, Yi; McLaughlin, Sherry; McNevin, John; Zhao, Hong; Wong, Kim; Stoddard, Julia N; Raugi, Dana; Sorensen, Stephanie; Genowati, Indira; Birditt, Brian; McKay, Angela; Diem, Kurt; Maust, Brandon S; Deng, Wenjie; Collier, Ann C; Stekler, Joanne D; McElrath, M Juliana; Mullins, James I

2011-08-01

HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected with HIV-1 subtype B. A total of 475 nearly full-length HIV-1 genome sequences were generated, representing on average 10 genomes per specimen at 2 to 12 visits over the first year of infection. Single founding variants with nearly homogeneous viral populations were detected in eight of the nine individuals who were enrolled during acute HIV-1 infection. Restriction to a single founder variant was not due to a lack of diversity in the transmitter as homogeneous populations were found in recipients from transmitters with chronic infection. Mutational patterns indicative of rapid viral population growth dominated during the first 5 weeks of infection and included a slight contraction of viral genetic diversity over the first 20 to 40 days. Subsequently, selection dominated, most markedly in env and nef. Mutants were detected in the first week and became consensus as early as day 21 after the onset of symptoms of primary HIV infection. We found multiple indications of cytotoxic T lymphocyte (CTL) escape mutations while reversions appeared limited. Putative escape mutations were often rapidly replaced with mutually exclusive mutations nearby, indicating the existence of a maturational escape process, possibly in adaptation to viral fitness constraints or to immune responses against new variants. We showed that establishment of HIV-1 infection is likely due to a biological mechanism that restricts transmission rather than to early adaptive evolution during acute infection. Furthermore, the diversity of HIV strains coupled with complex and individual-specific patterns of CTL escape did not reveal shared sequence characteristics of acute infection that could be harnessed for vaccine design.

Sites of Retroviral DNA Integration: From Basic Research to Clinical Applications

PubMed Central

Serrao, Erik; Engelman, Alan N.

2016-01-01

One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of the viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with HIV-1 can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or AIDS patients on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

Implications of segment mismatch for influenza A virus evolution

PubMed Central

White, Maria C.; Lowen, Anice C.

2018-01-01

Influenza A virus (IAV) is an RNA virus with a segmented genome. These viral properties allow for the rapid evolution of IAV under selective pressure, due to mutation occurring from error-prone replication and the exchange of gene segments within a co-infected cell, termed reassortment. Both mutation and reassortment give rise to genetic diversity, but constraints shape their impact on viral evolution: just as most mutations are deleterious, most reassortment events result in genetic incompatibilities. The phenomenon of segment mismatch encompasses both RNA- and protein-based incompatibilities between co-infecting viruses and results in the production of progeny viruses with fitness defects. Segment mismatch is an important determining factor of the outcomes of mixed IAV infections and has been addressed in multiple risk assessment studies undertaken to date. However, due to the complexity of genetic interactions among the eight viral gene segments, our understanding of segment mismatch and its underlying mechanisms remain incomplete. Here, we summarize current knowledge regarding segment mismatch and discuss the implications of this phenomenon for IAV reassortment and diversity. PMID:29244017

The role of sequence context, nucleotide pool balance and stress in 2′-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA

PubMed Central

Wang, Jin; Dong, Hongping; Chionh, Yok Hian; McBee, Megan E.; Sirirungruang, Sasilada; Cunningham, Richard P.; Shi, Pei-Yong; Dedon, Peter C.

2016-01-01

The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 103 to 105 ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections. PMID:27365049

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

PubMed Central

Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

2018-01-01

ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota. PMID:29925671

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment

PubMed Central

Moreno, Elena; Gallego, Isabel; Gregori, Josep; Lucía-Sanz, Adriana; Soria, María Eugenia; Castro, Victoria; Beach, Nathan M.; Manrubia, Susanna; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Gómez, Jordi; Gastaminza, Pablo

2017-01-01

ABSTRACT Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments. PMID:28275194

Merkel Cell Polyomavirus Exhibits Dominant Control of the Tumor Genome and Transcriptome in Virus-Associated Merkel Cell Carcinoma.

PubMed

Starrett, Gabriel J; Marcelus, Christina; Cantalupo, Paul G; Katz, Joshua P; Cheng, Jingwei; Akagi, Keiko; Thakuria, Manisha; Rabinowits, Guilherme; Wang, Linda C; Symer, David E; Pipas, James M; Harris, Reuben S; DeCaprio, James A

2017-01-03

Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations. A variety of mutagenic processes that shape the evolution of tumors are critical determinants of disease outcome. Here, we sequenced the entire genome of virus-positive and virus-negative primary Merkel cell carcinomas (MCCs), revealing distinct mutation spectra and corresponding expression profiles. Our studies highlight the strong effect that Merkel cell polyomavirus has on the divergent development of viral MCC compared to the somatic alterations that typically drive nonviral tumorigenesis. A more comprehensive understanding of the distinct mutagenic processes operative in viral and nonviral MCCs has implications for the effective treatment of these tumors. Copyright © 2017 Starrett et al.

Dengue virus replicates and accumulates in Aedes aegypti salivary glands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raquin, Vincent, E-mail: vincent.raquin@univ-lyon1

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidencemore » that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. - Highlights: •Strand-specific RT-qPCR allows accurate quantification of DENV (-) RNA in mosquito tissues. •Detection of DENV (-) RNA in salivary glands provides evidence of viral replication in this tissue. •Viral replication in salivary glands likely replenishes DENV genetic diversity prior to transmission.« less

Evolution of puma lentivirus in bobcats (Lynx rufus) and mountain lions (Puma concolor) in North America

USGS Publications Warehouse

Lee, Justin S.; Bevins, Sarah N.; Serieys, Laurel E.K.; Vickers, Winston; Logan, Ken A.; Aldredge, Mat; Boydston, Erin E.; Lyren, Lisa M.; McBride, Roy; Roelke-Parker, Melody; Pecon-Slattery, Jill; Troyer, Jennifer L.; Riley, Seth P.; Boyce, Walter M.; Crooks, Kevin R.; VandeWoude, Sue

2014-01-01

Mountain lions (Puma concolor) throughout North and South America are infected with puma lentivirus clade B (PLVB). A second, highly divergent lentiviral clade, PLVA, infects mountain lions in southern California and Florida. Bobcats (Lynx rufus) in these two geographic regions are also infected with PLVA, and to date, this is the only strain of lentivirus identified in bobcats. We sequenced full-length PLV genomes in order to characterize the molecular evolution of PLV in bobcats and mountain lions. Low sequence homology (88% average pairwise identity) and frequent recombination (1 recombination breakpoint per 3 isolates analyzed) were observed in both clades. Viral proteins have markedly different patterns of evolution; sequence homology and negative selection were highest in Gag and Pol and lowest in Vif and Env. A total of 1.7% of sites across the PLV genome evolve under positive selection, indicating that host-imposed selection pressure is an important force shaping PLV evolution. PLVA strains are highly spatially structured, reflecting the population dynamics of their primary host, the bobcat. In contrast, the phylogeography of PLVB reflects the highly mobile mountain lion, with diverse PLVB isolates cocirculating in some areas and genetically related viruses being present in populations separated by thousands of kilometers. We conclude that PLVA and PLVB are two different viral species with distinct feline hosts and evolutionary histories.

Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

PubMed

Smith, Richard H; Hallwirth, Claus V; Westerman, Michael; Hetherington, Nicola A; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B; Koonin, Eugene V; Agbandje-McKenna, Mavis; Kotin, Robert M; Alexander, Ian E

2016-07-05

Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

Viruses of invasive Argentine ants from the European Main supercolony: characterization, interactions and evolution.

PubMed

Viljakainen, Lumi; Holmberg, Ida; Abril, Sílvia; Jurvansuu, Jaana

2018-06-25

The Argentine ant (Linepithema humile) is a highly invasive pest, yet very little is known about its viruses. We analysed individual RNA-sequencing data from 48 Argentine ant queens to identify and characterisze their viruses. We discovered eight complete RNA virus genomes - all from different virus families - and one putative partial entomopoxvirus genome. Seven of the nine virus sequences were found from ant samples spanning 7 years, suggesting that these viruses may cause long-term infections within the super-colony. Although all nine viruses successfully infect Argentine ants, they have very different characteristics, such as genome organization, prevalence, loads, activation frequencies and rates of evolution. The eight RNA viruses constituted in total 23 different virus combinations which, based on statistical analysis, were non-random, suggesting that virus compatibility is a factor in infections. We also searched for virus sequences from New Zealand and Californian Argentine ant RNA-sequencing data and discovered that many of the viruses are found on different continents, yet some viruses are prevalent only in certain colonies. The viral loads described here most probably present a normal asymptomatic level of infection; nevertheless, detailed knowledge of Argentine ant viruses may enable the design of viral biocontrol methods against this pest.

Genomic approaches for understanding dengue: insights from the virus, vector, and host.

PubMed

Sim, Shuzhen; Hibberd, Martin L

2016-03-02

The incidence and geographic range of dengue have increased dramatically in recent decades. Climate change, rapid urbanization and increased global travel have facilitated the spread of both efficient mosquito vectors and the four dengue virus serotypes between population centers. At the same time, significant advances in genomics approaches have provided insights into host-pathogen interactions, immunogenetics, and viral evolution in both humans and mosquitoes. Here, we review these advances and the innovative treatment and control strategies that they are inspiring.

Innovations in Undergraduate Science Education: Going Viral.

PubMed

Hatfull, Graham F

2015-08-01

Bacteriophage discovery and genomics provides a powerful and effective platform for integrating missions in research and education. Implementation of the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program facilitates a broad impact by including a diverse array of schools, faculty, and students. The program generates new insights into the diversity and evolution of the bacteriophage population and presents a model for introducing first-year undergraduate students to discovery-based research experiences. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification.

PubMed

Sinclair, Robert M; Ravantti, Janne J; Bamford, Dennis H

2017-04-15

Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. Copyright © 2017 Sinclair et al.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification

PubMed Central

Sinclair, Robert M.; Ravantti, Janne J.

2017-01-01

ABSTRACT Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. PMID:28122979

APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

PubMed

Sato, Kei; Takeuchi, Junko S; Misawa, Naoko; Izumi, Taisuke; Kobayashi, Tomoko; Kimura, Yuichi; Iwami, Shingo; Takaori-Kondo, Akifumi; Hu, Wei-Shau; Aihara, Kazuyuki; Ito, Mamoru; An, Dong Sung; Pathak, Vinay K; Koyanagi, Yoshio

2014-10-01

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

Zika virus evolution and spread in the Americas

PubMed Central

Metsky, Hayden C.; Matranga, Christian B.; Wohl, Shirlee; Schaffner, Stephen F.; Freije, Catherine A.; Winnicki, Sarah M.; West, Kendra; Qu, James; Baniecki, Mary Lynn; Gladden-Young, Adrianne; Lin, Aaron E.; Tomkins-Tinch, Christopher H.; Ye, Simon H.; Park, Daniel J.; Luo, Cynthia Y.; Barnes, Kayla G.; Shah, Rickey R.; Chak, Bridget; Barbosa-Lima, Giselle; Delatorre, Edson; Vieira, Yasmine R.; Paul, Lauren M.; Tan, Amanda L.; Barcellona, Carolyn M.; Porcelli, Mario C.; Vasquez, Chalmers; Cannons, Andrew C.; Cone, Marshall R.; Hogan, Kelly N.; Kopp, Edgar W.; Anzinger, Joshua J.; Garcia, Kimberly F.; Parham, Leda A.; Gélvez Ramírez, Rosa M.; Miranda Montoya, Maria C.; Rojas, Diana P.; Brown, Catherine M.; Hennigan, Scott; Sabina, Brandon; Scotland, Sarah; Gangavarapu, Karthik; Grubaugh, Nathan D.; Oliveira, Glenn; Robles-Sikisaka, Refugio; Rambaut, Andrew; Gehrke, Lee; Smole, Sandra; Halloran, M. Elizabeth; Villar, Luis; Mattar, Salim; Lorenzana, Ivette; Cerbino-Neto, Jose; Valim, Clarissa; Degrave, Wim; Bozza, Patricia T.; Gnirke, Andreas; Andersen, Kristian G.; Isern, Sharon; Michael, Scott F.; Bozza, Fernando A.; Souza, Thiago M. L.; Bosch, Irene; Yozwiak, Nathan L.; MacInnis, Bronwyn L.; Sabeti, Pardis C.

2017-01-01

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention1,2, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests. PMID:28538734

A comprehensive and quantitative exploration of thousands of viral genomes

PubMed Central

Mahmoudabadi, Gita

2018-01-01

The complete assembly of viral genomes from metagenomic datasets (short genomic sequences gathered from environmental samples) has proven to be challenging, so there are significant blind spots when we view viral genomes through the lens of metagenomics. One approach to overcoming this problem is to leverage the thousands of complete viral genomes that are publicly available. Here we describe our efforts to assemble a comprehensive resource that provides a quantitative snapshot of viral genomic trends – such as gene density, noncoding percentage, and abundances of functional gene categories – across thousands of viral genomes. We have also developed a coarse-grained method for visualizing viral genome organization for hundreds of genomes at once, and have explored the extent of the overlap between bacterial and bacteriophage gene pools. Existing viral classification systems were developed prior to the sequencing era, so we present our analysis in a way that allows us to assess the utility of the different classification systems for capturing genomic trends. PMID:29624169

A comprehensive and quantitative exploration of thousands of viral genomes.

PubMed

Mahmoudabadi, Gita; Phillips, Rob

2018-04-19

The complete assembly of viral genomes from metagenomic datasets (short genomic sequences gathered from environmental samples) has proven to be challenging, so there are significant blind spots when we view viral genomes through the lens of metagenomics. One approach to overcoming this problem is to leverage the thousands of complete viral genomes that are publicly available. Here we describe our efforts to assemble a comprehensive resource that provides a quantitative snapshot of viral genomic trends - such as gene density, noncoding percentage, and abundances of functional gene categories - across thousands of viral genomes. We have also developed a coarse-grained method for visualizing viral genome organization for hundreds of genomes at once, and have explored the extent of the overlap between bacterial and bacteriophage gene pools. Existing viral classification systems were developed prior to the sequencing era, so we present our analysis in a way that allows us to assess the utility of the different classification systems for capturing genomic trends. © 2018, Mahmoudabadi et al.

Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution

PubMed Central

Pope, Welkin H.; Jacobs-Sera, Deborah; Russell, Daniel A.; Peebles, Craig L.; Al-Atrache, Zein; Alcoser, Turi A.; Alexander, Lisa M.; Alfano, Matthew B.; Alford, Samantha T.; Amy, Nichols E.; Anderson, Marie D.; Anderson, Alexander G.; Ang, Andrew A. S.; Ares, Manuel; Barber, Amanda J.; Barker, Lucia P.; Barrett, Jonathan M.; Barshop, William D.; Bauerle, Cynthia M.; Bayles, Ian M.; Belfield, Katherine L.; Best, Aaron A.; Borjon, Agustin; Bowman, Charles A.; Boyer, Christine A.; Bradley, Kevin W.; Bradley, Victoria A.; Broadway, Lauren N.; Budwal, Keshav; Busby, Kayla N.; Campbell, Ian W.; Campbell, Anne M.; Carey, Alyssa; Caruso, Steven M.; Chew, Rebekah D.; Cockburn, Chelsea L.; Cohen, Lianne B.; Corajod, Jeffrey M.; Cresawn, Steven G.; Davis, Kimberly R.; Deng, Lisa; Denver, Dee R.; Dixon, Breyon R.; Ekram, Sahrish; Elgin, Sarah C. R.; Engelsen, Angela E.; English, Belle E. V.; Erb, Marcella L.; Estrada, Crystal; Filliger, Laura Z.; Findley, Ann M.; Forbes, Lauren; Forsyth, Mark H.; Fox, Tyler M.; Fritz, Melissa J.; Garcia, Roberto; George, Zindzi D.; Georges, Anne E.; Gissendanner, Christopher R.; Goff, Shannon; Goldstein, Rebecca; Gordon, Kobie C.; Green, Russell D.; Guerra, Stephanie L.; Guiney-Olsen, Krysta R.; Guiza, Bridget G.; Haghighat, Leila; Hagopian, Garrett V.; Harmon, Catherine J.; Harmson, Jeremy S.; Hartzog, Grant A.; Harvey, Samuel E.; He, Siping; He, Kevin J.; Healy, Kaitlin E.; Higinbotham, Ellen R.; Hildebrandt, Erin N.; Ho, Jason H.; Hogan, Gina M.; Hohenstein, Victoria G.; Holz, Nathan A.; Huang, Vincent J.; Hufford, Ericka L.; Hynes, Peter M.; Jackson, Arrykka S.; Jansen, Erica C.; Jarvik, Jonathan; Jasinto, Paul G.; Jordan, Tuajuanda C.; Kasza, Tomas; Katelyn, Murray A.; Kelsey, Jessica S.; Kerrigan, Larisa A.; Khaw, Daryl; Kim, Junghee; Knutter, Justin Z.; Ko, Ching-Chung; Larkin, Gail V.; Laroche, Jennifer R.; Latif, Asma; Leuba, Kohana D.; Leuba, Sequoia I.; Lewis, Lynn O.; Loesser-Casey, Kathryn E.; Long, Courtney A.; Lopez, A. Javier; Lowery, Nicholas; Lu, Tina Q.; Mac, Victor; Masters, Isaac R.; McCloud, Jazmyn J.; McDonough, Molly J.; Medenbach, Andrew J.; Menon, Anjali; Miller, Rachel; Morgan, Brandon K.; Ng, Patrick C.; Nguyen, Elvis; Nguyen, Katrina T.; Nguyen, Emilie T.; Nicholson, Kaylee M.; Parnell, Lindsay A.; Peirce, Caitlin E.; Perz, Allison M.; Peterson, Luke J.; Pferdehirt, Rachel E.; Philip, Seegren V.; Pogliano, Kit; Pogliano, Joe; Polley, Tamsen; Puopolo, Erica J.; Rabinowitz, Hannah S.; Resiss, Michael J.; Rhyan, Corwin N.; Robinson, Yetta M.; Rodriguez, Lauren L.; Rose, Andrew C.; Rubin, Jeffrey D.; Ruby, Jessica A.; Saha, Margaret S.; Sandoz, James W.; Savitskaya, Judith; Schipper, Dale J.; Schnitzler, Christine E.; Schott, Amanda R.; Segal, J. Bradley; Shaffer, Christopher D.; Sheldon, Kathryn E.; Shepard, Erica M.; Shepardson, Jonathan W.; Shroff, Madav K.; Simmons, Jessica M.; Simms, Erika F.; Simpson, Brandy M.; Sinclair, Kathryn M.; Sjoholm, Robert L.; Slette, Ingrid J.; Spaulding, Blaire C.; Straub, Clark L.; Stukey, Joseph; Sughrue, Trevor; Tang, Tin-Yun; Tatyana, Lyons M.; Taylor, Stephen B.; Taylor, Barbara J.; Temple, Louise M.; Thompson, Jasper V.; Tokarz, Michael P.; Trapani, Stephanie E.; Troum, Alexander P.; Tsay, Jonathan; Tubbs, Anthony T.; Walton, Jillian M.; Wang, Danielle H.; Wang, Hannah; Warner, John R.; Weisser, Emilie G.; Wendler, Samantha C.; Weston-Hafer, Kathleen A.; Whelan, Hilary M.; Williamson, Kurt E.; Willis, Angelica N.; Wirtshafter, Hannah S.; Wong, Theresa W.; Wu, Phillip; Yang, Yun jeong; Yee, Brandon C.; Zaidins, David A.; Zhang, Bo; Zúniga, Melina Y.; Hendrix, Roger W.; Hatfull, Graham F.

2011-01-01

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists. PMID:21298013

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

PubMed

Pope, Welkin H; Jacobs-Sera, Deborah; Russell, Daniel A; Peebles, Craig L; Al-Atrache, Zein; Alcoser, Turi A; Alexander, Lisa M; Alfano, Matthew B; Alford, Samantha T; Amy, Nichols E; Anderson, Marie D; Anderson, Alexander G; Ang, Andrew A S; Ares, Manuel; Barber, Amanda J; Barker, Lucia P; Barrett, Jonathan M; Barshop, William D; Bauerle, Cynthia M; Bayles, Ian M; Belfield, Katherine L; Best, Aaron A; Borjon, Agustin; Bowman, Charles A; Boyer, Christine A; Bradley, Kevin W; Bradley, Victoria A; Broadway, Lauren N; Budwal, Keshav; Busby, Kayla N; Campbell, Ian W; Campbell, Anne M; Carey, Alyssa; Caruso, Steven M; Chew, Rebekah D; Cockburn, Chelsea L; Cohen, Lianne B; Corajod, Jeffrey M; Cresawn, Steven G; Davis, Kimberly R; Deng, Lisa; Denver, Dee R; Dixon, Breyon R; Ekram, Sahrish; Elgin, Sarah C R; Engelsen, Angela E; English, Belle E V; Erb, Marcella L; Estrada, Crystal; Filliger, Laura Z; Findley, Ann M; Forbes, Lauren; Forsyth, Mark H; Fox, Tyler M; Fritz, Melissa J; Garcia, Roberto; George, Zindzi D; Georges, Anne E; Gissendanner, Christopher R; Goff, Shannon; Goldstein, Rebecca; Gordon, Kobie C; Green, Russell D; Guerra, Stephanie L; Guiney-Olsen, Krysta R; Guiza, Bridget G; Haghighat, Leila; Hagopian, Garrett V; Harmon, Catherine J; Harmson, Jeremy S; Hartzog, Grant A; Harvey, Samuel E; He, Siping; He, Kevin J; Healy, Kaitlin E; Higinbotham, Ellen R; Hildebrandt, Erin N; Ho, Jason H; Hogan, Gina M; Hohenstein, Victoria G; Holz, Nathan A; Huang, Vincent J; Hufford, Ericka L; Hynes, Peter M; Jackson, Arrykka S; Jansen, Erica C; Jarvik, Jonathan; Jasinto, Paul G; Jordan, Tuajuanda C; Kasza, Tomas; Katelyn, Murray A; Kelsey, Jessica S; Kerrigan, Larisa A; Khaw, Daryl; Kim, Junghee; Knutter, Justin Z; Ko, Ching-Chung; Larkin, Gail V; Laroche, Jennifer R; Latif, Asma; Leuba, Kohana D; Leuba, Sequoia I; Lewis, Lynn O; Loesser-Casey, Kathryn E; Long, Courtney A; Lopez, A Javier; Lowery, Nicholas; Lu, Tina Q; Mac, Victor; Masters, Isaac R; McCloud, Jazmyn J; McDonough, Molly J; Medenbach, Andrew J; Menon, Anjali; Miller, Rachel; Morgan, Brandon K; Ng, Patrick C; Nguyen, Elvis; Nguyen, Katrina T; Nguyen, Emilie T; Nicholson, Kaylee M; Parnell, Lindsay A; Peirce, Caitlin E; Perz, Allison M; Peterson, Luke J; Pferdehirt, Rachel E; Philip, Seegren V; Pogliano, Kit; Pogliano, Joe; Polley, Tamsen; Puopolo, Erica J; Rabinowitz, Hannah S; Resiss, Michael J; Rhyan, Corwin N; Robinson, Yetta M; Rodriguez, Lauren L; Rose, Andrew C; Rubin, Jeffrey D; Ruby, Jessica A; Saha, Margaret S; Sandoz, James W; Savitskaya, Judith; Schipper, Dale J; Schnitzler, Christine E; Schott, Amanda R; Segal, J Bradley; Shaffer, Christopher D; Sheldon, Kathryn E; Shepard, Erica M; Shepardson, Jonathan W; Shroff, Madav K; Simmons, Jessica M; Simms, Erika F; Simpson, Brandy M; Sinclair, Kathryn M; Sjoholm, Robert L; Slette, Ingrid J; Spaulding, Blaire C; Straub, Clark L; Stukey, Joseph; Sughrue, Trevor; Tang, Tin-Yun; Tatyana, Lyons M; Taylor, Stephen B; Taylor, Barbara J; Temple, Louise M; Thompson, Jasper V; Tokarz, Michael P; Trapani, Stephanie E; Troum, Alexander P; Tsay, Jonathan; Tubbs, Anthony T; Walton, Jillian M; Wang, Danielle H; Wang, Hannah; Warner, John R; Weisser, Emilie G; Wendler, Samantha C; Weston-Hafer, Kathleen A; Whelan, Hilary M; Williamson, Kurt E; Willis, Angelica N; Wirtshafter, Hannah S; Wong, Theresa W; Wu, Phillip; Yang, Yun jeong; Yee, Brandon C; Zaidins, David A; Zhang, Bo; Zúniga, Melina Y; Hendrix, Roger W; Hatfull, Graham F

2011-01-27

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Short reads from honey bee (Apis sp.) sequencing projects reflect microbial associate diversity

PubMed Central

Hurst, Gregory D.D.

2017-01-01

High throughput (or ‘next generation’) sequencing has transformed most areas of biological research and is now a standard method that underpins empirical study of organismal biology, and (through comparison of genomes), reveals patterns of evolution. For projects focused on animals, these sequencing methods do not discriminate between the primary target of sequencing (the animal genome) and ‘contaminating’ material, such as associated microbes. A common first step is to filter out these contaminants to allow better assembly of the animal genome or transcriptome. Here, we aimed to assess if these ‘contaminations’ provide information with regard to biologically important microorganisms associated with the individual. To achieve this, we examined whether the short read data from Apis retrieved elements of its well established microbiome. To this end, we screened almost 1,000 short read libraries of honey bee (Apis sp.) DNA sequencing project for the presence of microbial sequences, and find sequences from known honey bee microbial associates in at least 11% of them. Further to this, we screened ∼500 Apis RNA sequencing libraries for evidence of viral infections, which were found to be present in about half of them. We then used the data to reconstruct draft genomes of three Apis associated bacteria, as well as several viral strains de novo. We conclude that ‘contamination’ in short read sequencing libraries can provide useful genomic information on microbial taxa known to be associated with the target organisms, and may even lead to the discovery of novel associations. Finally, we demonstrate that RNAseq samples from experiments commonly carry uneven viral loads across libraries. We note variation in viral presence and load may be a confounding feature of differential gene expression analyses, and as such it should be incorporated as a random factor in analyses. PMID:28717593

Short reads from honey bee (Apis sp.) sequencing projects reflect microbial associate diversity.

PubMed

Gerth, Michael; Hurst, Gregory D D

2017-01-01

High throughput (or 'next generation') sequencing has transformed most areas of biological research and is now a standard method that underpins empirical study of organismal biology, and (through comparison of genomes), reveals patterns of evolution. For projects focused on animals, these sequencing methods do not discriminate between the primary target of sequencing (the animal genome) and 'contaminating' material, such as associated microbes. A common first step is to filter out these contaminants to allow better assembly of the animal genome or transcriptome. Here, we aimed to assess if these 'contaminations' provide information with regard to biologically important microorganisms associated with the individual. To achieve this, we examined whether the short read data from Apis retrieved elements of its well established microbiome. To this end, we screened almost 1,000 short read libraries of honey bee ( Apis sp.) DNA sequencing project for the presence of microbial sequences, and find sequences from known honey bee microbial associates in at least 11% of them. Further to this, we screened ∼500 Apis RNA sequencing libraries for evidence of viral infections, which were found to be present in about half of them. We then used the data to reconstruct draft genomes of three Apis associated bacteria, as well as several viral strains de novo . We conclude that 'contamination' in short read sequencing libraries can provide useful genomic information on microbial taxa known to be associated with the target organisms, and may even lead to the discovery of novel associations. Finally, we demonstrate that RNAseq samples from experiments commonly carry uneven viral loads across libraries. We note variation in viral presence and load may be a confounding feature of differential gene expression analyses, and as such it should be incorporated as a random factor in analyses.

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

PubMed Central

Stewart, H.; Bingham, R.J.; White, S. J.; Dykeman, E. C.; Zothner, C.; Tuplin, A. K.; Stockley, P. G.; Twarock, R.; Harris, M.

2016-01-01

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly. PMID:26972799

Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

PubMed

Dembowski, Jill A; DeLuca, Neal A

2015-05-01

Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.

RNA Recombination In Vivo in the Absence of Viral Replication

PubMed Central

Gallei, Andreas; Pankraz, Alexander; Thiel, Heinz-Jürgen; Becher, Paul

2004-01-01

To study fundamental aspects of RNA recombination, an in vivo RNA recombination system was established. This system allowed the efficient generation of recombinant cytopathogenic pestiviruses after transfection of synthetic, nonreplicatable, subgenomic transcripts in cells infected with a replicating noncytopathogenic virus. Studies addressing the interplay between RNA recombination and replication revealed that cotransfection of noninfected cells with various pairs of nonreplicatable RNA derivatives also led to the emergence of recombinant viral genomes. Remarkably, homologous and nonhomologous recombination occurred between two overlapping transcripts, each lacking different essential parts of the viral RNA-dependent RNA polymerase (RdRp) gene. Apart from the generally accepted viral replicative copy choice recombination, our results prove the existence of a viral RdRp-independent mechanism of RNA recombination that occurs in vivo. It appears likely that such a mechanism not only contributes to the evolution of RNA viruses but also leads to the generation of recombinant cellular RNAs. PMID:15163720

Evolution of Foamy Viruses: The Most Ancient of All Retroviruses †

PubMed Central

Rethwilm, Axel; Bodem, Jochen

2013-01-01

Recent evidence indicates that foamy viruses (FVs) are the oldest retroviruses (RVs) that we know and coevolved with their hosts for several hundred million years. This coevolution may have contributed to the non-pathogenicity of FVs, an important factor in development of foamy viral vectors in gene therapy. However, various questions on the molecular evolution of FVs remain still unanswered. The analysis of the spectrum of animal species infected by exogenous FVs or harboring endogenous FV elements in their genome is pivotal. Furthermore, animal studies might reveal important issues, such as the identification of the FV in vivo target cells, which than require a detailed characterization, to resolve the molecular basis of the accuracy with which FVs copy their genome. The issues of the extent of FV viremia and of the nature of the virion genome (RNA vs. DNA) also need to be experimentally addressed. PMID:24072062

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

PubMed

McLaughlin, Paul J; Keegan, Liam P

2014-08-01

Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.

Field Monitoring of Avian Influenza Viruses: Whole-Genome Sequencing and Tracking of Neuraminidase Evolution Using 454 Pyrosequencing

PubMed Central

Croville, Guillaume; Soubies, Sébastien Mathieu; Barbieri, Johanna; Klopp, Christophe; Mariette, Jérôme; Bouchez, Olivier; Camus-Bouclainville, Christelle

2012-01-01

Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants. PMID:22718944

Evolution and Diversity in Human Herpes Simplex Virus Genomes

PubMed Central

Gatherer, Derek; Ochoa, Alejandro; Greenbaum, Benjamin; Dolan, Aidan; Bowden, Rory J.; Enquist, Lynn W.; Legendre, Matthieu; Davison, Andrew J.

2014-01-01

Herpes simplex virus 1 (HSV-1) causes a chronic, lifelong infection in >60% of adults. Multiple recent vaccine trials have failed, with viral diversity likely contributing to these failures. To understand HSV-1 diversity better, we comprehensively compared 20 newly sequenced viral genomes from China, Japan, Kenya, and South Korea with six previously sequenced genomes from the United States, Europe, and Japan. In this diverse collection of passaged strains, we found that one-fifth of the newly sequenced members share a gene deletion and one-third exhibit homopolymeric frameshift mutations (HFMs). Individual strains exhibit genotypic and potential phenotypic variation via HFMs, deletions, short sequence repeats, and single-nucleotide polymorphisms, although the protein sequence identity between strains exceeds 90% on average. In the first genome-scale analysis of positive selection in HSV-1, we found signs of selection in specific proteins and residues, including the fusion protein glycoprotein H. We also confirmed previous results suggesting that recombination has occurred with high frequency throughout the HSV-1 genome. Despite this, the HSV-1 strains analyzed clustered by geographic origin during whole-genome distance analysis. These data shed light on likely routes of HSV-1 adaptation to changing environments and will aid in the selection of vaccine antigens that are invariant worldwide. PMID:24227835

Genome segregation and packaging machinery in Acanthamoeba polyphaga mimivirus is reminiscent of bacterial apparatus.

PubMed

Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi; Kondabagil, Kiran

2014-06-01

Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses.

Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus

PubMed Central

Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi

2014-01-01

ABSTRACT Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses. PMID:24623441

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

PubMed

O'Neill, F J; Gao, Y; Xu, X

1993-11-01

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.

Micro-evolution of the hepatitis B virus genome in hepatitis B e-antigen-positive carriers: comparison of genotypes B and C at various immune stages.

PubMed

Liu, Chun-Jen; Chen, Ting-Chih; Chen, Pei-Jer; Wang, Hurng-Yi; Tseng, Tai-Chung; Cheng, Huei-Ru; Liu, Chen-Hua; Chen, Ding-Shinn; Kao, Jia-Horng

2015-01-01

Patients with hepatitis B virus (HBV) genotype B infection experience hepatitis B e-antigen (HBeAg) seroconversion at an earlier stage than do patients with genotype C infection. Therefore, this study investigated whether the differential phenotypes are related to HBV genomic evolution. Thirty-three HBeAg-positive patients with a mean follow-up of 3.1 years were enrolled: 16 at the immune tolerance stage (group I) and 17 at the immune clearance stage (group II). The evolution rates of paired viral genomes at enrollment and at the final follow-up in the full-length genome (μf), nonoverlapping regions (synonymous [μs] and nonsynonymous [μa]), and overlapping regions (μ) were calculated. The evolution rates were then compared according to serum alanine aminotransferase (ALT) levels and HBV genotype. The overall μf evolution rate was lower in group I than in group II (1.4 × 10(-5)  ± 3.3 × 10(-5) vs 1.2 × 10(-3)  ± 1.2 × 10(-3) nucleotide substitution/site/year, P < 0.001). We observed similar results for the μs, μa, and μ evolution rates. All evolution parameters were comparable between genotypes B and C. We determined a positive correlation between μa/y and the area under the average ALT time curve in genotype B (R(2)  = 0.6935, P < 0.0001), but not in genotype C (R(2)  = 0.1606, P = 0.124). The evolution rate of the HBV genome is higher at the immune clearance stage than at the immune tolerance stage. Host immune selection might play a role in triggering evolution of genotype B. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

The evolution of subtype B HIV-1 tat in the Netherlands during 1985-2012.

PubMed

van der Kuyl, Antoinette C; Vink, Monique; Zorgdrager, Fokla; Bakker, Margreet; Wymant, Chris; Hall, Matthew; Gall, Astrid; Blanquart, François; Berkhout, Ben; Fraser, Christophe; Cornelissen, Marion

2018-05-02

For the production of viral genomic RNA, HIV-1 is dependent on an early viral protein, Tat, which is required for high-level transcription. The quantity of viral RNA detectable in blood of HIV-1 infected individuals varies dramatically, and a factor involved could be the efficiency of Tat protein variants to stimulate RNA transcription. HIV-1 virulence, measured by set-point viral load, has been observed to increase over time in the Netherlands and elsewhere. Investigation of tat gene evolution in clinical isolates could discover a role of Tat in this changing virulence. A dataset of 291 Dutch HIV-1 subtype B tat genes, derived from full-length HIV-1 genome sequences from samples obtained between 1985-2012, was used to analyse the evolution of Tat. Twenty-two patient-derived tat genes, and the control Tat HXB2 were analysed for their capacity to stimulate expression of an LTR-luciferase reporter gene construct in diverse cell lines, as well as for their ability to complement a tat-defective HIV-1 LAI clone. Analysis of 291 historical tat sequences from the Netherlands showed ample amino acid (aa) variation between isolates, although no specific mutations were selected for over time. Of note, however, the encoded protein varied its length over the years through the loss or gain of stop codons in the second exon. In transmission clusters, a selection against the shorter Tat86 ORF was apparent in favour of the more common Tat101 version, likely due to negative selection against Tat86 itself, although random drift, transmission bottlenecks, or linkage to other variants could also explain the observation. There was no correlation between Tat length and set-point viral load; however, the number of non-intermediate variants in our study was small. In addition, variation in the length of Tat did not significantly change its capacity to stimulate transcription. From 1985 till 2012, variation in the length of the HIV-1 subtype B tat gene is increasingly found in the Dutch epidemic. However, as Tat proteins did not differ significantly in their capacity to stimulate transcription elongation in vitro, the increased HIV-1 virulence seen in recent years could not be linked to an evolving viral Tat protein. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Viral phylogenomics using an alignment-free method: A three-step approach to determine optimal length of k-mer

DOE PAGES

Zhang, Qian; Jun, Se -Ran; Leuze, Michael; ...

2017-01-19

The development of rapid, economical genome sequencing has shed new light on the classification of viruses. As of October 2016, the National Center for Biotechnology Information (NCBI) database contained >2 million viral genome sequences and a reference set of ~4000 viral genome sequences that cover a wide range of known viral families. Whole-genome sequences can be used to improve viral classification and provide insight into the viral tree of life . However, due to the lack of evolutionary conservation amongst diverse viruses, it is not feasible to build a viral tree of life using traditional phylogenetic methods based on conservedmore » proteins. In this study, we used an alignment-free method that uses k-mers as genomic features for a large-scale comparison of complete viral genomes available in RefSeq. To determine the optimal feature length, k (an essential step in constructing a meaningful dendrogram), we designed a comprehensive strategy that combines three approaches: (1) cumulative relative entropy, (2) average number of common features among genomes, and (3) the Shannon diversity index. This strategy was used to determine k for all 3,905 complete viral genomes in RefSeq. Lastly, the resulting dendrogram shows consistency with the viral taxonomy of the ICTV and the Baltimore classification of viruses.« less

Viral phylogenomics using an alignment-free method: A three-step approach to determine optimal length of k-mer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qian; Jun, Se -Ran; Leuze, Michael

The development of rapid, economical genome sequencing has shed new light on the classification of viruses. As of October 2016, the National Center for Biotechnology Information (NCBI) database contained >2 million viral genome sequences and a reference set of ~4000 viral genome sequences that cover a wide range of known viral families. Whole-genome sequences can be used to improve viral classification and provide insight into the viral tree of life . However, due to the lack of evolutionary conservation amongst diverse viruses, it is not feasible to build a viral tree of life using traditional phylogenetic methods based on conservedmore » proteins. In this study, we used an alignment-free method that uses k-mers as genomic features for a large-scale comparison of complete viral genomes available in RefSeq. To determine the optimal feature length, k (an essential step in constructing a meaningful dendrogram), we designed a comprehensive strategy that combines three approaches: (1) cumulative relative entropy, (2) average number of common features among genomes, and (3) the Shannon diversity index. This strategy was used to determine k for all 3,905 complete viral genomes in RefSeq. Lastly, the resulting dendrogram shows consistency with the viral taxonomy of the ICTV and the Baltimore classification of viruses.« less

Viral Phylogenomics Using an Alignment-Free Method: A Three-Step Approach to Determine Optimal Length of k-mer

PubMed Central

Zhang, Qian; Jun, Se-Ran; Leuze, Michael; Ussery, David; Nookaew, Intawat

2017-01-01

The development of rapid, economical genome sequencing has shed new light on the classification of viruses. As of October 2016, the National Center for Biotechnology Information (NCBI) database contained >2 million viral genome sequences and a reference set of ~4000 viral genome sequences that cover a wide range of known viral families. Whole-genome sequences can be used to improve viral classification and provide insight into the viral “tree of life”. However, due to the lack of evolutionary conservation amongst diverse viruses, it is not feasible to build a viral tree of life using traditional phylogenetic methods based on conserved proteins. In this study, we used an alignment-free method that uses k-mers as genomic features for a large-scale comparison of complete viral genomes available in RefSeq. To determine the optimal feature length, k (an essential step in constructing a meaningful dendrogram), we designed a comprehensive strategy that combines three approaches: (1) cumulative relative entropy, (2) average number of common features among genomes, and (3) the Shannon diversity index. This strategy was used to determine k for all 3,905 complete viral genomes in RefSeq. The resulting dendrogram shows consistency with the viral taxonomy of the ICTV and the Baltimore classification of viruses. PMID:28102365

A large outbreak of acute gastroenteritis in Shippensburg, Pennsylvania, 1972 revisited: evidence for common source exposure to a recombinant GII.Pg/GII.3 norovirus.

PubMed

Johnson, J A; Parra, G I; Levenson, E A; Green, K Y

2017-06-01

Historical outbreaks can be an important source of information in the understanding of norovirus evolution and epidemiology. Here, we revisit an outbreak of undiagnosed gastroenteritis that occurred in Shippensburg, Pennsylvania in 1972. Nearly 5000 people fell ill over the course of 10 days. Symptoms included diarrhea, vomiting, stomach cramps, and fever, lasting for a median of 24 h. Using current techniques, including next-generation sequencing of full-length viral genomic amplicons, we identified an unusual norovirus recombinant (GII.Pg/GII.3) in nine of 15 available stool samples from the outbreak. This particular recombinant virus has not been reported in recent decades, although GII.3 and GII.Pg genotypes have been detected individually in current epidemic strains. The consensus nucleotide sequences were nearly identical among the four viral genomes analysed, although each strain had three to seven positions in the genome with heterogenous non-synonymous nucleotide subpopulations. Two of these resulting amino acid polymorphisms were conserved in frequency among all four cases, consistent with common source exposure and successful transmission of a mixed viral population. Continued investigation of variant nucleotide populations and recombination events among ancestral norovirus strains such as the Shippensburg virus may provide unique insight into the origin of contemporary strains.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

PubMed Central

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-01-01

Abstract Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

Emerging complexities of APOBEC3G action on immunity and viral fitness during HIV infection and treatment.

PubMed

Monajemi, Mahdis; Woodworth, Claire F; Benkaroun, Jessica; Grant, Michael; Larijani, Mani

2012-04-30

The enzyme APOBEC3G (A3G) mutates the human immunodeficiency virus (HIV) genome by converting deoxycytidine (dC) to deoxyuridine (dU) on minus strand viral DNA during reverse transcription. A3G restricts viral propagation by degrading or incapacitating the coding ability of the HIV genome. Thus, this enzyme has been perceived as an innate immune barrier to viral replication whilst adaptive immunity responses escalate to effective levels. The discovery of A3G less than a decade ago led to the promise of new anti-viral therapies based on manipulation of its cellular expression and/or activity. The rationale for therapeutic approaches has been solidified by demonstration of the effectiveness of A3G in diminishing viral replication in cell culture systems of HIV infection, reports of its mutational footprint in virions from patients, and recognition of its unusually robust enzymatic potential in biochemical studies in vitro. Despite its effectiveness in various experimental systems, numerous recent studies have shown that the ability of A3G to combat HIV in the physiological setting is severely limited. In fact, it has become apparent that its mutational activity may actually enhance viral fitness by accelerating HIV evolution towards the evasion of both anti-viral drugs and the immune system. This body of work suggests that the role of A3G in HIV infection is more complex than heretofore appreciated and supports the hypothesis that HIV has evolved to exploit the action of this host factor. Here we present an overview of recent data that bring to light historical overestimation of A3G's standing as a strictly anti-viral agent. We discuss the limitations of experimental systems used to assess its activities as well as caveats in data interpretation.

Inherited Chromosomally Integrated Human Herpesvirus 6 Genomes Are Ancient, Intact, and Potentially Able To Reactivate from Telomeres

PubMed Central

Zhang, Enjie; Bell, Adam J.; Wilkie, Gavin S.; Suárez, Nicolás M.; Batini, Chiara; Veal, Colin D.; Armendáriz-Castillo, Isaac; Neumann, Rita; Cotton, Victoria E.; Huang, Yan; Porteous, David J.; Jarrett, Ruth F.; Davison, Andrew J.

2017-01-01

ABSTRACT The genomes of human herpesvirus 6A (HHV-6A) and HHV-6B have the capacity to integrate into telomeres, the essential capping structures of chromosomes that play roles in cancer and ageing. About 1% of people worldwide are carriers of chromosomally integrated HHV-6 (ciHHV-6), which is inherited as a genetic trait. Understanding the consequences of integration for the evolution of the viral genome, for the telomere, and for the risk of disease associated with carrier status is hampered by a lack of knowledge about ciHHV-6 genomes. Here, we report an analysis of 28 ciHHV-6 genomes and show that they are significantly divergent from the few modern nonintegrated HHV-6 strains for which complete sequences are currently available. In addition, ciHHV-6B genomes in Europeans are more closely related to each other than to ciHHV-6B genomes from China and Pakistan, suggesting regional variation of the trait. Remarkably, at least one group of European ciHHV-6B carriers has inherited the same ciHHV-6B genome, integrated in the same telomere allele, from a common ancestor estimated to have existed 24,500 ± 10,600 years ago. Despite the antiquity of some, and possibly most, germ line HHV-6 integrations, the majority of ciHHV-6B (95%) and ciHHV-6A (72%) genomes contain a full set of intact viral genes and therefore appear to have the capacity for viral gene expression and full reactivation. IMPORTANCE Inheritance of HHV-6A or HHV-6B integrated into a telomere occurs at a low frequency in most populations studied to date, but its characteristics are poorly understood. However, stratification of ciHHV-6 carriers in modern populations due to common ancestry is an important consideration for genome-wide association studies that aim to identify disease risks for these people. Here, we present full sequence analysis of 28 ciHHV-6 genomes and show that ciHHV-6B in many carriers with European ancestry most likely originated from ancient integration events in a small number of ancestors. We propose that ancient ancestral origins for ciHHV-6A and ciHHV-6B are also likely in other populations. Moreover, despite their antiquity, all of the ciHHV-6 genomes appear to retain the capacity to express viral genes, and most are predicted to be capable of full viral reactivation. These discoveries represent potentially important considerations in immunocompromised patients, in particular in organ transplantation and in stem cell therapy. PMID:28835501

Inherited Chromosomally Integrated Human Herpesvirus 6 Genomes Are Ancient, Intact, and Potentially Able To Reactivate from Telomeres.

PubMed

Zhang, Enjie; Bell, Adam J; Wilkie, Gavin S; Suárez, Nicolás M; Batini, Chiara; Veal, Colin D; Armendáriz-Castillo, Isaac; Neumann, Rita; Cotton, Victoria E; Huang, Yan; Porteous, David J; Jarrett, Ruth F; Davison, Andrew J; Royle, Nicola J

2017-11-15

The genomes of human herpesvirus 6A (HHV-6A) and HHV-6B have the capacity to integrate into telomeres, the essential capping structures of chromosomes that play roles in cancer and ageing. About 1% of people worldwide are carriers of chromosomally integrated HHV-6 (ciHHV-6), which is inherited as a genetic trait. Understanding the consequences of integration for the evolution of the viral genome, for the telomere, and for the risk of disease associated with carrier status is hampered by a lack of knowledge about ciHHV-6 genomes. Here, we report an analysis of 28 ciHHV-6 genomes and show that they are significantly divergent from the few modern nonintegrated HHV-6 strains for which complete sequences are currently available. In addition, ciHHV-6B genomes in Europeans are more closely related to each other than to ciHHV-6B genomes from China and Pakistan, suggesting regional variation of the trait. Remarkably, at least one group of European ciHHV-6B carriers has inherited the same ciHHV-6B genome, integrated in the same telomere allele, from a common ancestor estimated to have existed 24,500 ± 10,600 years ago. Despite the antiquity of some, and possibly most, germ line HHV-6 integrations, the majority of ciHHV-6B (95%) and ciHHV-6A (72%) genomes contain a full set of intact viral genes and therefore appear to have the capacity for viral gene expression and full reactivation. IMPORTANCE Inheritance of HHV-6A or HHV-6B integrated into a telomere occurs at a low frequency in most populations studied to date, but its characteristics are poorly understood. However, stratification of ciHHV-6 carriers in modern populations due to common ancestry is an important consideration for genome-wide association studies that aim to identify disease risks for these people. Here, we present full sequence analysis of 28 ciHHV-6 genomes and show that ciHHV-6B in many carriers with European ancestry most likely originated from ancient integration events in a small number of ancestors. We propose that ancient ancestral origins for ciHHV-6A and ciHHV-6B are also likely in other populations. Moreover, despite their antiquity, all of the ciHHV-6 genomes appear to retain the capacity to express viral genes, and most are predicted to be capable of full viral reactivation. These discoveries represent potentially important considerations in immunocompromised patients, in particular in organ transplantation and in stem cell therapy. Copyright © 2017 Zhang et al.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

PubMed

Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

2016-02-01

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics

DOE PAGES

Roux, Simon; Hawley, Alyse K.; Torres Beltran, Monica; ...

2014-08-29

Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus–host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186more » microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host's energy metabolism. Together, these results demonstrate closely coupled SUP05 virus–host co-evolutionary dynamics with the potential to modulate biogeochemical cycling in climate-critical and expanding OMZs.« less

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

PubMed

Moreno, Elena; Gallego, Isabel; Gregori, Josep; Lucía-Sanz, Adriana; Soria, María Eugenia; Castro, Victoria; Beach, Nathan M; Manrubia, Susanna; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M; Gómez, Jordi; Gastaminza, Pablo; Domingo, Esteban; Perales, Celia

2017-05-15

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments. Copyright © 2017 American Society for Microbiology.

Universal evolutionary selection for high dimensional silent patterns of information hidden in the redundancy of viral genetic code.

PubMed

Goz, Eli; Zafrir, Zohar; Tuller, Tamir

2018-04-30

Understanding how viruses co-evolve with their hosts and adapt various genomic level strategies in order to ensure their fitness may have essential implications in unveiling the secrets of viral evolution, and in developing new vaccines and therapeutic approaches. Here, based on a novel genomic analysis of 2,625 different viruses and 439 corresponding host organisms, we provide evidence of universal evolutionary selection for high dimensional 'silent' patterns of information hidden in the redundancy of viral genetic code. Our model suggests that long substrings of nucleotides in the coding regions of viruses from all classes, often also repeat in the corresponding viral hosts from all domains of life. Selection for these substrings cannot be explained only by such phenomena as codon usage bias, horizontal gene transfer, and the encoded proteins. Genes encoding structural proteins responsible for building the core of the viral particles were found to include more host-repeating substrings, and these substrings tend to appear in the middle parts of the viral coding regions. In addition, in human viruses these substrings tend to be enriched with motives related to transcription factors and RNA binding proteins. The host-repeating substrings are possibly related to the evolutionary pressure on the viruses to effectively interact with host's intracellular factors and to efficiently escape from the host's immune system. tamirtul@post.tau.ac.il (TT). Supplementary data are available at Bioinformatics online.

Single-Cell Analysis Uncovers Extensive Biological Noise in Poliovirus Replication

PubMed Central

Schulte, Michael B.

2014-01-01

ABSTRACT Viral infections often begin with a very small number of initiating particles. Accordingly, the outcome of an infection is likely to be affected by variability in the initial molecular interactions between virus and host. In this study, we investigated the range of outcomes upon infection of single cells. We isolated individual cells infected with poliovirus at low or high multiplicities of infection (MOI) and measured viral genomic replication and infectious viral progeny in each cell. We first determined that at 7 h postinfection, the ratio of positive to negative strands in individual cells varies from 5:1 to more than 190:1, with and average of 20:1, suggesting a significant variability in RNA synthesis. We further found that while virus genome production is higher in cells infected at a high multiplicity, the production of infectious particles is largely independent of the number of viruses infecting each cell. Strikingly, by correlating RNA and particle production within individual infections, we uncovered a significant contribution of stochastic noise to the outcome of infection. At low MOI, stochastic influences appear as kinetic effects which are most critical at the initial steps in infection. At high MOI, stochastic influences appear to dictate the virus's ability to harness cellular resources. We conclude that biological noise is a critical determinant of the overall productivity of viral infections. The distinct nature of stochasticity in the outcome of infection by low and high numbers of viral particles may have important implications for our understanding of the determinants of successful viral infections. IMPORTANCE By correlating genome and particle production in single-cell infections, we elucidated sources of noise in viral infections. When a cell was infected by only a single infectious particle, variation in the kinetics of the initial steps of replication contributed significantly to the overall productivity of the infection. Additionally, variation in the distribution of subcellular resources impacted infections initiated by one or many infectious particles. We also observed that when a cell was infected with multiple particles, more genomes were produced, while particle production was hindered by an apparent cellular resource limit. Understanding variations in viral infections may illuminate the dynamics of infection and pathogenesis and has implications for virus adaptation and evolution. PMID:24648454

Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae

PubMed Central

Huang, Youhua; Huang, Xiaohong; Liu, Hong; Gong, Jie; Ouyang, Zhengliang; Cui, Huachun; Cao, Jianhao; Zhao, Yingtao; Wang, Xiujie; Jiang, Yulin; Qin, Qiwei

2009-01-01

Background Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis. Results We determined the complete nucleotide sequence of the STIV genome using 454 Life Science sequencing technology. The STIV genome is 105 890 bp in length with a base composition of 55.1% G+C. Computer assisted analysis revealed that the STIV genome contains 105 potential open reading frames (ORFs), which encode polypeptides ranging from 40 to 1,294 amino acids and 20 microRNA candidates. Among the putative proteins, 20 share homology with the ancestral proteins of the nuclear and cytoplasmic large DNA viruses (NCLDVs). Comparative genomic analysis showed that STIV has the highest degree of sequence conservation and a colinear arrangement of genes with frog virus 3 (FV3), followed by Tiger frog virus (TFV), Ambystoma tigrinum virus (ATV), Singapore grouper iridovirus (SGIV), Grouper iridovirus (GIV) and other iridovirus isolates. Phylogenetic analysis based on conserved core genes and complete genome sequence of STIV with other virus genomes was performed. Moreover, analysis of the gene gain-and-loss events in the family Iridoviridae suggested that the genes encoded by iridoviruses have evolved for favoring adaptation to different natural host species. Conclusion This study has provided the complete genome sequence of STIV. Phylogenetic analysis suggested that STIV and FV3 are strains of the same viral species belonging to the Ranavirus genus in the Iridoviridae family. Given virus-host co-evolution and the phylogenetic relationship among vertebrates from fish to reptiles, we propose that iridovirus might transmit between reptiles and amphibians and that STIV and FV3 are strains of the same viral species in the Ranavirus genus. PMID:19439104

Co-Circulation and Evolution of Polioviruses and Species C Enteroviruses in a District of Madagascar

PubMed Central

Rakoto-Andrianarivelo, Mala; Guillot, Sophie; Iber, Jane; Balanant, Jean; Blondel, Bruno; Riquet, Franck; Martin, Javier; Kew, Olen; Randriamanalina, Bakolalao; Razafinimpiasa, Lalatiana; Rousset, Dominique; Delpeyroux, Francis

2007-01-01

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5′-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3′-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3Dpol coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity. PMID:18085822

Pan-vertebrate comparative genomics unmasks retrovirus macroevolution.

PubMed

Hayward, Alexander; Cornwallis, Charlie K; Jern, Patric

2015-01-13

Although extensive research has demonstrated host-retrovirus microevolutionary dynamics, it has been difficult to gain a deeper understanding of the macroevolutionary patterns of host-retrovirus interactions. Here we use recent technological advances to infer broad patterns in retroviral diversity, evolution, and host-virus relationships by using a large-scale phylogenomic approach using endogenous retroviruses (ERVs). Retroviruses insert a proviral DNA copy into the host cell genome to produce new viruses. ERVs are provirus insertions in germline cells that are inherited down the host lineage and consequently present a record of past host-viral associations. By mining ERVs from 65 host genomes sampled across vertebrate diversity, we uncover a great diversity of ERVs, indicating that retroviral sequences are much more prevalent and widespread across vertebrates than previously appreciated. The majority of ERV clades that we recover do not contain known retroviruses, implying either that retroviral lineages are highly transient over evolutionary time or that a considerable number of retroviruses remain to be identified. By characterizing the distribution of ERVs, we show that no major vertebrate lineage has escaped retroviral activity and that retroviruses are extreme host generalists, having an unprecedented ability for rampant host switching among distantly related vertebrates. In addition, we examine whether the distribution of ERVs can be explained by host factors predicted to influence viral transmission and find that internal fertilization has a pronounced effect on retroviral colonization of host genomes. By capturing the mode and pattern of retroviral evolution and contrasting ERV diversity with known retroviral diversity, our study provides a cohesive framework to understand host-virus coevolution better.

Pan-vertebrate comparative genomics unmasks retrovirus macroevolution

PubMed Central

Hayward, Alexander; Cornwallis, Charlie K.; Jern, Patric

2015-01-01

Although extensive research has demonstrated host-retrovirus microevolutionary dynamics, it has been difficult to gain a deeper understanding of the macroevolutionary patterns of host–retrovirus interactions. Here we use recent technological advances to infer broad patterns in retroviral diversity, evolution, and host–virus relationships by using a large-scale phylogenomic approach using endogenous retroviruses (ERVs). Retroviruses insert a proviral DNA copy into the host cell genome to produce new viruses. ERVs are provirus insertions in germline cells that are inherited down the host lineage and consequently present a record of past host–viral associations. By mining ERVs from 65 host genomes sampled across vertebrate diversity, we uncover a great diversity of ERVs, indicating that retroviral sequences are much more prevalent and widespread across vertebrates than previously appreciated. The majority of ERV clades that we recover do not contain known retroviruses, implying either that retroviral lineages are highly transient over evolutionary time or that a considerable number of retroviruses remain to be identified. By characterizing the distribution of ERVs, we show that no major vertebrate lineage has escaped retroviral activity and that retroviruses are extreme host generalists, having an unprecedented ability for rampant host switching among distantly related vertebrates. In addition, we examine whether the distribution of ERVs can be explained by host factors predicted to influence viral transmission and find that internal fertilization has a pronounced effect on retroviral colonization of host genomes. By capturing the mode and pattern of retroviral evolution and contrasting ERV diversity with known retroviral diversity, our study provides a cohesive framework to understand host–virus coevolution better. PMID:25535393

Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

DOE PAGES

Burstein, David; Sun, Christine L.; Brown, Christopher T.; ...

2016-02-03

Here, current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth’s ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. Wemore » correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.« less

Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burstein, David; Sun, Christine L.; Brown, Christopher T.

Here, current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth’s ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. Wemore » correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.« less

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes

PubMed Central

Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

2014-01-01

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. PMID:25071804

Gene repertoire of amoeba-associated giant viruses.

PubMed

Colson, Philippe; Raoult, Didier

2010-01-01

Acanthamoeba polyphaga mimivirus, Marseillevirus, and Sputnik, a virophage, are intra-amoebal viruses that have been isolated from water collected in cooling towers. They have provided fascinating data and have raised exciting questions about viruses definition and evolution. Mimivirus and Marseillevirus have been classified in the nucleo-cytoplasmic large DNA viruses (NCLDVs) class. Their genomes are the largest and fifth largest viral genomes sequenced so far. The gene repertoire of these amoeba-associated viruses can be divided into four groups: the core genome, genes acquired by lateral gene transfer, duplicated genes, and ORFans. Open reading frames (ORFs) that have homologs in the NCLDVs core gene set represent 2.9 and 6.1% of the Mimivirus and Marseillevirus gene contents, respectively. A substantial proportion of the Mimivirus, Marseillevirus and Sputnik ORFs exhibit sequence similarities to homologs found in bacteria, archaea, eukaryotes or viruses. The large amount of chimeric genes in these viral genomes might have resulted from acquisitions by lateral gene transfers, implicating sympatric bacteria and viruses with an intra-amoebal lifestyle. In addition, lineage-specific gene expansion may have played a major role in the genome shaping. Altogether, the data so far accumulated on amoeba-associated giant viruses are a powerful incentive to isolate and study additional strains to gain better understanding of their pangenome. Copyright 2010 S. Karger AG, Basel.

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties ▿

PubMed Central

Sánchez-Navarro, Jesús; Fajardo, Thor; Zicca, Stefania; Pallás, Vicente; Stavolone, Livia

2010-01-01

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway. PMID:20130061

Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus.

PubMed

Xin, Xiu; Wang, Hailong; Han, Lingling; Wang, Mingzhen; Fang, Hui; Hao, Yao; Li, Jiadai; Zhang, Hu; Zheng, Congyi; Shen, Chao

2018-05-01

Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G 2 /M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell. IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells. Copyright © 2018 American Society for Microbiology.

The great billion-year war between ribosome- and capsid-encoding organisms (cells and viruses) as the major source of evolutionary novelties.

PubMed

Forterre, Patrick; Prangishvili, David

2009-10-01

Our conceptions on the origin, nature, and role of viruses have been shaken recently by several independent lines of research. There are many reasons to believe now that viruses are more ancient than modern cells and have always been more abundant and diverse than their cellular targets. Viruses can be defined as capsid-encoding organisms that transform their "host" cell into a viral factory. If capsid-encoding organisms (viruses) and ribosome-encoding organisms (cells) are the major types of living entities on our planet, it seems logical to conclude that their conflict has been a major engine of biological evolution (in the framework of natural selection). In particular, many novelties first selected in the viral world might have been transferred to cells as a consequence of the continuous flow of viral genes into cellular genomes. We discuss recent observations and hypotheses suggesting that viruses have played a major role at different stages of biological evolution, such as the RNA to DNA transition, the origin of the eukaryotic nucleus, or, alternatively, the origin of unique features in multicellular macrobes.

Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

PubMed Central

Bonvicini, Francesca; Manaresi, Elisabetta; Di Furio, Francesca; De Falco, Luisa; Gallinella, Giorgio

2012-01-01

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression. The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections. The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19. PMID:22413013

Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion.

PubMed

Fulton, Benjamin O; Sachs, David; Schwarz, Megan C; Palese, Peter; Evans, Matthew J

2017-08-01

The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus. IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses. Copyright © 2017 American Society for Microbiology.

Three novel Pseudomonas phages isolated from composting provide insights into the evolution and diversity of tailed phages.

PubMed

Amgarten, Deyvid; Martins, Layla Farage; Lombardi, Karen Cristina; Antunes, Luciana Principal; de Souza, Ana Paula Silva; Nicastro, Gianlucca Gonçalves; Kitajima, Elliott Watanabe; Quaggio, Ronaldo Bento; Upton, Chris; Setubal, João Carlos; da Silva, Aline Maria

2017-05-04

Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. The results about the newly discovered and described phages contribute to the understanding of tailed bacteriophage diversity, evolution, and role in the complex composting environment.

Evolution and the complexity of bacteriophages.

PubMed

Serwer, Philip

2007-03-13

The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine which, if any, bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamics of homolog evolution. This hypothesis is an explanation of evolutionary leaps in general. If accurate, it will assist both understanding and influencing the evolution of microbes and their communities. Analysis of evolutionary complexity increase for at least prokaryotes should include analysis of genomes of long-genome bacteriophages.

Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1,000 Genomes Project.

PubMed

Santpere, Gabriel; Darre, Fleur; Blanco, Soledad; Alcami, Antonio; Villoslada, Pablo; Mar Albà, M; Navarro, Arcadi

2014-04-01

Most people in the world (∼90%) are infected by the Epstein-Barr virus (EBV), which establishes itself permanently in B cells. Infection by EBV is related to a number of diseases including infectious mononucleosis, multiple sclerosis, and different types of cancer. So far, only seven complete EBV strains have been described, all of them coming from donors presenting EBV-related diseases. To perform a detailed comparative genomic analysis of EBV including, for the first time, EBV strains derived from healthy individuals, we reconstructed EBV sequences infecting lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project. As strain B95-8 was used to transform B cells to obtain LCLs, it is always present, but a specific deletion in its genome sets it apart from natural EBV strains. After studying hundreds of individuals, we determined the presence of natural EBV in at least 10 of them and obtained a set of variants specific to wild-type EBV. By mapping the natural EBV reads into the EBV reference genome (NC007605), we constructed nearly complete wild-type viral genomes from three individuals. Adding them to the five disease-derived EBV genomic sequences available in the literature, we performed an in-depth comparative genomic analysis. We found that latency genes harbor more nucleotide diversity than lytic genes and that six out of nine latency-related genes, as well as other genes involved in viral attachment and entry into host cells, packaging, and the capsid, present the molecular signature of accelerated protein evolution rates, suggesting rapid host-parasite coevolution.

Are endogenous feline leukemia viruses really endogenous?

PubMed

Stewart, H; Jarrett, O; Hosie, M J; Willett, B J

2011-10-15

Full length endogenous feline leukemia virus (FeLV) proviruses exist within the genomes of many breeds of domestic cat raising the possibility that they may also exist in a transmissible exogenous form. Such viruses would share receptor usage with the recombinant FeLV-B subgroup, a viral subgroup that arises in vivo by recombination between exogenous subgroup A virus (FeLV-A) and endogenous FeLV. Accordingly, all isolates of FeLV-B made to date have contained a "helper" FeLV-A, consistent with their recombinatorial origin. In order to assess whether endogenous viruses are transmitted between cats, we examined primary isolates of FeLV for which the viral subgroup had been determined for the presence of a subgroup B virus that lacked an FeLV-A. Here we describe the identification of two primary field isolates of FeLV (2518 and 4314) that appeared to contain subgroup B virus only by classical interference assays, raising the possibility of between-host transmission of endogenous FeLV. Sequencing of the env gene and U3 region of the 3' long terminal repeat (LTR) confirmed that both viral genomes contained endogenous viral env genes. However the viral 3' LTRs appeared exogenous in origin with a putative 3' recombination breakpoint residing at the 3' end of the env gene. Further, the FeLV-2518 virions also co-packaged a truncated FeLV-A genome containing a defective env gene, termed FeLV-2518(A) whilst no helper subgroup A viral genome was detected in virions of FeLV-4314. The acquisition of an exogenous LTR by the endogenous FeLV in 4314 may have allowed a recombinant FeLV variant to outgrow an exogenous FeLV-A virus that was presumably present during first infection. Given time, a similar evolution may also occur within the 2518 isolate. The data suggest that endogenous FeLVs may be mobilised by acquisition of exogenous LTRs yielding novel viruses that type biologically as FeLV-B. Copyright © 2011 Elsevier B.V. All rights reserved.

Influenza A virus evolution and spatio-temporal dynamics in Eurasian wild birds: a phylogenetic and phylogeographical study of whole-genome sequence data

PubMed Central

Lewis, Nicola S.; Verhagen, Josanne H.; Javakhishvili, Zurab; Russell, Colin A.; Lexmond, Pascal; Westgeest, Kim B.; Bestebroer, Theo M.; Halpin, Rebecca A.; Lin, Xudong; Ransier, Amy; Fedorova, Nadia B.; Stockwell, Timothy B.; Latorre-Margalef, Neus; Olsen, Björn; Smith, Gavin; Bahl, Justin; Wentworth, David E.; Waldenström, Jonas; Fouchier, Ron A. M.

2015-01-01

Low pathogenic avian influenza A viruses (IAVs) have a natural host reservoir in wild waterbirds and the potential to spread to other host species. Here, we investigated the evolutionary, spatial and temporal dynamics of avian IAVs in Eurasian wild birds. We used whole-genome sequences collected as part of an intensive long-term Eurasian wild bird surveillance study, and combined this genetic data with temporal and spatial information to explore the virus evolutionary dynamics. Frequent reassortment and co-circulating lineages were observed for all eight genomic RNA segments over time. There was no apparent species-specific effect on the diversity of the avian IAVs. There was a spatial and temporal relationship between the Eurasian sequences and significant viral migration of avian IAVs from West Eurasia towards Central Eurasia. The observed viral migration patterns differed between segments. Furthermore, we discuss the challenges faced when analysing these surveillance and sequence data, and the caveats to be borne in mind when drawing conclusions from the apparent results of such analyses. PMID:25904147

Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus.

PubMed

Ansari, M Azim; Pedergnana, Vincent; L C Ip, Camilla; Magri, Andrea; Von Delft, Annette; Bonsall, David; Chaturvedi, Nimisha; Bartha, Istvan; Smith, David; Nicholson, George; McVean, Gilean; Trebes, Amy; Piazza, Paolo; Fellay, Jacques; Cooke, Graham; Foster, Graham R; Hudson, Emma; McLauchlan, John; Simmonds, Peter; Bowden, Rory; Klenerman, Paul; Barnes, Eleanor; Spencer, Chris C A

2017-05-01

Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control.

[Molecular evolution of the tick-borne encephalitis and Powassan viruses].

PubMed

Subbotina, E L; Loktev, V B

2012-01-01

The problem of emerging viruses, their genetic diversity and viral evolution in nature are attracting more attention. The phylogenetic analysis and evaluationary rate estimation were made for pathogenic flaviviruses such as tick-borne encephalitis virus (TBEV) and Powassan (PV) circulated in natural foci in Russia. 47 nucleotide sequences of encoded protein E of the TBEV and 17 sequences of NS5 genome region of the PV have been used. It was found that the rate of accumulation of nucleotide substitutions for E genome region of TBEV was approximately 1.4 x 10(-4) and 5.4 x 10(-5) substitutions per site per year for NS5 genome region of PV. The ratio of non-synonymous nucleotide substitutions to synonymous substitution (dN/dS) for viral sequences were estimated of 0.049 for TBEV and 0.098 for PV. Maximum value dN/dS was 0.201-0.220 for sub-cluster of Russian and Canadian strains of PV and the minimum - 0.024 for cluster of Russian and Chinese strains of Far Eastern genotype TBEV. Evaluation of time intervals of evolutionary events associated with these viruses showed that European subtype TBEV are diverged from all-TBEV ancestor within approximately 2750 years and the Siberian and Far Eastern subtypes are emerged about 2250 years ago. The PV was introduced into natural foci of the Primorsky Krai of Russia only about 70 years ago and PV is a very close to Canadian strains of PV. Evolutionary picture for PV in North America is similar to evolution of Siberian and Far Eastern subtypes TBEV in Asia. The divergence time for main genetic groups of TBEV and PV are correlated with historical periods of warming and cooling. These allow to propose a hypothesis that climate changes were essential to the evolution of the flaviviruses in the past millenniums.

Viral dark matter and virus–host interactions resolved from publicly available microbial genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roux, Simon; Hallam, Steven J.; Woyke, Tanja

The ecological importance of viruses is now widely recognized, yet our limited knowledge of viral sequence space and virus–host interactions precludes accurate prediction of their roles and impacts. In this study, we mined publicly available bacterial and archaeal genomic data sets to identify 12,498 high-confidence viral genomes linked to their microbial hosts. These data augment public data sets 10-fold, provide first viral sequences for 13 new bacterial phyla including ecologically abundant phyla, and help taxonomically identify 7–38% of ‘unknown’ sequence space in viromes. Genome- and network-based classification was largely consistent with accepted viral taxonomy and suggested that (i) 264 newmore » viral genera were identified (doubling known genera) and (ii) cross-taxon genomic recombination is limited. Further analyses provided empirical data on extrachromosomal prophages and coinfection prevalences, as well as evaluation of in silico virus–host linkage predictions. Together these findings illustrate the value of mining viral signal from microbial genomes.« less

Viral dark matter and virus-host interactions resolved from publicly available microbial genomes.

PubMed

Roux, Simon; Hallam, Steven J; Woyke, Tanja; Sullivan, Matthew B

2015-07-22

The ecological importance of viruses is now widely recognized, yet our limited knowledge of viral sequence space and virus-host interactions precludes accurate prediction of their roles and impacts. In this study, we mined publicly available bacterial and archaeal genomic data sets to identify 12,498 high-confidence viral genomes linked to their microbial hosts. These data augment public data sets 10-fold, provide first viral sequences for 13 new bacterial phyla including ecologically abundant phyla, and help taxonomically identify 7-38% of 'unknown' sequence space in viromes. Genome- and network-based classification was largely consistent with accepted viral taxonomy and suggested that (i) 264 new viral genera were identified (doubling known genera) and (ii) cross-taxon genomic recombination is limited. Further analyses provided empirical data on extrachromosomal prophages and coinfection prevalences, as well as evaluation of in silico virus-host linkage predictions. Together these findings illustrate the value of mining viral signal from microbial genomes.

Viral dark matter and virus–host interactions resolved from publicly available microbial genomes

DOE PAGES

Roux, Simon; Hallam, Steven J.; Woyke, Tanja; ...

2015-07-22

The ecological importance of viruses is now widely recognized, yet our limited knowledge of viral sequence space and virus–host interactions precludes accurate prediction of their roles and impacts. In this study, we mined publicly available bacterial and archaeal genomic data sets to identify 12,498 high-confidence viral genomes linked to their microbial hosts. These data augment public data sets 10-fold, provide first viral sequences for 13 new bacterial phyla including ecologically abundant phyla, and help taxonomically identify 7–38% of ‘unknown’ sequence space in viromes. Genome- and network-based classification was largely consistent with accepted viral taxonomy and suggested that (i) 264 newmore » viral genera were identified (doubling known genera) and (ii) cross-taxon genomic recombination is limited. Further analyses provided empirical data on extrachromosomal prophages and coinfection prevalences, as well as evaluation of in silico virus–host linkage predictions. Together these findings illustrate the value of mining viral signal from microbial genomes.« less

Extensive Gene Remodeling in the Viral World: New Evidence for Nongradual Evolution in the Mobilome Network

PubMed Central

Jachiet, Pierre-Alain; Colson, Philippe; Lopez, Philippe; Bapteste, Eric

2014-01-01

Complex nongradual evolutionary processes such as gene remodeling are difficult to model, to visualize, and to investigate systematically. Despite these challenges, the creation of composite (or mosaic) genes by combination of genetic segments from unrelated gene families was established as an important adaptive phenomena in eukaryotic genomes. In contrast, almost no general studies have been conducted to quantify composite genes in viruses. Although viral genome mosaicism has been well-described, the extent of gene mosaicism and its rules of emergence remain largely unexplored. Applying methods from graph theory to inclusive similarity networks, and using data from more than 3,000 complete viral genomes, we provide the first demonstration that composite genes in viruses are 1) functionally biased, 2) involved in key aspects of the arm race between cells and viruses, and 3) can be classified into two distinct types of composite genes in all viral classes. Beyond the quantification of the widespread recombination of genes among different viruses of the same class, we also report a striking sharing of genetic information between viruses of different classes and with different nucleic acid types. This latter discovery provides novel evidence for the existence of a large and complex mobilome network, which appears partly bound by the sharing of genetic information and by the formation of composite genes between mobile entities with different genetic material. Considering that there are around 10E31 viruses on the planet, gene remodeling appears as a hugely significant way of generating and moving novel sequences between different kinds of organisms on Earth. PMID:25104113

Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

PubMed Central

Chiu, Ya-Fang; Sugden, Arthur U.

2017-01-01

Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning and viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected. PMID:28696226

Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

DOE PAGES

Chiu, Ya-Fang; Sugden, Arthur U.; Fox, Kathryn; ...

2017-07-10

Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning andmore » viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.« less

Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiu, Ya-Fang; Sugden, Arthur U.; Fox, Kathryn

Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning andmore » viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.« less

Functional RNA elements in the dengue virus genome.

PubMed

Gebhard, Leopoldo G; Filomatori, Claudia V; Gamarnik, Andrea V

2011-09-01

Dengue virus (DENV) genome amplification is a process that involves the viral RNA, cellular and viral proteins, and a complex architecture of cellular membranes. The viral RNA is not a passive template during this process; it plays an active role providing RNA signals that act as promoters, enhancers and/or silencers of the replication process. RNA elements that modulate RNA replication were found at the 5' and 3' UTRs and within the viral coding sequence. The promoter for DENV RNA synthesis is a large stem loop structure located at the 5' end of the genome. This structure specifically interacts with the viral polymerase NS5 and promotes RNA synthesis at the 3' end of a circularized genome. The circular conformation of the viral genome is mediated by long range RNA-RNA interactions that span thousands of nucleotides. Recent studies have provided new information about the requirement of alternative, mutually exclusive, structures in the viral RNA, highlighting the idea that the viral genome is flexible and exists in different conformations. In this article, we describe elements in the promoter SLA and other RNA signals involved in NS5 polymerase binding and activity, and provide new ideas of how dynamic secondary and tertiary structures of the viral RNA participate in the viral life cycle.

Adenovirus Core Protein VII Protects the Viral Genome from a DNA Damage Response at Early Times after Infection▿

PubMed Central

Karen, Kasey A.; Hearing, Patrick

2011-01-01

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity. PMID:21345950

Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone

PubMed Central

Park, Daniel J.; Dudas, Gytis; Wohl, Shirlee; Goba, Augustine; Whitmer, Shannon L.M.; Andersen, Kristian G.; Sealfon, Rachel S.; Ladner, Jason T.; Kugelman, Jeffrey R.; Matranga, Christian B.; Winnicki, Sarah M.; Qu, James; Gire, Stephen K.; Gladden-Young, Adrianne; Jalloh, Simbirie; Nosamiefan, Dolo; Yozwiak, Nathan L.; Moses, Lina M.; Jiang, Pan-Pan; Lin, Aaron E.; Schaffner, Stephen F.; Bird, Brian; Towner, Jonathan; Mamoh, Mambu; Gbakie, Michael; Kanneh, Lansana; Kargbo, David; Massally, James L.B.; Kamara, Fatima K.; Konuwa, Edwin; Sellu, Josephine; Jalloh, Abdul A.; Mustapha, Ibrahim; Foday, Momoh; Yillah, Mohamed; Erickson, Bobbie R.; Sealy, Tara; Blau, Dianna; Paddock, Christopher; Brault, Aaron; Amman, Brian; Basile, Jane; Bearden, Scott; Belser, Jessica; Bergeron, Eric; Campbell, Shelley; Chakrabarti, Ayan; Dodd, Kimberly; Flint, Mike; Gibbons, Aridth; Goodman, Christin; Klena, John; McMullan, Laura; Morgan, Laura; Russell, Brandy; Salzer, Johanna; Sanchez, Angela; Wang, David; Jungreis, Irwin; Tomkins-Tinch, Christopher; Kislyuk, Andrey; Lin, Michael F.; Chapman, Sinead; MacInnis, Bronwyn; Matthews, Ashley; Bochicchio, James; Hensley, Lisa E.; Kuhn, Jens H.; Nusbaum, Chad; Schieffelin, John S.; Birren, Bruce W.; Forget, Marc; Nichol, Stuart T.; Palacios, Gustavo F.; Ndiaye, Daouda; Happi, Christian; Gevao, Sahr M.; Vandi, Mohamed A.; Kargbo, Brima; Holmes, Edward C.; Bedford, Trevor; Gnirke, Andreas; Ströher, Ute; Rambaut, Andrew; Garry, Robert F.; Sabeti, Pardis C.

2015-01-01

Summary The 2013–2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the region and describe viral evolution during prolonged human-to-human transmission. PMID:26091036

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

PubMed

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-06-02

Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

TIA-1 and TIAR interact with 5'-UTR of enterovirus 71 genome and facilitate viral replication.

PubMed

Wang, Xiaohui; Wang, Huanru; Li, Yixuan; Jin, Yu; Chu, Ying; Su, Airong; Wu, Zhiwei

2015-10-16

Enterovirus 71 is one of the major causative pathogens of HFMD in children. Upon infection, the viral RNA is translated in an IRES-dependent manner and requires several host factors for effective replication. Here, we found that T-cell-restricted intracellular antigen 1 (TIA-1), and TIA-1 related protein (TIAR) were translocated from nucleus to cytoplasm after EV71 infection and localized to the sites of viral replication. We found that TIA-1 and TIAR can facilitate EV71 replication by enhancing the viral genome synthesis in host cells. We demonstrated that both proteins bound to the stem-loop I of 5'-UTR of viral genome and improved the stability of viral genomic RNA. Our results suggest that TIA-1 and TIAR are two new host factors that interact with 5-UTR of EV71 genome and positively regulate viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Unexpected Inheritance: Multiple Integrations of Ancient Bornavirus and Ebolavirus/Marburgvirus Sequences in Vertebrate Genomes

PubMed Central

Belyi, Vladimir A.; Levine, Arnold J.; Skalka, Anna Marie

2010-01-01

Vertebrate genomes contain numerous copies of retroviral sequences, acquired over the course of evolution. Until recently they were thought to be the only type of RNA viruses to be so represented, because integration of a DNA copy of their genome is required for their replication. In this study, an extensive sequence comparison was conducted in which 5,666 viral genes from all known non-retroviral families with single-stranded RNA genomes were matched against the germline genomes of 48 vertebrate species, to determine if such viruses could also contribute to the vertebrate genetic heritage. In 19 of the tested vertebrate species, we discovered as many as 80 high-confidence examples of genomic DNA sequences that appear to be derived, as long ago as 40 million years, from ancestral members of 4 currently circulating virus families with single strand RNA genomes. Surprisingly, almost all of the sequences are related to only two families in the Order Mononegavirales: the Bornaviruses and the Filoviruses, which cause lethal neurological disease and hemorrhagic fevers, respectively. Based on signature landmarks some, and perhaps all, of the endogenous virus-like DNA sequences appear to be LINE element-facilitated integrations derived from viral mRNAs. The integrations represent genes that encode viral nucleocapsid, RNA-dependent-RNA-polymerase, matrix and, possibly, glycoproteins. Integrations are generally limited to one or very few copies of a related viral gene per species, suggesting that once the initial germline integration was obtained (or selected), later integrations failed or provided little advantage to the host. The conservation of relatively long open reading frames for several of the endogenous sequences, the virus-like protein regions represented, and a potential correlation between their presence and a species' resistance to the diseases caused by these pathogens, are consistent with the notion that their products provide some important biological advantage to the species. In addition, the viruses could also benefit, as some resistant species (e.g. bats) may serve as natural reservoirs for their persistence and transmission. Given the stringent limitations imposed in this informatics search, the examples described here should be considered a low estimate of the number of such integration events that have persisted over evolutionary time scales. Clearly, the sources of genetic information in vertebrate genomes are much more diverse than previously suspected. PMID:20686665

Unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes.

PubMed

Belyi, Vladimir A; Levine, Arnold J; Skalka, Anna Marie

2010-07-29

Vertebrate genomes contain numerous copies of retroviral sequences, acquired over the course of evolution. Until recently they were thought to be the only type of RNA viruses to be so represented, because integration of a DNA copy of their genome is required for their replication. In this study, an extensive sequence comparison was conducted in which 5,666 viral genes from all known non-retroviral families with single-stranded RNA genomes were matched against the germline genomes of 48 vertebrate species, to determine if such viruses could also contribute to the vertebrate genetic heritage. In 19 of the tested vertebrate species, we discovered as many as 80 high-confidence examples of genomic DNA sequences that appear to be derived, as long ago as 40 million years, from ancestral members of 4 currently circulating virus families with single strand RNA genomes. Surprisingly, almost all of the sequences are related to only two families in the Order Mononegavirales: the Bornaviruses and the Filoviruses, which cause lethal neurological disease and hemorrhagic fevers, respectively. Based on signature landmarks some, and perhaps all, of the endogenous virus-like DNA sequences appear to be LINE element-facilitated integrations derived from viral mRNAs. The integrations represent genes that encode viral nucleocapsid, RNA-dependent-RNA-polymerase, matrix and, possibly, glycoproteins. Integrations are generally limited to one or very few copies of a related viral gene per species, suggesting that once the initial germline integration was obtained (or selected), later integrations failed or provided little advantage to the host. The conservation of relatively long open reading frames for several of the endogenous sequences, the virus-like protein regions represented, and a potential correlation between their presence and a species' resistance to the diseases caused by these pathogens, are consistent with the notion that their products provide some important biological advantage to the species. In addition, the viruses could also benefit, as some resistant species (e.g. bats) may serve as natural reservoirs for their persistence and transmission. Given the stringent limitations imposed in this informatics search, the examples described here should be considered a low estimate of the number of such integration events that have persisted over evolutionary time scales. Clearly, the sources of genetic information in vertebrate genomes are much more diverse than previously suspected.

An experimental evaluation of drug-induced mutational meltdown as an antiviral treatment strategy.

PubMed

Bank, Claudia; Renzette, Nicholas; Liu, Ping; Matuszewski, Sebastian; Shim, Hyunjin; Foll, Matthieu; Bolon, Daniel N A; Zeldovich, Konstantin B; Kowalik, Timothy F; Finberg, Robert W; Wang, Jennifer P; Jensen, Jeffrey D

2016-11-01

The rapid evolution of drug resistance remains a critical public health concern. The treatment of influenza A virus (IAV) has proven particularly challenging, due to the ability of the virus to develop resistance against current antivirals and vaccines. Here, we evaluate a novel antiviral drug therapy, favipiravir, for which the mechanism of action in IAV involves an interaction with the viral RNA-dependent RNA polymerase resulting in an effective increase in the viral mutation rate. We used an experimental evolution framework, combined with novel population genetic method development for inference from time-sampled data, to evaluate the effectiveness of favipiravir against IAV. Evaluating whole genome polymorphism data across 15 time points under multiple drug concentrations and in controls, we present the first evidence for the ability of IAV populations to effectively adapt to low concentrations of favipiravir. In contrast, under high concentrations, we observe population extinction, indicative of mutational meltdown. We discuss the observed dynamics with respect to the evolutionary forces at play and emphasize the utility of evolutionary theory to inform drug development. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

PubMed

Bierle, Craig J; Anderholm, Kaitlyn M; Wang, Jian Ben; McVoy, Michael A; Schleiss, Mark R

2016-08-01

The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

VirSorter: mining viral signal from microbial genomic data.

PubMed

Roux, Simon; Enault, Francois; Hurwitz, Bonnie L; Sullivan, Matthew B

2015-01-01

Viruses of microbes impact all ecosystems where microbes drive key energy and substrate transformations including the oceans, humans and industrial fermenters. However, despite this recognized importance, our understanding of viral diversity and impacts remains limited by too few model systems and reference genomes. One way to fill these gaps in our knowledge of viral diversity is through the detection of viral signal in microbial genomic data. While multiple approaches have been developed and applied for the detection of prophages (viral genomes integrated in a microbial genome), new types of microbial genomic data are emerging that are more fragmented and larger scale, such as Single-cell Amplified Genomes (SAGs) of uncultivated organisms or genomic fragments assembled from metagenomic sequencing. Here, we present VirSorter, a tool designed to detect viral signal in these different types of microbial sequence data in both a reference-dependent and reference-independent manner, leveraging probabilistic models and extensive virome data to maximize detection of novel viruses. Performance testing shows that VirSorter's prophage prediction capability compares to that of available prophage predictors for complete genomes, but is superior in predicting viral sequences outside of a host genome (i.e., from extrachromosomal prophages, lytic infections, or partially assembled prophages). Furthermore, VirSorter outperforms existing tools for fragmented genomic and metagenomic datasets, and can identify viral signal in assembled sequence (contigs) as short as 3kb, while providing near-perfect identification (>95% Recall and 100% Precision) on contigs of at least 10kb. Because VirSorter scales to large datasets, it can also be used in "reverse" to more confidently identify viral sequence in viral metagenomes by sorting away cellular DNA whether derived from gene transfer agents, generalized transduction or contamination. Finally, VirSorter is made available through the iPlant Cyberinfrastructure that provides a web-based user interface interconnected with the required computing resources. VirSorter thus complements existing prophage prediction softwares to better leverage fragmented, SAG and metagenomic datasets in a way that will scale to modern sequencing. Given these features, VirSorter should enable the discovery of new viruses in microbial datasets, and further our understanding of uncultivated viral communities across diverse ecosystems.

VirSorter: mining viral signal from microbial genomic data

PubMed Central

Roux, Simon; Enault, Francois; Hurwitz, Bonnie L.

2015-01-01

Viruses of microbes impact all ecosystems where microbes drive key energy and substrate transformations including the oceans, humans and industrial fermenters. However, despite this recognized importance, our understanding of viral diversity and impacts remains limited by too few model systems and reference genomes. One way to fill these gaps in our knowledge of viral diversity is through the detection of viral signal in microbial genomic data. While multiple approaches have been developed and applied for the detection of prophages (viral genomes integrated in a microbial genome), new types of microbial genomic data are emerging that are more fragmented and larger scale, such as Single-cell Amplified Genomes (SAGs) of uncultivated organisms or genomic fragments assembled from metagenomic sequencing. Here, we present VirSorter, a tool designed to detect viral signal in these different types of microbial sequence data in both a reference-dependent and reference-independent manner, leveraging probabilistic models and extensive virome data to maximize detection of novel viruses. Performance testing shows that VirSorter’s prophage prediction capability compares to that of available prophage predictors for complete genomes, but is superior in predicting viral sequences outside of a host genome (i.e., from extrachromosomal prophages, lytic infections, or partially assembled prophages). Furthermore, VirSorter outperforms existing tools for fragmented genomic and metagenomic datasets, and can identify viral signal in assembled sequence (contigs) as short as 3kb, while providing near-perfect identification (>95% Recall and 100% Precision) on contigs of at least 10kb. Because VirSorter scales to large datasets, it can also be used in “reverse” to more confidently identify viral sequence in viral metagenomes by sorting away cellular DNA whether derived from gene transfer agents, generalized transduction or contamination. Finally, VirSorter is made available through the iPlant Cyberinfrastructure that provides a web-based user interface interconnected with the required computing resources. VirSorter thus complements existing prophage prediction softwares to better leverage fragmented, SAG and metagenomic datasets in a way that will scale to modern sequencing. Given these features, VirSorter should enable the discovery of new viruses in microbial datasets, and further our understanding of uncultivated viral communities across diverse ecosystems. PMID:26038737

Supramolecular Architecture of the Coronavirus Particle.

PubMed

Neuman, B W; Buchmeier, M J

2016-01-01

Coronavirus particles serve three fundamentally important functions in infection. The virion provides the means to deliver the viral genome across the plasma membrane of a host cell. The virion is also a means of escape for newly synthesized genomes. Lastly, the virion is a durable vessel that protects the genome on its journey between cells. This review summarizes the available X-ray crystallography, NMR, and cryoelectron microscopy structural data for coronavirus structural proteins, and looks at the role of each of the major structural proteins in virus entry and assembly. The potential wider conservation of the nucleoprotein fold identified in the Arteriviridae and Coronaviridae families and a speculative model for the evolution of corona-like virus architecture are discussed. © 2016 Elsevier Inc. All rights reserved.

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

PubMed Central

Lowry, Kym; Woodman, Andrew; Cook, Jonathan; Evans, David J.

2014-01-01

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses. PMID:24945141

Subversion of cytokine networks by virally encoded decoy receptors

PubMed Central

Epperson, Megan L.; Lee, Chung A.; Fremont, Daved H.

2012-01-01

Summary During the course of evolution, viruses have captured or created a diverse array of open reading frames that encode for proteins that serve to evade and sabotage the host innate and adaptive immune responses, which would otherwise lead to their elimination. These viral genomes are some of the best textbooks of immunology ever written. The established arsenal of immunomodulatory proteins encoded by viruses is large and growing and includes specificities for virtually all known inflammatory pathways and targets. The focus of this review is on herpes and poxvirus-encoded cytokine and chemokine binding proteins that serve to undermine the coordination of host immune surveillance. Structural and mechanistic studies of these decoy receptors have provided a wealth of information, not only about viral pathogenesis but also about the inner workings of cytokine signaling networks. PMID:23046131

Changes in the EV-A71 Genome through Recombination and Spontaneous Mutations: Impact on Virulence.

PubMed

Mandary, Madiiha Bibi; Poh, Chit Laa

2018-06-12

Enterovirus 71 (EV-A71) is a major etiological agent of hand, foot and mouth disease (HFMD) that mainly affects young children less than five years old. The onset of severe HFMD is due to neurological complications bringing about acute flaccid paralysis and pulmonary oedema. In this review, we address how genetic events such as recombination and spontaneous mutations could change the genomic organization of EV-A71, leading to an impact on viral virulence. An understanding of the recombination mechanism of the poliovirus and non-polio enteroviruses will provide further evidence of the emergence of novel strains responsible for fatal HFMD outbreaks. We aim to see if the virulence of EV-A71 is contributed solely by the presence of fatal strains or is due to the co-operation of quasispecies within a viral population. The phenomenon of quasispecies within the poliovirus is discussed to reflect viral fitness, virulence and its implications for EV-A71. Ultimately, this review gives an insight into the evolution patterns of EV-A71 by looking into its recombination history and how spontaneous mutations would affect its virulence.

Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics

PubMed Central

Roux, Simon; Hawley, Alyse K; Torres Beltran, Monica; Scofield, Melanie; Schwientek, Patrick; Stepanauskas, Ramunas; Woyke, Tanja; Hallam, Steven J; Sullivan, Matthew B

2014-01-01

Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus–host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host's energy metabolism. Together, these results demonstrate closely coupled SUP05 virus–host co-evolutionary dynamics with the potential to modulate biogeochemical cycling in climate-critical and expanding OMZs. DOI: http://dx.doi.org/10.7554/eLife.03125.001 PMID:25171894

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lang, Daniel; Ullrich, Kristian K.; Murat, Florent

Here, the draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome–scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene– and TE–rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono–centric with peaks of a class of Copia elements potentially coinciding with centromeres. Genemore » body methylation is evident in 5.7% of the protein–coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure–based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant–specific cell growth and tissue organization. The P. patens genome lacks the TE–rich pericentromeric and gene–rich distal regions typical for most flowering plant genomes. More non–seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.« less

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution

DOE PAGES

Lang, Daniel; Ullrich, Kristian K.; Murat, Florent; ...

2017-12-13

Here, the draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome–scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene– and TE–rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono–centric with peaks of a class of Copia elements potentially coinciding with centromeres. Genemore » body methylation is evident in 5.7% of the protein–coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure–based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant–specific cell growth and tissue organization. The P. patens genome lacks the TE–rich pericentromeric and gene–rich distal regions typical for most flowering plant genomes. More non–seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.« less

Dengue Virus Genome Uncoating Requires Ubiquitination.

PubMed

Byk, Laura A; Iglesias, Néstor G; De Maio, Federico A; Gebhard, Leopoldo G; Rossi, Mario; Gamarnik, Andrea V

2016-06-28

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade regarding molecular aspects of the fusion step, but little is known about the events that follow this process, which leads to viral RNA release from the nucleocapsid. Here, we investigated the fate of nucleocapsid components (capsid protein and viral genome) during the infection process and found that capsid is degraded by the ubiquitin-proteasome system. However, in contrast to that observed for other RNA and DNA viruses, dengue virus capsid degradation was not responsible for genome uncoating. Interestingly, we found that dengue virus genome release requires a nondegradative ubiquitination step. These results provide the first insights into dengue virus uncoating and present new opportunities for antiviral intervention. Copyright © 2016 Byk et al.

Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

PubMed Central

Stenglein, Mark D.; Jacobson, Elliott R.; Wozniak, Edward J.; Wellehan, James F. X.; Kincaid, Anne; Gordon, Marcus; Porter, Brian F.; Baumgartner, Wes; Stahl, Scott; Kelley, Karen; Towner, Jonathan S.

2014-01-01

ABSTRACT A severe, sometimes fatal respiratory disease has been observed in captive ball pythons (Python regius) since the late 1990s. In order to better understand this disease and its etiology, we collected case and control samples and performed pathological and diagnostic analyses. Electron micrographs revealed filamentous virus-like particles in lung epithelial cells of sick animals. Diagnostic testing for known pathogens did not identify an etiologic agent, so unbiased metagenomic sequencing was performed. Abundant nidovirus-like sequences were identified in cases and were used to assemble the genome of a previously unknown virus in the order Nidovirales. The nidoviruses, which were not previously known to infect nonavian reptiles, are a diverse order that includes important human and veterinary pathogens. The presence of the viral RNA was confirmed in all diseased animals (n = 8) but was not detected in healthy pythons or other snakes (n = 57). Viral RNA levels were generally highest in the lung and other respiratory tract tissues. The 33.5-kb viral genome is the largest RNA genome yet described and shares canonical characteristics with other nidovirus genomes, although several features distinguish this from related viruses. This virus, which we named ball python nidovirus (BPNV), will likely establish a new genus in Torovirinae subfamily. The identification of a novel nidovirus in reptiles contributes to our understanding of the biology and evolution of related viruses, and its association with lung disease in pythons is a promising step toward elucidating an etiology for this long-standing veterinary disease. PMID:25205093

Flavivirus and Filovirus EvoPrinters: New alignment tools for the comparative analysis of viral evolution.

PubMed

Brody, Thomas; Yavatkar, Amarendra S; Park, Dong Sun; Kuzin, Alexander; Ross, Jermaine; Odenwald, Ward F

2017-06-01

Flavivirus and Filovirus infections are serious epidemic threats to human populations. Multi-genome comparative analysis of these evolving pathogens affords a view of their essential, conserved sequence elements as well as progressive evolutionary changes. While phylogenetic analysis has yielded important insights, the growing number of available genomic sequences makes comparisons between hundreds of viral strains challenging. We report here a new approach for the comparative analysis of these hemorrhagic fever viruses that can superimpose an unlimited number of one-on-one alignments to identify important features within genomes of interest. We have adapted EvoPrinter alignment algorithms for the rapid comparative analysis of Flavivirus or Filovirus sequences including Zika and Ebola strains. The user can input a full genome or partial viral sequence and then view either individual comparisons or generate color-coded readouts that superimpose hundreds of one-on-one alignments to identify unique or shared identity SNPs that reveal ancestral relationships between strains. The user can also opt to select a database genome in order to access a library of pre-aligned genomes of either 1,094 Flaviviruses or 460 Filoviruses for rapid comparative analysis with all database entries or a select subset. Using EvoPrinter search and alignment programs, we show the following: 1) superimposing alignment data from many related strains identifies lineage identity SNPs, which enable the assessment of sublineage complexity within viral outbreaks; 2) whole-genome SNP profile screens uncover novel Dengue2 and Zika recombinant strains and their parental lineages; 3) differential SNP profiling identifies host cell A-to-I hyper-editing within Ebola and Marburg viruses, and 4) hundreds of superimposed one-on-one Ebola genome alignments highlight ultra-conserved regulatory sequences, invariant amino acid codons and evolutionarily variable protein-encoding domains within a single genome. EvoPrinter allows for the assessment of lineage complexity within Flavivirus or Filovirus outbreaks, identification of recombinant strains, highlights sequences that have undergone host cell A-to-I editing, and identifies unique input and database SNPs within highly conserved sequences. EvoPrinter's ability to superimpose alignment data from hundreds of strains onto a single genome has allowed us to identify unique Zika virus sublineages that are currently spreading in South, Central and North America, the Caribbean, and in China. This new set of integrated alignment programs should serve as a useful addition to existing tools for the comparative analysis of these viruses.

Viral dark matter and virus–host interactions resolved from publicly available microbial genomes

PubMed Central

Roux, Simon; Hallam, Steven J; Woyke, Tanja; Sullivan, Matthew B

2015-01-01

The ecological importance of viruses is now widely recognized, yet our limited knowledge of viral sequence space and virus–host interactions precludes accurate prediction of their roles and impacts. In this study, we mined publicly available bacterial and archaeal genomic data sets to identify 12,498 high-confidence viral genomes linked to their microbial hosts. These data augment public data sets 10-fold, provide first viral sequences for 13 new bacterial phyla including ecologically abundant phyla, and help taxonomically identify 7–38% of ‘unknown’ sequence space in viromes. Genome- and network-based classification was largely consistent with accepted viral taxonomy and suggested that (i) 264 new viral genera were identified (doubling known genera) and (ii) cross-taxon genomic recombination is limited. Further analyses provided empirical data on extrachromosomal prophages and coinfection prevalences, as well as evaluation of in silico virus–host linkage predictions. Together these findings illustrate the value of mining viral signal from microbial genomes. DOI: http://dx.doi.org/10.7554/eLife.08490.001 PMID:26200428

Phylogenetic Diversity and Genotypical Complexity of H9N2 Influenza A Viruses Revealed by Genomic Sequence Analysis

PubMed Central

Dong, Guoying; Luo, Jing; Zhang, Hong; Wang, Chengmin; Duan, Mingxing; Deliberto, Thomas Jude; Nolte, Dale Louis; Ji, Guangju; He, Hongxuan

2011-01-01

H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A–G). Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses. PMID:21386964

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

PubMed Central

Sampath, Rangarajan; Russell, Kevin L.; Massire, Christian; Eshoo, Mark W.; Harpin, Vanessa; Blyn, Lawrence B.; Melton, Rachael; Ivy, Cristina; Pennella, Thuy; Li, Feng; Levene, Harold; Hall, Thomas A.; Libby, Brian; Fan, Nancy; Walcott, Demetrius J.; Ranken, Raymond; Pear, Michael; Schink, Amy; Gutierrez, Jose; Drader, Jared; Moore, David; Metzgar, David; Addington, Lynda; Rothman, Richard; Gaydos, Charlotte A.; Yang, Samuel; St. George, Kirsten; Fuschino, Meghan E.; Dean, Amy B.; Stallknecht, David E.; Goekjian, Ginger; Yingst, Samuel; Monteville, Marshall; Saad, Magdi D.; Whitehouse, Chris A.; Baldwin, Carson; Rudnick, Karl H.; Hofstadler, Steven A.; Lemon, Stanley M.; Ecker, David J.

2007-01-01

Background Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. Methods and Principal Findings Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. Conclusion/Significance Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance. PMID:17534439

Bovine Enteroviruses as Indicators of Fecal Contamination

PubMed Central

Ley, Victoria; Higgins, James; Fayer, Ronald

2002-01-01

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies. PMID:12089028

Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

PubMed Central

Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

2011-01-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands. PMID:21908672

Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

PubMed

Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

2011-11-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

PubMed

Abdelwhab, E M; Hassan, M K; Abdel-Moneim, A S; Naguib, M M; Mostafa, A; Hussein, I T M; Arafa, A; Erfan, A M; Kilany, W H; Agour, M G; El-Kanawati, Z; Hussein, H A; Selim, A A; Kholousy, S; El-Naggar, H; El-Zoghby, E F; Samy, A; Iqbal, M; Eid, A; Ibraheem, E M; Pleschka, S; Veits, J; Nasef, S A; Beer, M; Mettenleiter, T C; Grund, C; Ali, M M; Harder, T C; Hafez, H M

2016-06-01

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years. Copyright © 2016 Elsevier B.V. All rights reserved.

FY09 Final Report for LDRD Project: Understanding Viral Quasispecies Evolution through Computation and Experiment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, C

2009-11-12

In FY09 they will (1) complete the implementation, verification, calibration, and sensitivity and scalability analysis of the in-cell virus replication model; (2) complete the design of the cell culture (cell-to-cell infection) model; (3) continue the research, design, and development of their bioinformatics tools: the Web-based structure-alignment-based sequence variability tool and the functional annotation of the genome database; (4) collaborate with the University of California at San Francisco on areas of common interest; and (5) submit journal articles that describe the in-cell model with simulations and the bioinformatics approaches to evaluation of genome variability and fitness.

Evolution of 2009 H1N1 influenza viruses during the pandemic correlates with increased viral pathogenicity and transmissibility in the ferret model.

PubMed

Otte, Anna; Marriott, Anthony C; Dreier, Carola; Dove, Brian; Mooren, Kyra; Klingen, Thorsten R; Sauter, Martina; Thompson, Katy-Anne; Bennett, Allan; Klingel, Karin; van Riel, Debby; McHardy, Alice C; Carroll, Miles W; Gabriel, Gülsah

2016-06-24

There is increasing evidence that 2009 pandemic H1N1 influenza viruses have evolved after pandemic onset giving rise to severe epidemics in subsequent waves. However, it still remains unclear which viral determinants might have contributed to disease severity after pandemic initiation. Here, we show that distinct mutations in the 2009 pandemic H1N1 virus genome have occurred with increased frequency after pandemic declaration. Among those, a mutation in the viral hemagglutinin was identified that increases 2009 pandemic H1N1 virus binding to human-like α2,6-linked sialic acids. Moreover, these mutations conferred increased viral replication in the respiratory tract and elevated respiratory droplet transmission between ferrets. Thus, our data show that 2009 H1N1 influenza viruses have evolved after pandemic onset giving rise to novel virus variants that enhance viral replicative fitness and respiratory droplet transmission in a mammalian animal model. These findings might help to improve surveillance efforts to assess the pandemic risk by emerging influenza viruses.

Non-viral delivery of genome-editing nucleases for gene therapy.

PubMed

Wang, M; Glass, Z A; Xu, Q

2017-03-01

Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.

PubMed

Andersen, Kristian G; Shapiro, B Jesse; Matranga, Christian B; Sealfon, Rachel; Lin, Aaron E; Moses, Lina M; Folarin, Onikepe A; Goba, Augustine; Odia, Ikponmwonsa; Ehiane, Philomena E; Momoh, Mambu; England, Eleina M; Winnicki, Sarah; Branco, Luis M; Gire, Stephen K; Phelan, Eric; Tariyal, Ridhi; Tewhey, Ryan; Omoniwa, Omowunmi; Fullah, Mohammed; Fonnie, Richard; Fonnie, Mbalu; Kanneh, Lansana; Jalloh, Simbirie; Gbakie, Michael; Saffa, Sidiki; Karbo, Kandeh; Gladden, Adrianne D; Qu, James; Stremlau, Matthew; Nekoui, Mahan; Finucane, Hilary K; Tabrizi, Shervin; Vitti, Joseph J; Birren, Bruce; Fitzgerald, Michael; McCowan, Caryn; Ireland, Andrea; Berlin, Aaron M; Bochicchio, James; Tazon-Vega, Barbara; Lennon, Niall J; Ryan, Elizabeth M; Bjornson, Zach; Milner, Danny A; Lukens, Amanda K; Broodie, Nisha; Rowland, Megan; Heinrich, Megan; Akdag, Marjan; Schieffelin, John S; Levy, Danielle; Akpan, Henry; Bausch, Daniel G; Rubins, Kathleen; McCormick, Joseph B; Lander, Eric S; Günther, Stephan; Hensley, Lisa; Okogbenin, Sylvanus; Schaffner, Stephen F; Okokhere, Peter O; Khan, S Humarr; Grant, Donald S; Akpede, George O; Asogun, Danny A; Gnirke, Andreas; Levin, Joshua Z; Happi, Christian T; Garry, Robert F; Sabeti, Pardis C

2015-08-13

The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Clinical sequencing uncovers origins and evolution of Lassa virus

PubMed Central

Andersen, Kristian G.; Shapiro, B. Jesse; Matranga, Christian B.; Sealfon, Rachel; Lin, Aaron E.; Moses, Lina M.; Folarin, Onikepe A.; Goba, Augustine; Odia, Ikponmwonsa; Ehiane, Philomena E.; Momoh, Mambu; England, Eleina M.; Winnicki, Sarah; Branco, Luis M.; Gire, Stephen K.; Phelan, Eric; Tariyal, Ridhi; Tewhey, Ryan; Omoniwa, Omowunmi; Fullah, Mohammed; Fonnie, Richard; Fonnie, Mbalu; Kanneh, Lansana; Jalloh, Simbirie; Gbakie, Michael; Saffa, Sidiki; Karbo, Kandeh; Gladden, Adrianne D.; Qu, James; Stremlau, Matthew; Nekoui, Mahan; Finucane, Hilary K.; Tabrizi, Shervin; Vitti, Joseph J.; Birren, Bruce; Fitzgerald, Michael; McCowan, Caryn; Ireland, Andrea; Berlin, Aaron M.; Bochicchio, James; Tazon-Vega, Barbara; Lennon, Niall J.; Ryan, Elizabeth M.; Bjornson, Zach; Milner, Danny A.; Lukens, Amanda K.; Broodie, Nisha; Rowland, Megan; Heinrich, Megan; Akdag, Marjan; Schieffelin, John S.; Levy, Danielle; Akpan, Henry; Bausch, Daniel G.; Rubins, Kathleen; McCormick, Joseph B.; Lander, Eric S.; Günther, Stephan; Hensley, Lisa; Okogbenin, Sylvanus; Schaffner, Stephen F.; Okokhere, Peter O.; Khan, S. Humarr; Grant, Donald S.; Akpede, George O.; Asogun, Danny A.; Gnirke, Andreas; Levin, Joshua Z.; Happi, Christian T.; Garry, Robert F.; Sabeti, Pardis C.

2015-01-01

Summary The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us how little is known about biosafety level-4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. PMID:26276630

Multiplex Reverse Transcription-PCR for Simultaneous Surveillance of Influenza A and B Viruses

PubMed Central

Zhou, Bin; Barnes, John R.; Sessions, October M.; Chou, Tsui-Wen; Wilson, Malania; Stark, Thomas J.; Volk, Michelle; Spirason, Natalie; Halpin, Rebecca A.; Kamaraj, Uma Sangumathi; Ding, Tao; Stockwell, Timothy B.; Ghedin, Elodie; Barr, Ian G.

2017-01-01

ABSTRACT Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms. PMID:28978683

Role of Host-Driven Mutagenesis in Determining Genome Evolution of Sigma Virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster.

PubMed

Piontkivska, Helen; Matos, Luis F; Paul, Sinu; Scharfenberg, Brian; Farmerie, William G; Miyamoto, Michael M; Wayne, Marta L

2016-10-05

Sigma virus (DMelSV) is ubiquitous in natural populations of Drosophila melanogaster. Host-mediated, selective RNA editing of adenosines to inosines (ADAR) may contribute to control of viral infection by preventing transcripts from being transported into the cytoplasm or being translated accurately; or by increasing the viral genomic mutation rate. Previous PCR-based studies showed that ADAR mutations occur in DMelSV at low frequency. Here we use SOLiD TM deep sequencing of flies from a single host population from Athens, GA, USA to comprehensively evaluate patterns of sequence variation in DMelSV with respect to ADAR. GA dinucleotides, which are weak targets of ADAR, are strongly overrepresented in the positive strand of the virus, consistent with selection to generate ADAR resistance on this complement of the transient, double-stranded RNA intermediate in replication and transcription. Potential ADAR sites in a worldwide sample of viruses are more likely to be "resistant" if the sites do not vary among samples. Either variable sites are less constrained and hence are subject to weaker selection than conserved sites, or the variation is driven by ADAR. We also find evidence of mutations segregating within hosts, hereafter referred to as hypervariable sites. Some of these sites were variable only in one or two flies (i.e., rare); others were shared by four or even all five of the flies (i.e., common). Rare and common hypervariable sites were indistinguishable with respect to susceptibility to ADAR; however, polymorphism in rare sites were more likely to be consistent with the action of ADAR than in common ones, again suggesting that ADAR is deleterious to the virus. Thus, in DMelSV, host mutagenesis is constraining viral evolution both within and between hosts. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Dengue Virus Genome Uncoating Requires Ubiquitination

PubMed Central

Byk, Laura A.; Iglesias, Néstor G.; De Maio, Federico A.; Gebhard, Leopoldo G.; Rossi, Mario

2016-01-01

ABSTRACT The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. PMID:27353759

Single-virus genomics reveals hidden cosmopolitan and abundant viruses

PubMed Central

Martinez-Hernandez, Francisco; Fornas, Oscar; Lluesma Gomez, Monica; Bolduc, Benjamin; de la Cruz Peña, Maria Jose; Martínez, Joaquín Martínez; Anton, Josefa; Gasol, Josep M.; Rosselli, Riccardo; Rodriguez-Valera, Francisco; Sullivan, Matthew B.; Acinas, Silvia G.; Martinez-Garcia, Manuel

2017-01-01

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies. PMID:28643787

Evolution of endogenous sequences of banana streak virus: what can we learn from banana (Musa sp.) evolution?

PubMed

Gayral, Philippe; Blondin, Laurence; Guidolin, Olivier; Carreel, Françoise; Hippolyte, Isabelle; Perrier, Xavier; Iskra-Caruana, Marie-Line

2010-07-01

Endogenous plant pararetroviruses (EPRVs) are viral sequences of the family Caulimoviridae integrated into the nuclear genome of numerous plant species. The ability of some endogenous sequences of Banana streak viruses (eBSVs) in the genome of banana (Musa sp.) to induce infections just like the virus itself was recently demonstrated (P. Gayral et al., J. Virol. 83:6697-6710, 2008). Although eBSVs probably arose from accidental events, infectious eBSVs constitute an extreme case of parasitism, as well as a newly described strategy for vertical virus transmission in plants. We investigated the early evolutionary stages of infectious eBSV for two distinct BSV species-GF (BSGFV) and Imové (BSImV)-through the study of their distribution, insertion polymorphism, and structure evolution among selected banana genotypes representative of the diversity of 60 wild Musa species and genotypes. To do so, the historical frame of host evolution was analyzed by inferring banana phylogeny from two chloroplast regions-matK and trnL-trnF-as well as from the nuclear genome, using 19 microsatellite loci. We demonstrated that both BSV species integrated recently in banana evolution, circa 640,000 years ago. The two infectious eBSVs were subjected to different selective pressures and showed distinct levels of rearrangement within their final structure. In addition, the molecular phylogenies of integrated and nonintegrated BSVs enabled us to establish the phylogenetic origins of eBSGFV and eBSImV.

Rapid one-step construction of a Middle East Respiratory Syndrome (MERS-CoV) infectious clone system by homologous recombination.

PubMed

Nikiforuk, Aidan M; Leung, Anders; Cook, Bradley W M; Court, Deborah A; Kobasa, Darwyn; Theriault, Steven S

2016-10-01

Viral Infectious clone systems serve as robust platforms to study viral gene or replicative function by reverse genetics, formulate vaccines and adapt a wild type-virus to an animal host. Since the development of the first viral infectious clone system for the poliovirus, novel strategies of viral genome construction have allowed for the assembly of viral genomes across the identified viral families. However, the molecular profiles of some viruses make their genome more difficult to construct than others. Two factors that affect the difficulty of infectious clone construction are genome length and genome complexity. This work examines the available strategies for overcoming the obstacles of assembling the long and complex RNA genomes of coronaviruses and reports one-step construction of an infectious clone system for the Middle East Respiratory Syndrome coronavirus (MERS-CoV) by homologous recombination in S. cerevisiae. Future use of this methodology will shorten the time between emergence of a novel viral pathogen and construction of an infectious clone system. Completion of a viral infectious clone system facilitates further study of a virus's biology, improvement of diagnostic tests, vaccine production and the screening of antiviral compounds. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

A Cluster of Paralytic Poliomyelitis Cases Due to Transmission of Slightly Diverged Sabin 2 Vaccine Poliovirus

PubMed Central

Korotkova, Ekaterina A.; Gmyl, Anatoly P.; Yakovenko, Maria L.; Ivanova, Olga E.; Eremeeva, Tatyana P.; Kozlovskaya, Liubov I.; Shakaryan, Armen K.; Lipskaya, Galina Y.; Parshina, Irina L.; Loginovskikh, Nataliya V.; Morozova, Nadezhda S.

2016-01-01

ABSTRACT Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July to August 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features. (i) Until this outbreak, Sabin-like viruses (in distinction to more markedly evolved vaccine-derived polioviruses [VDPVs]) were reported to cause only sporadic cases of VAPP. Consequently, VAPP cases were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPVs in epidemiological terms. (ii) The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity. (iii) The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events. (iv) The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking, and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems. IMPORTANCE The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: those caused by slightly diverged (Sabin-like) viruses on the one hand and those caused by significantly diverged VDPVs on the other. This classification is based on the number of mutations in the viral genome region encoding a viral structural protein. Until now, only sporadic poliomyelitis cases due to Sabin-like polioviruses had been described, and in distinction from the VDPV-triggered outbreaks, they did not require broad-scale epidemiological responses. Here, an unusual outbreak of poliomyelitis caused by a Sabin-like virus is reported, which had an exceptionally high disease/infection ratio. This outbreak blurred the borderline between Sabin-like polioviruses and VDPVs both in pathogenicity and in the kind of responses required, as well as underscoring important gaps in understanding the pathogenicity, epidemiology, and evolution of vaccine-derived polioviruses. PMID:27099315

A Cluster of Paralytic Poliomyelitis Cases Due to Transmission of Slightly Diverged Sabin 2 Vaccine Poliovirus.

PubMed

Korotkova, Ekaterina A; Gmyl, Anatoly P; Yakovenko, Maria L; Ivanova, Olga E; Eremeeva, Tatyana P; Kozlovskaya, Liubov I; Shakaryan, Armen K; Lipskaya, Galina Y; Parshina, Irina L; Loginovskikh, Nataliya V; Morozova, Nadezhda S; Agol, Vadim I

2016-07-01

Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July to August 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features. (i) Until this outbreak, Sabin-like viruses (in distinction to more markedly evolved vaccine-derived polioviruses [VDPVs]) were reported to cause only sporadic cases of VAPP. Consequently, VAPP cases were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPVs in epidemiological terms. (ii) The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity. (iii) The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events. (iv) The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking, and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems. The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: those caused by slightly diverged (Sabin-like) viruses on the one hand and those caused by significantly diverged VDPVs on the other. This classification is based on the number of mutations in the viral genome region encoding a viral structural protein. Until now, only sporadic poliomyelitis cases due to Sabin-like polioviruses had been described, and in distinction from the VDPV-triggered outbreaks, they did not require broad-scale epidemiological responses. Here, an unusual outbreak of poliomyelitis caused by a Sabin-like virus is reported, which had an exceptionally high disease/infection ratio. This outbreak blurred the borderline between Sabin-like polioviruses and VDPVs both in pathogenicity and in the kind of responses required, as well as underscoring important gaps in understanding the pathogenicity, epidemiology, and evolution of vaccine-derived polioviruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Viruses in close associations with free-living amoebae.

PubMed

Scheid, Patrick

2015-11-01

As both groups of organisms, free-living amoebae (FLA) and viruses, can be found in aquatic environments side by side, it appears obvious that there are multiple interactions with respect to host-endocytobiont relationships. Several relationships between viruses and protozoan hosts are described and it was the discovery of the so called "giant viruses," associated with amoebae, which gave another dimension to these interactions. Mimiviruses, Pandoraviruses and Pithoviruses are examples for interesting viral endocytobionts within FLA. In the Mimivirus viral factories, viral DNA undergoes replication and transcription, and the DNA is prepared to be packed in procapsids. Theses Mimivirus factories can be considered as efficient "production lines" where, at any given moment, all stages of viral generation including membrane biogenesis, capsid assembly and genome encapsidation, are occurring concomitantly. There are some hints that similar replication factories are involved as well during the Pandoravirus development. Some scientists favour the assumption that the giant viruses have received many of their genes from their hosts or from sympatric occurring endocytobionts via lateral gene transfer. This hypothesis would mean that this type of transfer has been an important process in the evolution of genomes in the context of the intracellular parasitic or endocytobiotic lifestyle. In turn, that would migitate against hypothesizing development of a new branch in the tree of life. Based on the described scenarios to explain the presence of genes related to translation, it is also possible that earlier ancestors of today's DNA viruses were involved in the origin of eukaryotes. That possibly could in turn support the idea that cellular organisms could have evolved from viruses with growing autarkic properties. In future we expect the discovery of further (giant) viruses within free-living amoebae and other protozoa through genomic, transcriptomic and proteomic analyses.

Detection of porcine circovirus type 2 in pigs imported from Indonesia.

PubMed

Manokaran, Gayathri; Lin, Yueh-Nuo; Soh, Moi-Lien; Lim, Elizabeth Ai-Sim; Lim, Chee-Wee; Tan, Boon-Huan

2008-11-25

We have detected the presence of porcine circovirus (PCV) type 2 in Indonesian pigs imported to Singapore for food consumption. A total of three viral isolates were identified, and to genetically characterise them further, their full genomes were sequenced. Each genome showed a typical organization of PCV type 2, with the three isolates sharing similar genome lengths of 1767 nucleotide (nt) at high nt identities of 99.8-100%, further indicating that the viral isolates were quite homogeneous. Sequence analysis further revealed that the ORF2 genes contain the nt sequence CCCCGC (from nt position 262 to 267) that was previously reported to be associated with PCV type 2, group 1C. The phylogenetic tree was constructed for the ORF2 genes, and the PCV type 2 isolates distributed into two distinctive groups. The Indonesian PCV type 2 clustered tightly with one China isolate, accession number AY035820, as a sub-cluster in group 1C. The sequence and phylogenetic analyses both confirmed that the three Indonesian PCV type 2 isolates belong to group 1C, and that the genetic changes for the three Indonesian isolates were very stable, possibly due to the low-scale evolution.

Functional hierarchy of two L domains in porcine endogenous retrovirus (PERV) that influence release and infectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcucci, Katherine T.; Kellogg School of Science and Technology, Scripps Research Institute, La Jolla, CA, 92037; Martina, Yuri

2008-06-05

The porcine endogenous retrovirus (PERV) Gag protein contains two late (L) domain motifs, PPPY and P(F/S)AP. Using viral release assays we demonstrate that PPPY is the dominant L domain involved in PERV release. PFAP represents a novel retroviral L domain variant and is defined by abnormal viral assembly phenotypes visualized by electron microscopy and attenuation of early PERV release as measured by viral genomes. PSAP is functionally dominant over PFAP in early PERV release. PSAP virions are 3.5-fold more infectious in vitro by TCID{sub 50} and in vivo results in more RNA positive tissues and higher levels of proviral DNAmore » using our human PERV-A receptor (HuPAR-2) transgenic mouse model [Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U., Wilson, C.A., Patience, C., Salomon, D.R., 2006. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. J. Virol. 80 (7), 3135-3146]. The functional hierarchies displayed by PERV L domains, demonstrates that L domain selection in viral evolution exists to promote efficient viral assembly, release and infectivity in the virus-host context.« less

Evidence of translation efficiency adaptation of the coding regions of the bacteriophage lambda.

PubMed

Goz, Eli; Mioduser, Oriah; Diament, Alon; Tuller, Tamir

2017-08-01

Deciphering the way gene expression regulatory aspects are encoded in viral genomes is a challenging mission with ramifications related to all biomedical disciplines. Here, we aimed to understand how the evolution shapes the bacteriophage lambda genes by performing a high resolution analysis of ribosomal profiling data and gene expression related synonymous/silent information encoded in bacteriophage coding regions.We demonstrated evidence of selection for distinct compositions of synonymous codons in early and late viral genes related to the adaptation of translation efficiency to different bacteriophage developmental stages. Specifically, we showed that evolution of viral coding regions is driven, among others, by selection for codons with higher decoding rates; during the initial/progressive stages of infection the decoding rates in early/late genes were found to be superior to those in late/early genes, respectively. Moreover, we argued that selection for translation efficiency could be partially explained by adaptation to Escherichia coli tRNA pool and the fact that it can change during the bacteriophage life cycle.An analysis of additional aspects related to the expression of viral genes, such as mRNA folding and more complex/longer regulatory signals in the coding regions, is also reported. The reported conclusions are likely to be relevant also to additional viruses. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Infection cycles of large DNA viruses: emerging themes and underlying questions.

PubMed

Mutsafi, Yael; Fridmann-Sirkis, Yael; Milrot, Elad; Hevroni, Liron; Minsky, Abraham

2014-10-01

The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these 'nuclear-like' organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the 'stargate' portal that is used for genome release. Such a 'division of labor' is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Consolidation of molecular testing in clinical virology.

PubMed

Scagnolari, Carolina; Turriziani, Ombretta; Monteleone, Katia; Pierangeli, Alessandra; Antonelli, Guido

2017-04-01

The development of quantitative methods for the detection of viral nucleic acids have significantly improved our ability to manage disease progression and to assess the efficacy of antiviral treatment. Moreover, major advances in molecular technologies during the last decade have allowed the identification of new host genetic markers associated with antiviral drug response but have also strongly revolutionized the way we see and perform virus diagnostics in the coming years. Areas covered: In this review, we describe the history and development of virology diagnostic methods, dedicating particular emphasis on the gradual evolution and recent advances toward the introduction of multiparametric platforms for the syndromic diagnosis. In parallel, we outline the consolidation of viral genome quantification practice in different clinical settings. Expert commentary: More rapid, accurate and affordable molecular technology can be predictable with particular emphasis on emerging techniques (next generation sequencing, digital PCR, point of care testing and syndromic diagnosis) to simplify viral diagnosis in the next future.

Frequent inter-species transmission and geographic subdivision in avian influenza viruses from wild birds.

PubMed

Chen, Rubing; Holmes, Edward C

2009-01-05

Revealing the factors that shape the genetic structure of avian influenza viruses (AIVs) in wild bird populations is essential to understanding their evolution. However, the relationship between epidemiological dynamics and patterns of genetic diversity in AIV is not well understood, especially at the continental scale. To address this question, we undertook a phylogeographic analysis of complete genome sequences of AIV sampled from wild birds in North America. In particular, we asked whether host species, geographic location or sampling time played the major role in shaping patterns of viral genetic diversity. Strikingly, our analysis revealed no strong species effect, yet a significant viral clustering by time and place of sampling, as well as the circulation of multiple viral lineages in single locations. These results suggest that AIVs can readily infect many of the bird species that share breeding/feeding areas.

Effects of cellular differentiation, chromosomal integration and 5-aza-2'-deoxycytidine treatment on human papillomavirus-16 DNA methylation in cultured cell lines.

PubMed

Kalantari, Mina; Lee, Denis; Calleja-Macias, Itzel E; Lambert, Paul F; Bernard, Hans-Ulrich

2008-05-10

Human papillomavirus-16 (HPV-16) genomes in cell culture and in situ are affected by polymorphic methylation patterns, which can repress the viral transcription. In order to understand some of the underlying mechanisms, we investigated changes of the methylation of HPV-16 DNA in cell cultures in response to cellular differentiation, to recombination with cellular DNA, and to an inhibitor of methylation. Undifferentiated W12E cells, derived from a precancerous lesion, contained extrachromosomal HPV-16 DNA with a sporadically methylated enhancer-promoter segment. Upon W12E cell differentiation, the viral DNA was demethylated, suggesting a link between differentiation and the epigenetic state of HPV-16 DNA. The viral genomes present in two W12I clones, in which individual copies of the HPV-16 genome have integrated into cellular DNA (type 1 integrants), were unmethylated, akin to that seen in the cervical carcinoma cell line SiHa (also a type 1 integrant). This finding is consistent with hypomethylation being necessary for continued viral gene expression. In contrast, two of three type 2 integrant W12I clones, containing concatemers of HPV-16 genomes integrated into the cellular DNA contained hypermethylated viral DNA, as observed in the cervical carcinoma cell line CaSki (also a type 2 integrant). A third, type 2, W12I clone, interestingly with fewer copies of the viral genome, contained unmethylated HPV-16 genomes. Epithelial differentiation of W12I clones did not lead to demethylation of chromosomally integrated viral genomes as was seen for extrachromosomal HPV-16 DNA in W12E clones. Hypomethylation of CaSki cells in the presence of the DNA methylation inhibitor 5-aza-2'-deoxycytidine reduced the cellular viability, possibly as a consequence of toxic effects of an excess of HPV-16 gene products. Our data support a model wherein (i) the DNA methylation state of extrachromosomal HPV16 replicons and epithelial differentiation are inversely coupled during the viral life cycle, (ii) integration of the viral genome into the host chromosome events leads to an alteration in methylation patterns on the viral genome that is dependent upon the type of integration event and possibly copy number, and (iii) integration universally results in the viral DNA becoming refractory to changes in methylation state upon cellular differentiation that are observed with extrachromosomal HPV-16 genomes.

In silico and ex vivo approaches indicate immune pressure on capsid and non-capsid regions of coxsackie B viruses in the human system.

PubMed

Kundu, Rhiannon; Knight, Robin; Dunga, Meenakshi; Peakman, Mark

2018-01-01

Coxsackie B Virus (CBV) infection has been linked to the aetiology of type 1 diabetes (T1D) and vaccination has been proposed as prophylaxis for disease prevention. Serum neutralising antibodies and the presence of viral protein and RNA in tissues have been common tools to examine this potential disease relationship, whilst the role of anti-CBV cytotoxic T cell responses and their targets have not been studied. To address this knowledge gap, we augmented conventional HLA-binding predictive algorithm-based epitope discovery by cross-referencing epitopes with sites of positive natural selection within the CBV3 viral genome, identified using mixed effects models of evolution. Eight epitopes for the common MHC class I allele HLA-A*0201 occur at sites that appear to be positively selected. Furthermore, such epitopes span the viral genome, indicating that effective anti-viral responses may not be restricted to the capsid region. To assess the spectrum of IFNy responses in non-diabetic subjects and recently diagnosed type 1 diabetes (T1D) patients, we stimulated PBMC ex vivo with pools of synthetic peptides based on component-restricted sequences identified in silico. We found responders were more likely to recognize multiple rather than a single CBV peptide pool, indicating that the natural course of infection results in multiple targets for effector memory responses, rather than immunodominant epitopes or viral components. The finding that anti-CBV CD8 T cell immunity is broadly targeted has implications for vaccination strategies and studies on the pathogenesis of CBV-linked diseases.

Structure of Ljungan virus provides insight into genome packaging of this picornavirus

NASA Astrophysics Data System (ADS)

Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Porta, Claudine; Wenham, Hannah; Ekström, Jens-Ola; Panjwani, Anusha; Knowles, Nick J.; Kotecha, Abhay; Siebert, C. Alistair; Lindberg, A. Michael; Fry, Elizabeth E.; Rao, Zihe; Tuthill, Tobias J.; Stuart, David I.

2015-10-01

Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

Structure of Ljungan virus provides insight into genome packaging of this picornavirus.

PubMed

Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Porta, Claudine; Wenham, Hannah; Ekström, Jens-Ola; Panjwani, Anusha; Knowles, Nick J; Kotecha, Abhay; Siebert, C Alistair; Lindberg, A Michael; Fry, Elizabeth E; Rao, Zihe; Tuthill, Tobias J; Stuart, David I

2015-10-08

Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes

PubMed Central

Gilbert, C.; Meik, J. M.; Dashevsky, D.; Card, D. C.; Castoe, T. A.; Schaack, S.

2014-01-01

We report the discovery of endogenous viral elements (EVEs) from Hepadnaviridae, Bornaviridae and Circoviridae in the speckled rattlesnake, Crotalus mitchellii, the first viperid snake for which a draft whole genome sequence assembly is available. Analysis of the draft assembly reveals genome fragments from the three virus families were inserted into the genome of this snake over the past 50 Myr. Cross-species PCR screening of orthologous loci and computational scanning of the python and king cobra genomes reveals that circoviruses integrated most recently (within the last approx. 10 Myr), whereas bornaviruses and hepadnaviruses integrated at least approximately 13 and approximately 50 Ma, respectively. This is, to our knowledge, the first report of circo-, borna- and hepadnaviruses in snakes and the first characterization of non-retroviral EVEs in non-avian reptiles. Our study provides a window into the historical dynamics of viruses in these host lineages and shows that their evolution involved multiple host-switches between mammals and reptiles. PMID:25080342

A complete ancient RNA genome: identification, reconstruction and evolutionary history of archaeological Barley Stripe Mosaic Virus

PubMed Central

Smith, Oliver; Clapham, Alan; Rose, Pam; Liu, Yuan; Wang, Jun; Allaby, Robin G.

2014-01-01

The origins of many plant diseases appear to be recent and associated with the rise of domestication, the spread of agriculture or recent global movements of crops. Distinguishing between these possibilities is problematic because of the difficulty of determining rates of molecular evolution over short time frames. Heterochronous approaches using recent and historical samples show that plant viruses exhibit highly variable and often rapid rates of molecular evolution. The accuracy of estimated evolution rates and age of origin can be greatly improved with the inclusion of older molecular data from archaeological material. Here we present the first reconstruction of an archaeological RNA genome, which is of Barley Stripe Mosaic Virus (BSMV) isolated from barley grain ~750 years of age. Phylogenetic analysis of BSMV that includes this genome indicates the divergence of BSMV and its closest relative prior to this time, most likely around 2000 years ago. However, exclusion of the archaeological data results in an apparently much more recent origin of the virus that postdates even the archaeological sample. We conclude that this viral lineage originated in the Near East or North Africa, and spread to North America and East Asia with their hosts along historical trade routes. PMID:24499968

BRD4 is associated with raccoon polyomavirus genome and mediates viral gene transcription and maintenance of a stem cell state in neuroglial tumour cells.

PubMed

Church, Molly E; Estrada, Marko; Leutenegger, Christian M; Dela Cruz, Florante N; Pesavento, Patricia A; Woolard, Kevin D

2016-11-01

Polyomavirus infection often results in persistence of the viral genome with little or no virion production. However, infection of certain cell types can result in high viral gene transcription and either cytolysis or neoplastic transformation. While infection by polyomavirus is common in humans and many animals, major questions regarding viral persistence of most polyomaviruses remain unanswered. Specifically, identification of target cells for viral infection and the mechanisms polyomaviruses employ to maintain viral genomes within cells are important not only in ascribing causality to polyomaviruses in disease, but in understanding specific mechanisms by which they cause disease. Here, we characterize the cell of origin in raccoon polyomavirus (RacPyV)-associated neuroglial brain tumours as a neural stem cell. Moreover, we identify an association between the viral genome and the host cell bromodomain protein, BRD4, which is involved in numerous cellular functions, including cell cycle progression, differentiation of stem cells, tethering of persistent DNA viruses, and regulation of viral and host-cell gene transcription. We demonstrate that inhibition of BRD4 by the small molecule inhibitors (+)-JQ1 and IBET-151 (GSK1210151A) results in reduced RacPyV genome within cells in vitro, as well as significant reduction of viral gene transcripts LT and VP1, highlighting its importance in both maintenance of the viral genome and in driving oncogenic transformation by RacPyV. This work implicates BRD4 as a central protein involved in RacPyV neuroglial tumour cell proliferation and in the maintenance of a stem cell state.

Infection cycles of large DNA viruses: Emerging themes and underlying questions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mutsafi, Yael, E-mail: yael.mutsafi@weizmann.ac.il; Fridmann-Sirkis, Yael; Milrot, Elad

The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in themore » crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these ‘nuclear-like’ organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the ‘stargate’ portal that is used for genome release. Such a ‘division of labor’ is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. - Highlights: • The discovery of giant DNA viruses blurs the distinction between viruses and cells. • Mimivirus and Vaccinia replicate exclusively in their host cytoplasm. • Mimivirus genome is delivered through a unique portal coined the Stargate. • Generation of Mimivirus internal membrane proceeds through a novel pathway.« less

Structure, sequence and expression of the hepatitis delta (δ) viral genome

NASA Astrophysics Data System (ADS)

Wang, Kang-Sheng; Choo, Qui-Lim; Weiner, Amy J.; Ou, Jing-Hsiung; Najarian, Richard C.; Thayer, Richard M.; Mullenbach, Guy T.; Denniston, Katherine J.; Gerin, John L.; Houghton, Michael

1986-10-01

Biochemical and electron microscopic data indicate that the human hepatitis δ viral agent contains a covalently closed circular and single-stranded RNA genome that has certain similarities with viroid-like agents from plants. The sequence of the viral genome (1,678 nucleotides) has been determined and an open reading frame within the complementary strand has been shown to encode an antigen that binds specifically to antisera from patients with chronic hepatitis δ viral infections.

Exploration of sequence space as the basis of viral RNA genome segmentation.

PubMed

Moreno, Elena; Ojosnegros, Samuel; García-Arriaza, Juan; Escarmís, Cristina; Domingo, Esteban; Perales, Celia

2014-05-06

The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp.

PubMed

Kim, Saet-Byul; Kang, Won-Hee; Huy, Hoang Ngoc; Yeom, Seon-In; An, Jeong-Tak; Kim, Seungill; Kang, Min-Young; Kim, Hyun Jung; Jo, Yeong Deuk; Ha, Yeaseong; Choi, Doil; Kang, Byoung-Cheorl

2017-01-01

Plants have evolved hundreds of nucleotide-binding and leucine-rich domain proteins (NLRs) as potential intracellular immune receptors, but the evolutionary mechanism leading to the ability to recognize specific pathogen effectors is elusive. Here, we cloned Pvr4 (a Potyvirus resistance gene in Capsicum annuum) and Tsw (a Tomato spotted wilt virus resistance gene in Capsicum chinense) via a genome-based approach using independent segregating populations. The genes both encode typical NLRs and are located at the same locus on pepper chromosome 10. Despite the fact that these two genes recognize completely different viral effectors, the genomic structures and coding sequences of the two genes are strikingly similar. Phylogenetic studies revealed that these two immune receptors diverged from a progenitor gene of a common ancestor. Our results suggest that sequence variations caused by gene duplication and neofunctionalization may underlie the evolution of the ability to specifically recognize different effectors. These findings thereby provide insight into the divergent evolution of plant immune receptors. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Ancient papillomavirus-host co-speciation in Felidae

PubMed Central

Rector, Annabel; Lemey, Philippe; Tachezy, Ruth; Mostmans, Sara; Ghim, Shin-Je; Van Doorslaer, Koenraad; Roelke, Melody; Bush, Mitchell; Montali, Richard J; Joslin, Janis; Burk, Robert D; Jenson, Alfred B; Sundberg, John P; Shapiro, Beth; Van Ranst, Marc

2007-01-01

Background Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1. Results The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 × 10-8 (95% confidence interval 1.32 × 10-8 to 2.47 × 10-8) nucleotide substitutions per site per year for the viral coding genome. Conclusion Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence. PMID:17430578

Single-virus genomics reveals hidden cosmopolitan and abundant viruses

DOE PAGES

Martinez-Hernandez, Francisco; Fornas, Oscar; Lluesma Gomez, Monica; ...

2017-06-23

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and thatmore » microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.« less

Single-virus genomics reveals hidden cosmopolitan and abundant viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez-Hernandez, Francisco; Fornas, Oscar; Lluesma Gomez, Monica

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and thatmore » microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.« less

Molecular characterization of occult hepatitis B virus in genotype E-infected subjects.

PubMed

Zahn, Astrid; Li, Chengyao; Danso, Kwabena; Candotti, Daniel; Owusu-Ofori, Shirley; Temple, Jillian; Allain, Jean-Pierre

2008-02-01

Occult hepatitis B virus (HBV) infection (OBI), defined as the presence of HBV DNA without detectable HBV surface antigen (HBsAg), is frequent in west Africa, where genotype E is prevalent. The prevalence of OBI in 804 blood donors and 1368 pregnant women was 1.7 and 1.5%, respectively. Nine of 32 OBI carriers were evaluated with HBV serology, viral load and complete HBV genome sequence of two to five clones. All samples except one were anti-HBV core antigen-positive and three contained antibodies against HBsAg (anti-HBs). All strains were of genotype E and formed quasispecies with 0.20-1.28% intra-sample sequence variation. Few uncommon mutations (absent in 23 genotype E reference sequences) were found across the entire genome. Two mutations in the core region encoded truncated or abnormal capsid protein, potentially affecting viral production, but were probably rescued by non-mutated variants, as found in one clone. No evidence of escape mutants was found in anti-HBs-carrying samples, as the 'a' region was consistently wild type. OBI carriers constitute approximately 10% of all HBV DNA-viraemic adult Ghanaians. OBI carriers appear as a disparate group, with a very low viral load in common, but multiple origins reflecting decades of natural evolution in an area essentially devoid of human intervention.

The impact of HIV-1 genetic diversity on the efficacy of a combinatorial RNAi-based gene therapy.

PubMed

Herrera-Carrillo, E; Berkhout, B

2015-06-01

A hurdle for human immunodeficiency virus (HIV-1) therapy is the genomic diversity of circulating viruses and the possibility that drug-resistant virus variants are selected. Although RNA interference (RNAi) is a powerful tool to stably inhibit HIV-1 replication by the expression of antiviral short hairpin RNAs (shRNAs) in transduced T cells, this approach is also vulnerable to pre-existing genetic variation and the development of viral resistance through mutation. To prevent viral escape, we proposed to combine multiple shRNAs against important regions of the HIV-1 RNA genome, which should ideally be conserved in all HIV-1 subtypes. The vulnerability of RNAi therapy to viral escape has been studied for a single subtype B strain, but it is unclear whether the antiviral shRNAs can inhibit diverse virus isolates and subtypes, including drug-resistant variants that could be present in treated patients. To determine the breadth of the RNAi gene therapy approach, we studied the susceptibility of HIV-1 subtypes A-E and drug-resistant variants. In addition, we monitored the evolution of HIV-1 escape variants. We demonstrate that the combinatorial RNAi therapy is highly effective against most isolates, supporting the future testing of this gene therapy in appropriate in vivo models.

Hypotheses of cancer weakening and origin.

PubMed

Chan, John Cheung Yuen

2015-01-01

Approximately 2.7 billion years ago, cyanobacteria began producing oxygen by photosynthesis. Any free oxygen they produced was chemically captured by dissolved iron or organic matter. There was no ozone layer to protect living species against the radiation from space. Eukaryotic cells lived in water, under hypoxic environments, and metabolized glucose by fermentation. The Great Oxygenation Event (GOE) describes the point when oxygen sinks became saturated. This massive oxygenation of the Earth occurred approximately half a billion years ago. Species that evolved after the GOE are characterized by aerobic metabolism. Mammals evolved approximately a few hundred million years ago, with the ancient eukaryotic genes deeply embedded in their genome. Many genes have been exchanged by horizontal gene transfer (HGT) throughout the history of cellular evolution. Mammals have been invaded by viruses, and while viral genetic relics are embedded in mammalian junk genes, not all junk genes are genetic relics of viruses. These viral relics have been inactivated through evolution and have little impact on mammalian life. However, there is evidence to suggest that these viral genetic relics are linked to cancer. This hypothesis states that cancer develops when cell reproduction becomes defective because of the active involvement of viral genes, in a process similar to genetic engineering. Cancer cells are amalgamations of genetically modified organisms (GMOs). There are two main groups in cancer development. One group of cells arises by genetic engineering of a viral genetic relic, such as endogenous retroviruses (ERVs), which evolved after oxygenation of the atmosphere. This group is referred to here as genetically modified organisms from viral genes (GMOV). GMOVs may be inhibited by anticancer drugs. The second group arises by engineering of the genes of ancient eukaryotes, which existed prior to the oxygenation of the Earth. This second group is referred to as genetically modified organisms from ancient eukaryotic genes (GMOE). The GMOE group lives in hypoxic environments and metabolizes glucose by fermentation. GMOEs represent advanced cancer, which proliferate aggressively and are resistant to DNA damage. It has been demonstrated that as an ERV becomes more prevalent in a mammalian genome, the possibility that the mammal will develop cancer increases. The hypothesis also states that most cancers have their origins in GMOV by the incorporation of viral genes from junk genes. As the cancer progresses, further subgroups of cancer GMOs will develop. If the cancer advances even further, the GMOE could eventually develop prior to late-stage cancer. Because the genes of ancient eukaryotes have enhanced innate immunity, GMOE will eventually prevail over the weaker GMOV during cancer subgroup competition. Hence, cancer development is mainly determined by genes in the mammalian genome. An inherent weakness of cancer cells is their dependence on glucose and iron. Furthermore, they cannot tolerate physical disturbance. Ancient gene GMOs can be treated with a combination of mechanical vibration using glucose-coated magnetic nanoparticles and strengthening of the immune system. Herein, I suggest trials for verifying this hypothesis.

Hypotheses of Cancer Weakening and Origin

PubMed Central

CHAN, John Cheung Yuen

2015-01-01

Approximately 2.7 billion years ago, cyanobacteria began producing oxygen by photosynthesis. Any free oxygen they produced was chemically captured by dissolved iron or organic matter. There was no ozone layer to protect living species against the radiation from space. Eukaryotic cells lived in water, under hypoxic environments, and metabolized glucose by fermentation. The Great Oxygenation Event (GOE) describes the point when oxygen sinks became saturated. This massive oxygenation of the Earth occurred approximately half a billion years ago. Species that evolved after the GOE are characterized by aerobic metabolism. Mammals evolved approximately a few hundred million years ago, with the ancient eukaryotic genes deeply embedded in their genome. Many genes have been exchanged by horizontal gene transfer (HGT) throughout the history of cellular evolution. Mammals have been invaded by viruses, and while viral genetic relics are embedded in mammalian junk genes, not all junk genes are genetic relics of viruses. These viral relics have been inactivated through evolution and have little impact on mammalian life. However, there is evidence to suggest that these viral genetic relics are linked to cancer. This hypothesis states that cancer develops when cell reproduction becomes defective because of the active involvement of viral genes, in a process similar to genetic engineering. Cancer cells are amalgamations of genetically modified organisms (GMOs). There are two main groups in cancer development. One group of cells arises by genetic engineering of a viral genetic relic, such as endogenous retroviruses (ERVs), which evolved after oxygenation of the atmosphere. This group is referred to here as genetically modified organisms from viral genes (GMOV). GMOVs may be inhibited by anticancer drugs. The second group arises by engineering of the genes of ancient eukaryotes, which existed prior to the oxygenation of the Earth. This second group is referred to as genetically modified organisms from ancient eukaryotic genes (GMOE). The GMOE group lives in hypoxic environments and metabolizes glucose by fermentation. GMOEs represent advanced cancer, which proliferate aggressively and are resistant to DNA damage. It has been demonstrated that as an ERV becomes more prevalent in a mammalian genome, the possibility that the mammal will develop cancer increases. The hypothesis also states that most cancers have their origins in GMOV by the incorporation of viral genes from junk genes. As the cancer progresses, further subgroups of cancer GMOs will develop. If the cancer advances even further, the GMOE could eventually develop prior to late-stage cancer. Because the genes of ancient eukaryotes have enhanced innate immunity, GMOE will eventually prevail over the weaker GMOV during cancer subgroup competition. Hence, cancer development is mainly determined by genes in the mammalian genome. An inherent weakness of cancer cells is their dependence on glucose and iron. Furthermore, they cannot tolerate physical disturbance. Ancient gene GMOs can be treated with a combination of mechanical vibration using glucose-coated magnetic nanoparticles and strengthening of the immune system. Herein, I suggest trials for verifying this hypothesis. PMID:25874009

Adaptive evolution influences the infectious dose of MERS-CoV necessary to achieve severe respiratory disease.

PubMed

Douglas, Madeline G; Kocher, Jacob F; Scobey, Trevor; Baric, Ralph S; Cockrell, Adam S

2018-04-01

We recently established a mouse model (288-330 +/+ ) that developed acute respiratory disease resembling human pathology following infection with a high dose (5 × 10 6 PFU) of mouse-adapted MERS-CoV (icMERSma1). Although this high dose conferred fatal respiratory disease in mice, achieving similar pathology at lower viral doses may more closely reflect naturally acquired infections. Through continued adaptive evolution of icMERSma1 we generated a novel mouse-adapted MERS-CoV (maM35c4) capable of achieving severe respiratory disease at doses between 10 3 and 10 5 PFU. Novel mutations were identified in the maM35c4 genome that may be responsible for eliciting etiologies of acute respiratory distress syndrome at 10-1000 fold lower viral doses. Importantly, comparative genetics of the two mouse-adapted MERS strains allowed us to identify specific mutations that remained fixed through an additional 20 cycles of adaptive evolution. Our data indicate that the extent of MERS-CoV adaptation determines the minimal infectious dose required to achieve severe respiratory disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a Novel Enterovirus Species in Rhesus Macaque in China.

PubMed

Ao, Yuan-Yun; Yu, Jie-Mei; Zhang, Cui-Yuan; Xin, Yun-Yun; Li, Li-Li; Duan, Zhao-Jun

2016-06-22

Recent studies of Enterovirus (EV) in nonhuman primates (NHPs), which could act as a source of future emerging human viral diseases, have boosted interest in the search for novel EVs. Here, a highly divergent strain of EV, tentatively named SEV-gx, was identified by viral metagenomic analysis from stool samples of rhesus macaques in China. In total, 27 of 280 (9.6%) faecal samples from rhesus macaques were positive for SEV-gx. Its complete genomic sequence is 7,367 nucleotide (nt). Genomic analyses showed that it has a standard genomic organisation for EVs, being more closely related to EV-J strains (approximately 54.0%, 43.0-44.1%, 52.3-55.2%, 61.1-62.7% and 64.0% amino acids identity in polyprotein, P1, P2 and P3 and combined 2C/3CD regions, respectively). It was also shown to have genome characteristics typical of EVs. Phylogenetic analysis of P1, 2C and 3CD aa indicated that SEV-gx can be classified as a distinct cluster in the EVs. All of this evidence demonstrates SEV-gx is a novel species (tentatively named EV-K) in the EV genus, which contributes to our understanding of the genetic diversity and evolution of EVs. Further studies are needed to investigate the potential pathogenicity of SEV-gx in NHPs and humans.

Mechanisms of inhibition of viral replication in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1990-01-01

We have made a number of interesting observations of importance to the fields of virology and plant molecular biology. Topics include the genome of cucumber mosaic virus (CMV), recombination of the CMV genome, transgenic plants and viral movement genes, mapping resistance breakage sequences in the tomato mosaic virus (TMV) genome, and mapping pathogeneticity domains and viral RNA heterogeneity. 1 fig., 1 tab.

High-efficiency dual labeling of influenza virus for single-virus imaging.

PubMed

Liu, Shu-Lin; Tian, Zhi-Quan; Zhang, Zhi-Ling; Wu, Qiu-Mei; Zhao, Hai-Su; Ren, Bin; Pang, Dai-Wen

2012-11-01

Many viruses invade host cells by entering the cells and releasing their genome for replication, which are remarkable incidents for viral infection. Therefore, the viral internal and external components should be simultaneously labeled and dynamically tracked at single-virus level for further understanding viral infection mechanisms. However, most of the previously reported methods have very low labeling efficiency and require considerable time and effort, which is laborious and inconvenient for researchers. In this work, we report a general strategy to high-efficiently label viral envelope and genome for single-virus imaging with quantum dots (QDs) and Syto 82, respectively. It was found that nearly all viral envelopes could be labeled with QDs with superior stability, which makes it possible to realize global and long-term tracking of single virus in individual cells. Effectively labeling their genome with Syto 82, about 90% of QDs-labeled viruses could be used to monitor the viral genome signal, which may provide valuable information for deeply studying viral genome transport. This is very important and meaningful to investigate the viral infection mechanism. Our labeling strategy has advantage in commonality, convenience and efficiency, which is expected to be widely used in biological research. Copyright © 2012 Elsevier Ltd. All rights reserved.

Orchestrating the Selection and Packaging of Genomic RNA by Retroviruses: An Ensemble of Viral and Host Factors

PubMed Central

Kaddis Maldonado, Rebecca J.; Parent, Leslie J.

2016-01-01

Infectious retrovirus particles contain two copies of unspliced viral RNA that serve as the viral genome. Unspliced retroviral RNA is transcribed in the nucleus by the host RNA polymerase II and has three potential fates: (1) it can be spliced into subgenomic messenger RNAs (mRNAs) for the translation of viral proteins; or it can remain unspliced to serve as either (2) the mRNA for the translation of Gag and Gag–Pol; or (3) the genomic RNA (gRNA) that is packaged into virions. The Gag structural protein recognizes and binds the unspliced viral RNA to select it as a genome, which is selected in preference to spliced viral RNAs and cellular RNAs. In this review, we summarize the current state of understanding about how retroviral packaging is orchestrated within the cell and explore potential new mechanisms based on recent discoveries in the field. We discuss the cis-acting elements in the unspliced viral RNA and the properties of the Gag protein that are required for their interaction. In addition, we discuss the role of host factors in influencing the fate of the newly transcribed viral RNA, current models for how retroviruses distinguish unspliced viral mRNA from viral genomic RNA, and the possible subcellular sites of genomic RNA dimerization and selection by Gag. Although this review centers primarily on the wealth of data available for the alpharetrovirus Rous sarcoma virus, in which a discrete RNA packaging sequence has been identified, we have also summarized the cis- and trans-acting factors as well as the mechanisms governing gRNA packaging of other retroviruses for comparison. PMID:27657110

Ins and Outs of Multipartite Positive-Strand RNA Plant Viruses: Packaging versus Systemic Spread

PubMed Central

Dall’Ara, Mattia; Ratti, Claudio; Bouzoubaa, Salah E.; Gilmer, David

2016-01-01

Viruses possessing a non-segmented genome require a specific recognition of their nucleic acid to ensure its protection in a capsid. A similar feature exists for viruses having a segmented genome, usually consisting of viral genomic segments joined together into one viral entity. While this appears as a rule for animal viruses, the majority of segmented plant viruses package their genomic segments individually. To ensure a productive infection, all viral particles and thereby all segments have to be present in the same cell. Progression of the virus within the plant requires as well a concerted genome preservation to avoid loss of function. In this review, we will discuss the “life aspects” of chosen phytoviruses and argue for the existence of RNA-RNA interactions that drive the preservation of viral genome integrity while the virus progresses in the plant. PMID:27548199

Prophage-mediated defense against viral attack and viral counter-defense

PubMed Central

Dedrick, Rebekah M.; Jacobs-Sera, Deborah; Guerrero Bustamante, Carlos A.; Garlena, Rebecca A.; Mavrich, Travis N.; Pope, Welkin H.; Reyes, Juan C Cervantes; Russell, Daniel A.; Adair, Tamarah; Alvey, Richard; Bonilla, J. Alfred; Bricker, Jerald S.; Brown, Bryony R.; Byrnes, Deanna; Cresawn, Steven G.; Davis, William B.; Dickson, Leon A.; Edgington, Nicholas P.; Findley, Ann M.; Golebiewska, Urszula; Grose, Julianne H.; Hayes, Cory F.; Hughes, Lee E.; Hutchison, Keith W.; Isern, Sharon; Johnson, Allison A.; Kenna, Margaret A.; Klyczek, Karen K.; Mageeney, Catherine M.; Michael, Scott F.; Molloy, Sally D.; Montgomery, Matthew T.; Neitzel, James; Page, Shallee T.; Pizzorno, Marie C.; Poxleitner, Marianne K.; Rinehart, Claire A.; Robinson, Courtney J.; Rubin, Michael R.; Teyim, Joseph N.; Vazquez, Edwin; Ware, Vassie C.; Washington, Jacqueline; Hatfull, Graham F.

2017-01-01

Temperate phages are common and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses infecting mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity, and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages reveals at least five distinct prophage-expressed viral defense systems that interfere with infection of lytic and temperate phages that are either closely-related (homotypic defense) or unrelated (heterotypic defense). Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defense systems include a single-subunit restriction system, a heterotypic exclusion system, and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival, and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, that acts as a highly effective counter-defense system. Prophage-mediated viral defense offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defense promotes phage co-evolution. PMID:28067906

Lethal mutagenesis: targeting the mutator phenotype in cancer.

PubMed

Fox, Edward J; Loeb, Lawrence A

2010-10-01

The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method.

PubMed

Dou, Dan; Hernández-Neuta, Iván; Wang, Hao; Östbye, Henrik; Qian, Xiaoyan; Thiele, Swantje; Resa-Infante, Patricia; Kouassi, Nancy Mounogou; Sender, Vicky; Hentrich, Karina; Mellroth, Peter; Henriques-Normark, Birgitta; Gabriel, Gülsah; Nilsson, Mats; Daniels, Robert

2017-07-05

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Diverse mechanisms evolved by DNA viruses to inhibit early host defenses

PubMed Central

Sheng, Xinlei; Song, Bokai; Cristea, Ileana M.

2016-01-01

In mammalian cells, early defenses against infection by pathogens are mounted through a complex network of signaling pathways shepherded by immune-modulatory pattern-recognition receptors. As obligate parasites, the survival of viruses is dependent upon the evolutionary acquisition of mechanisms that tactfully dismantle and subvert the cellular intrinsic and innate immune responses. Here, we review the diverse mechanisms by which viruses that accommodate DNA genomes are able to circumvent activation of cellular immunity. We start by discussing viral manipulation of host defense protein levels by either transcriptional regulation or protein degradation. We next review viral strategies used to repurpose or inhibit these cellular immune factors by molecular hijacking or by regulating their post-translational modification status. Additionally, we explore the infection-induced temporal modulation of apoptosis to facilitate viral replication and spread. Lastly, the co-evolution of viruses with their hosts is highlighted by the acquisition of elegant mechanisms for suppressing host defenses via viral mimicry of host factors. In closing, we present a perspective on how characterizing these viral evasion tactics both broadens the understanding of virus-host interactions and reveals essential functions of the immune system at the molecular level. This knowledge is critical in understanding the sources of viral pathogenesis, as well as for the design of antiviral therapeutics and autoimmunity treatments. PMID:27650455

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

PubMed

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

2017-08-15

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication. Copyright © 2017 American Society for Microbiology.

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

PubMed Central

Frattini, M G; Lim, H B; Laimins, L A

1996-01-01

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8610168

Insights into origin and evolution of α-proteobacterial gene transfer agents

PubMed Central

Shakya, Migun; Soucy, Shannon M

2017-01-01

Abstract Several bacterial and archaeal lineages produce nanostructures that morphologically resemble small tailed viruses, but, unlike most viruses, contain apparently random pieces of the host genome. Since these elements can deliver the packaged DNA to other cells, they were dubbed gene transfer agents (GTAs). Because many genes involved in GTA production have viral homologs, it has been hypothesized that the GTA ancestor was a virus. Whether GTAs represent an atypical virus, a defective virus, or a virus co-opted by the prokaryotes for some function, remains to be elucidated. To evaluate these possibilities, we examined the distribution and evolutionary histories of genes that encode a GTA in the α-proteobacterium Rhodobacter capsulatus (RcGTA). We report that although homologs of many individual RcGTA genes are abundant across bacteria and their viruses, RcGTA-like genomes are mainly found in one subclade of α-proteobacteria. When compared with the viral homologs, genes of the RcGTA-like genomes evolve significantly slower, and do not have higher %A+T nucleotides than their host chromosomes. Moreover, they appear to reside in stable regions of the bacterial chromosomes that are generally conserved across taxonomic orders. These findings argue against RcGTA being an atypical or a defective virus. Our phylogenetic analyses suggest that RcGTA ancestor likely originated in the lineage that gave rise to contemporary α-proteobacterial orders Rhizobiales, Rhodobacterales, Caulobacterales, Parvularculales, and Sphingomonadales, and since that time the RcGTA-like element has co-evolved with its host chromosomes. Such evolutionary history is compatible with maintenance of these elements by bacteria due to some selective advantage. As for many other prokaryotic traits, horizontal gene transfer played a substantial role in the evolution of RcGTA-like elements, not only in shaping its genome components within the orders, but also in occasional dissemination of RcGTA-like regions across the orders and even to different bacterial phyla. PMID:29250433

Insights into origin and evolution of α-proteobacterial gene transfer agents.

PubMed

Shakya, Migun; Soucy, Shannon M; Zhaxybayeva, Olga

2017-07-01

Several bacterial and archaeal lineages produce nanostructures that morphologically resemble small tailed viruses, but, unlike most viruses, contain apparently random pieces of the host genome. Since these elements can deliver the packaged DNA to other cells, they were dubbed gene transfer agents (GTAs). Because many genes involved in GTA production have viral homologs, it has been hypothesized that the GTA ancestor was a virus. Whether GTAs represent an atypical virus, a defective virus, or a virus co-opted by the prokaryotes for some function, remains to be elucidated. To evaluate these possibilities, we examined the distribution and evolutionary histories of genes that encode a GTA in the α-proteobacterium Rhodobacter capsulatus (RcGTA). We report that although homologs of many individual RcGTA genes are abundant across bacteria and their viruses, RcGTA-like genomes are mainly found in one subclade of α-proteobacteria. When compared with the viral homologs, genes of the RcGTA-like genomes evolve significantly slower, and do not have higher %A+T nucleotides than their host chromosomes. Moreover, they appear to reside in stable regions of the bacterial chromosomes that are generally conserved across taxonomic orders. These findings argue against RcGTA being an atypical or a defective virus. Our phylogenetic analyses suggest that RcGTA ancestor likely originated in the lineage that gave rise to contemporary α-proteobacterial orders Rhizobiales , Rhodobacterales , Caulobacterales , Parvularculales , and Sphingomonadales , and since that time the RcGTA-like element has co-evolved with its host chromosomes. Such evolutionary history is compatible with maintenance of these elements by bacteria due to some selective advantage. As for many other prokaryotic traits, horizontal gene transfer played a substantial role in the evolution of RcGTA-like elements, not only in shaping its genome components within the orders, but also in occasional dissemination of RcGTA-like regions across the orders and even to different bacterial phyla.

Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo.

PubMed

Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L; Stavans, Joel

2014-05-20

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

PubMed

Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2013-06-01

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

PubMed Central

Li, Linlin; Deng, Xutao; Mee, Edward T.; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D.; Delwart, Eric

2014-01-01

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries on the efficiency of viral detection and virus genome coverage were compared. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces. PMID:25497414

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

PubMed

Goya, Stephanie; Valinotto, Laura E; Tittarelli, Estefania; Rojo, Gabriel L; Nabaes Jodar, Mercedes S; Greninger, Alexander L; Zaiat, Jonathan J; Marti, Marcelo A; Mistchenko, Alicia S; Viegas, Mariana

2018-01-01

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1-3 (HPIV1-3) were also obtained with the selected optimal methodology.

Components of Adenovirus Genome Packaging

PubMed Central

Ahi, Yadvinder S.; Mittal, Suresh K.

2016-01-01

Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

An Example of Genetically Distinct HIV Type 1 Variants in Cerebrospinal Fluid and Plasma During Suppressive Therapy

PubMed Central

Dahl, Viktor; Gisslen, Magnus; Hagberg, Lars; Peterson, Julia; Shao, Wei; Spudich, Serena; Price, Richard W.; Palmer, Sarah

2014-01-01

We sequenced the genome of human immunodeficiency virus type 1 (HIV-1) recovered from 70 cerebrospinal fluid (CSF) specimens and 29 plasma samples and corresponding samples obtained before treatment initiation from 17 subjects receiving suppressive therapy. More CSF sequences than plasma sequences were hypermutants. We determined CSF sequences and plasma sequences in specimens obtained from 2 subjects after treatment initiation. In one subject, we found genetically distinct CSF and plasma sequences, indicating that they came from HIV-1 from 2 different compartments, one potentially the central nervous system, during suppressive therapy. In addition, there was little evidence of viral evolution in the CSF during therapy, suggesting that continuous virus replication is not the major cause of viral persistence in the central nervous system. PMID:24338353

An example of genetically distinct HIV type 1 variants in cerebrospinal fluid and plasma during suppressive therapy.

PubMed

Dahl, Viktor; Gisslen, Magnus; Hagberg, Lars; Peterson, Julia; Shao, Wei; Spudich, Serena; Price, Richard W; Palmer, Sarah

2014-05-15

We sequenced the genome of human immunodeficiency virus type 1 (HIV-1) recovered from 70 cerebrospinal fluid (CSF) specimens and 29 plasma samples and corresponding samples obtained before treatment initiation from 17 subjects receiving suppressive therapy. More CSF sequences than plasma sequences were hypermutants. We determined CSF sequences and plasma sequences in specimens obtained from 2 subjects after treatment initiation. In one subject, we found genetically distinct CSF and plasma sequences, indicating that they came from HIV-1 from 2 different compartments, one potentially the central nervous system, during suppressive therapy. In addition, there was little evidence of viral evolution in the CSF during therapy, suggesting that continuous virus replication is not the major cause of viral persistence in the central nervous system.

Modelling Hepatitis B Virus Antiviral Therapy and Drug Resistant Mutant Strains

NASA Astrophysics Data System (ADS)

Bernal, Julie; Dix, Trevor; Allison, Lloyd; Bartholomeusz, Angeline; Yuen, Lilly

Despite the existence of vaccines, the Hepatitis B virus (HBV) is still a serious global health concern. HBV targets liver cells. It has an unusual replication process involving an RNA pre-genome that the reverse transcriptase domain of the viral polymerase protein translates into viral DNA. The reverse transcription process is error prone and together with the high replication rates of the virus, allows the virus to exist as a heterogeneous population of mutants, known as a quasispecies, that can adapt and become resistant to antiviral therapy. This study presents an individual-based model of HBV inside an artificial liver, and associated blood serum, undergoing antiviral therapy. This model aims to provide insights into the evolution of the HBV quasispecies and the individual contribution of HBV mutations in the outcome of therapy.

Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene.

PubMed

Gergen, Janina; Coulon, Flora; Creneguy, Alison; Elain-Duret, Nathan; Gutierrez, Alejandra; Pinkenburg, Olaf; Verhoeyen, Els; Anegon, Ignacio; Nguyen, Tuan Huy; Halary, Franck Albert; Haspot, Fabienne

2018-01-01

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

Hybridization capture reveals evolution and conservation across the entire Koala retrovirus genome.

PubMed

Tsangaras, Kyriakos; Siracusa, Matthew C; Nikolaidis, Nikolas; Ishida, Yasuko; Cui, Pin; Vielgrader, Hanna; Helgen, Kristofer M; Roca, Alfred L; Greenwood, Alex D

2014-01-01

The koala retrovirus (KoRV) is the only retrovirus known to be in the midst of invading the germ line of its host species. Hybridization capture and next generation sequencing were used on modern and museum DNA samples of koala (Phascolarctos cinereus) to examine ca. 130 years of evolution across the full KoRV genome. Overall, the entire proviral genome appeared to be conserved across time in sequence, protein structure and transcriptional binding sites. A total of 138 polymorphisms were detected, of which 72 were found in more than one individual. At every polymorphic site in the museum koalas, one of the character states matched that of modern KoRV. Among non-synonymous polymorphisms, radical substitutions involving large physiochemical differences between amino acids were elevated in env, potentially reflecting anti-viral immune pressure or avoidance of receptor interference. Polymorphisms were not detected within two functional regions believed to affect infectivity. Host sequences flanking proviral integration sites were also captured; with few proviral loci shared among koalas. Recently described variants of KoRV, designated KoRV-B and KoRV-J, were not detected in museum samples, suggesting that these variants may be of recent origin.

Hybridization Capture Reveals Evolution and Conservation across the Entire Koala Retrovirus Genome

PubMed Central

Ishida, Yasuko; Cui, Pin; Vielgrader, Hanna; Helgen, Kristofer M.; Roca, Alfred L.; Greenwood, Alex D.

2014-01-01

The koala retrovirus (KoRV) is the only retrovirus known to be in the midst of invading the germ line of its host species. Hybridization capture and next generation sequencing were used on modern and museum DNA samples of koala (Phascolarctos cinereus) to examine ca. 130 years of evolution across the full KoRV genome. Overall, the entire proviral genome appeared to be conserved across time in sequence, protein structure and transcriptional binding sites. A total of 138 polymorphisms were detected, of which 72 were found in more than one individual. At every polymorphic site in the museum koalas, one of the character states matched that of modern KoRV. Among non-synonymous polymorphisms, radical substitutions involving large physiochemical differences between amino acids were elevated in env, potentially reflecting anti-viral immune pressure or avoidance of receptor interference. Polymorphisms were not detected within two functional regions believed to affect infectivity. Host sequences flanking proviral integration sites were also captured; with few proviral loci shared among koalas. Recently described variants of KoRV, designated KoRV-B and KoRV-J, were not detected in museum samples, suggesting that these variants may be of recent origin. PMID:24752422

Persistence of an Oncogenic Papillomavirus Genome Requires cis Elements from the Viral Transcriptional Enhancer

PubMed Central

Van Doorslaer, Koenraad; Chen, Dan; Chapman, Sandra; Khan, Jameela

2017-01-01

ABSTRACT Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication. PMID:29162712

Horizontal acquisition of transposable elements and viral sequences: patterns and consequences.

PubMed

Gilbert, Clément; Feschotte, Cédric

2018-04-01

It is becoming clear that most eukaryotic transposable elements (TEs) owe their evolutionary success in part to horizontal transfer events, which enable them to invade new species. Recent large-scale studies are beginning to unravel the mechanisms and ecological factors underlying this mode of transmission. Viruses are increasingly recognized as vectors in the process but also as a direct source of genetic material horizontally acquired by eukaryotic organisms. Because TEs and endogenous viruses are major catalysts of variation and innovation in genomes, we argue that horizontal inheritance has had a more profound impact in eukaryotic evolution than is commonly appreciated. To support this proposal, we compile a list of examples, including some previously unrecognized, whereby new host functions and phenotypes can be directly attributed to horizontally acquired TE or viral sequences. We predict that the number of examples will rapidly grow in the future as the prevalence of horizontal transfer in the life cycle of TEs becomes even more apparent, firmly establishing this form of non-Mendelian inheritance as a consequential facet of eukaryotic evolution. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evolutionary characteristics of morbilliviruses during serial passages in vitro: Gradual attenuation of virus virulence.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Zou, Yanli; Liu, Shan; Wang, Zhiliang

2016-08-01

The genus Morbillivirus is classified into the family Paramyxoviridae, and is composed of 6 members, namely measles virus (MV), rinderpest virus (RPV), peste-des-petits-ruminants virus (PPRV), canine distemper virus (CDV), phocine distemper virus (PDV) and cetacean morbillivirus (CeMV). The MV, RPV, PPRV and CDV have been successfully attenuated through their serial passages in vitro for the production of live vaccines. It has been demonstrated that the morbilliviral virulence in animals was progressively attenuated with their consecutive passages in vitro. However, only a few reports were involved in explanation of an attenuation-related mechanism on them until many years after the establishment of a quasispecies theory. RNA virus quasispecies arise from rapid evolution of viruses with high mutation rate during genomic replication, and play an important role in gradual loss of viral virulence by serial passages. Here, we overviewed the development of live-attenuated vaccine strains against morbilliviruses by consecutive passages in vitro, and further discussed a related mechanism concerning the relationship between virulence attenuation and viral evolution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Computational Approaches to Viral Evolution and Rational Vaccine Design

NASA Astrophysics Data System (ADS)

Bhattacharya, Tanmoy

2006-10-01

Viral pandemics, including HIV, are a major health concern across the world. Experimental techniques available today have uncovered a great wealth of information about how these viruses infect, grow, and cause disease; as well as how our body attempts to defend itself against them. Nevertheless, due to the high variability and fast evolution of many of these viruses, the traditional method of developing vaccines by presenting a heuristically chosen strain to the body fails and an effective intervention strategy still eludes us. A large amount of carefully curated genomic data on a number of these viruses are now available, often annotated with disease and immunological context. The availability of parallel computers has now made it possible to carry out a systematic analysis of this data within an evolutionary framework. I will describe, as an example, how computations on such data has allowed us to understand the origins and diversification of HIV, the causative agent of AIDS. On the practical side, computations on the same data is now being used to inform choice or defign of optimal vaccine strains.

Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA

PubMed Central

LeBlanc, Jason; Weil, Jason; Beemon, Karen

2013-01-01

After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases. PMID:23754689

Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution

PubMed Central

Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander

2013-01-01

Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the ‘long-term evolution experiment’. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins. PMID:23783573

Genomic adaptation of the ISA virus to Salmo salar codon usage

PubMed Central

2013-01-01

Background The ISA virus (ISAV) is an Orthomyxovirus whose genome encodes for at least 10 proteins. Low protein identity and lack of genetic tools have hampered the study of the molecular mechanism behind its virulence. It has been shown that viral codon usage controls several processes such as translational efficiency, folding, tuning of protein expression, antigenicity and virulence. Despite this, the possible role that adaptation to host codon usage plays in virulence and viral evolution has not been studied in ISAV. Methods Intergenomic adaptation between viral and host genomes was calculated using the codon adaptation index score with EMBOSS software and the Kazusa database. Classification of host genes according to GeneOnthology was performed using Blast2go. A non parametric test was applied to determine the presence of significant correlations among CAI, mortality and time. Results Using the codon adaptation index (CAI) score, we found that the encoding genes for nucleoprotein, matrix protein M1 and antagonist of Interferon I signaling (NS1) are the ISAV genes that are more adapted to host codon usage, in agreement with their requirement for production of viral particles and inactivation of antiviral responses. Comparison to host genes showed that ISAV shares CAI values with less than 0.45% of Salmo salar genes. GeneOntology classification of host genes showed that ISAV genes share CAI values with genes from less than 3% of the host biological process, far from the 14% shown by Influenza A viruses and closer to the 5% shown by Influenza B and C. As well, we identified a positive correlation (p<0.05) between CAI values of a virus and the duration of the outbreak disease in given salmon farms, as well as a weak relationship between codon adaptation values of PB1 and the mortality rates of a set of ISA viruses. Conclusions Our analysis shows that ISAV is the least adapted viral Salmo salar pathogen and Orthomyxovirus family member less adapted to host codon usage, avoiding the general behavior of host genes. This is probably due to its recent emergence among farmed Salmon populations. PMID:23829271

Genomic adaptation of the ISA virus to Salmo salar codon usage.

PubMed

Tello, Mario; Vergara, Francisco; Spencer, Eugenio

2013-07-05

The ISA virus (ISAV) is an Orthomyxovirus whose genome encodes for at least 10 proteins. Low protein identity and lack of genetic tools have hampered the study of the molecular mechanism behind its virulence. It has been shown that viral codon usage controls several processes such as translational efficiency, folding, tuning of protein expression, antigenicity and virulence. Despite this, the possible role that adaptation to host codon usage plays in virulence and viral evolution has not been studied in ISAV. Intergenomic adaptation between viral and host genomes was calculated using the codon adaptation index score with EMBOSS software and the Kazusa database. Classification of host genes according to GeneOnthology was performed using Blast2go. A non parametric test was applied to determine the presence of significant correlations among CAI, mortality and time. Using the codon adaptation index (CAI) score, we found that the encoding genes for nucleoprotein, matrix protein M1 and antagonist of Interferon I signaling (NS1) are the ISAV genes that are more adapted to host codon usage, in agreement with their requirement for production of viral particles and inactivation of antiviral responses. Comparison to host genes showed that ISAV shares CAI values with less than 0.45% of Salmo salar genes. GeneOntology classification of host genes showed that ISAV genes share CAI values with genes from less than 3% of the host biological process, far from the 14% shown by Influenza A viruses and closer to the 5% shown by Influenza B and C. As well, we identified a positive correlation (p<0.05) between CAI values of a virus and the duration of the outbreak disease in given salmon farms, as well as a weak relationship between codon adaptation values of PB1 and the mortality rates of a set of ISA viruses. Our analysis shows that ISAV is the least adapted viral Salmo salar pathogen and Orthomyxovirus family member less adapted to host codon usage, avoiding the general behavior of host genes. This is probably due to its recent emergence among farmed Salmon populations.

Full-length genomic characterization and molecular evolution of canine parvovirus in China.

PubMed

Zhou, Ling; Tang, Qinghai; Shi, Lijun; Kong, Miaomiao; Liang, Lin; Mao, Qianqian; Bu, Bin; Yao, Lunguang; Zhao, Kai; Cui, Shangjin; Leal, Élcio

2016-06-01

Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China.

Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes.

PubMed

Gilbert, C; Meik, J M; Dashevsky, D; Card, D C; Castoe, T A; Schaack, S

2014-09-22

We report the discovery of endogenous viral elements (EVEs) from Hepadnaviridae, Bornaviridae and Circoviridae in the speckled rattlesnake, Crotalus mitchellii, the first viperid snake for which a draft whole genome sequence assembly is available. Analysis of the draft assembly reveals genome fragments from the three virus families were inserted into the genome of this snake over the past 50 Myr. Cross-species PCR screening of orthologous loci and computational scanning of the python and king cobra genomes reveals that circoviruses integrated most recently (within the last approx. 10 Myr), whereas bornaviruses and hepadnaviruses integrated at least approximately 13 and approximately 50 Ma, respectively. This is, to our knowledge, the first report of circo-, borna- and hepadnaviruses in snakes and the first characterization of non-retroviral EVEs in non-avian reptiles. Our study provides a window into the historical dynamics of viruses in these host lineages and shows that their evolution involved multiple host-switches between mammals and reptiles. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data.

PubMed

de Andrade, Roberto R S; Vaslin, Maite F S

2014-03-07

Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data

PubMed Central

2014-01-01

Background Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. Methods In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. Results The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. Conclusions SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. Availability and implementation SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/. PMID:24607237

Single cell genomics of uncultured marine alveolates shows paraphyly of basal dinoflagellates.

PubMed

Strassert, Jürgen F H; Karnkowska, Anna; Hehenberger, Elisabeth; Del Campo, Javier; Kolisko, Martin; Okamoto, Noriko; Burki, Fabien; Janouškovec, Jan; Poirier, Camille; Leonard, Guy; Hallam, Steven J; Richards, Thomas A; Worden, Alexandra Z; Santoro, Alyson E; Keeling, Patrick J

2018-01-01

Marine alveolates (MALVs) are diverse and widespread early-branching dinoflagellates, but most knowledge of the group comes from a few cultured species that are generally not abundant in natural samples, or from diversity analyses of PCR-based environmental SSU rRNA gene sequences. To more broadly examine MALV genomes, we generated single cell genome sequences from seven individually isolated cells. Genes expected of heterotrophic eukaryotes were found, with interesting exceptions like presence of proteorhodopsin and vacuolar H + -pyrophosphatase. Phylogenetic analysis of concatenated SSU and LSU rRNA gene sequences provided strong support for the paraphyly of MALV lineages. Dinoflagellate viral nucleoproteins were found only in MALV groups that branched as sister to dinokaryotes. Our findings indicate that multiple independent origins of several characteristics early in dinoflagellate evolution, such as a parasitic life style, underlie the environmental diversity of MALVs, and suggest they have more varied trophic modes than previously thought.

Tibrogargan and Coastal Plains rhabdoviruses: genomic characterization, evolution of novel genes and seroprevalence in Australian livestock.

PubMed

Gubala, Aneta; Davis, Steven; Weir, Richard; Melville, Lorna; Cowled, Chris; Boyle, David

2011-09-01

Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle in Australia and TIBV has also been isolated from the biting midge Culicoides brevitarsis. Complete genomic sequencing revealed that the viruses share a novel genome structure within the family Rhabdoviridae, each virus containing two additional putative genes between the matrix protein (M) and glycoprotein (G) genes and one between the G and viral RNA polymerase (L) genes. The predicted novel protein products are highly diverged at the sequence level but demonstrate clear conservation of secondary structure elements, suggesting conservation of biological functions. Phylogenetic analyses showed that TIBV and CPV form an independent group within the 'dimarhabdovirus supergroup'. Although no disease has been observed in association with these viruses, antibodies were detected at high prevalence in cattle and buffalo in northern Australia, indicating the need for disease monitoring and further study of this distinctive group of viruses.

Recoding method that removes inhibitory sequences and improves HIV gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rabadan, Raul; Krasnitz, Michael; Robins, Harlan

The invention relates to inhibitory nucleotide signal sequences or "INS" sequences in the genomes of lentiviruses. In particular the invention relates to the AGG motif present in all viral genomes. The AGG motif may have an inhibitory effect on a virus, for example by reducing the levels of, or maintaining low steady-state levels of, viral RNAs in host cells, and inducing and/or maintaining in viral latency. In one aspect, the invention provides vaccines that contain, or are produced from, viral nucleic acids in which the AGG sequences have been mutated. In another aspect, the invention provides methods and compositions formore » affecting the function of the AGG motif, and methods for identifying other INS sequences in viral genomes.« less

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

PubMed Central

Fadrosh, Douglas W.; Andrews-Pfannkoch, Cynthia; Williamson, Shannon J.

2011-01-01

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community’s genetic signature. Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography. PMID:21989424

Viral Genome DataBase: storing and analyzing genes and proteins from complete viral genomes.

PubMed

Hiscock, D; Upton, C

2000-05-01

The Viral Genome DataBase (VGDB) contains detailed information of the genes and predicted protein sequences from 15 completely sequenced genomes of large (&100 kb) viruses (2847 genes). The data that is stored includes DNA sequence, protein sequence, GenBank and user-entered notes, molecular weight (MW), isoelectric point (pI), amino acid content, A + T%, nucleotide frequency, dinucleotide frequency and codon use. The VGDB is a mySQL database with a user-friendly JAVA GUI. Results of queries can be easily sorted by any of the individual parameters. The software and additional figures and information are available at http://athena.bioc.uvic.ca/genomes/index.html .

Comparative analysis of viral RNA signatures on different RIG-I-like receptors

PubMed Central

Sanchez David, Raul Y; Combredet, Chantal; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Coppée, Jean-Yves; Mura, Marie; Guerbois Galla, Mathilde; Despres, Philippe; Tangy, Frédéric; Komarova, Anastassia V

2016-01-01

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection. DOI: http://dx.doi.org/10.7554/eLife.11275.001 PMID:27011352

Genome Cyclization as Strategy for Flavivirus RNA Replication

PubMed Central

Villordo, Sergio M.; Gamarnik, Andrea V.

2017-01-01

Long-range and local RNA-RNA contacts in viral RNA genomes result in tertiary structures that modulate the function of enhancers, promoters, and silencers during translation, RNA replication, and encapsidation. In the case of flaviviruses, the presence of inverted complementary sequences at the 5′ and 3′ ends of the genome mediate long-range RNA interactions and RNA cyclization. The circular conformation of flavivirus genomes was demonstrated to be essential for RNA amplification. New ideas about the mechanisms by which circular genomes participate in flavivirus replication have emerged in the last few years. Here, we will describe the latest information about cis-acting elements involved in flavivirus genome cyclization, RNA promoter elements required for viral polymerase recognition, and how these elements together coordinate viral RNA synthesis. PMID:18703097

The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

PubMed

Angly, Florent E; Willner, Dana; Prieto-Davó, Alejandra; Edwards, Robert A; Schmieder, Robert; Vega-Thurber, Rebecca; Antonopoulos, Dionysios A; Barott, Katie; Cottrell, Matthew T; Desnues, Christelle; Dinsdale, Elizabeth A; Furlan, Mike; Haynes, Matthew; Henn, Matthew R; Hu, Yongfei; Kirchman, David L; McDole, Tracey; McPherson, John D; Meyer, Folker; Miller, R Michael; Mundt, Egbert; Naviaux, Robert K; Rodriguez-Mueller, Beltran; Stevens, Rick; Wegley, Linda; Zhang, Lixin; Zhu, Baoli; Rohwer, Forest

2009-12-01

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

Some basic properties of immune selection.

PubMed

Iwasa, Yoh; Michor, Franziska; Nowak, Martin

2004-07-21

We analyze models for the evolutionary dynamics of viral or other infectious agents within a host. We study how the invasion of a new strain affects the composition and diversity of the viral population. We show that--under strain-specific immunity--the equilibrium abundance of uninfected cells declines during viral evolution. In addition, for cytotoxic immunity the absolute force of infection, and for non-cytotoxic immunity the absolute cellular virulence increases during viral evolution. We prove global stability by means of Lyapunov functions. These unidirectional trends of virus evolution under immune selection do not hold for general cross-reactive immune responses, which introduce frequency-dependent selection among viral strains. Therefore, appropriate cross-reactive immunity can lead to a viral evolution within a host which limits the extent of the disease.

Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing.

PubMed

Xu, Chenxi; Sun, Xuepeng; Taylor, Angela; Jiao, Chen; Xu, Yimin; Cai, Xiaofeng; Wang, Xiaoli; Ge, Chenhui; Pan, Guanghui; Wang, Quanxi; Fei, Zhangjun; Wang, Quanhua

2017-06-01

Tomato is a major vegetable crop that has tremendous popularity. However, viral disease is still a major factor limiting tomato production. Here, we report the tomato virome identified through sequencing small RNAs of 170 field-grown samples collected in China. A total of 22 viruses were identified, including both well-documented and newly detected viruses. The tomato viral community is dominated by a few species, and they exhibit polymorphisms and recombination in the genomes with cold spots and hot spots. Most samples were coinfected by multiple viruses, and the majority of identified viruses are positive-sense single-stranded RNA viruses. Evolutionary analysis of one of the most dominant tomato viruses, Tomato yellow leaf curl virus (TYLCV), predicts its origin and the time back to its most recent common ancestor. The broadly sampled data have enabled us to identify several unreported viruses in tomato, including a completely new virus, which has a genome of ∼13.4 kb and groups with aphid-transmitted viruses in the genus Cytorhabdovirus Although both DNA and RNA viruses can trigger the biogenesis of virus-derived small interfering RNAs (vsiRNAs), we show that features such as length distribution, paired distance, and base selection bias of vsiRNA sequences reflect different plant Dicer-like proteins and Argonautes involved in vsiRNA biogenesis. Collectively, this study offers insights into host-virus interaction in tomato and provides valuable information to facilitate the management of viral diseases. IMPORTANCE Tomato is an important source of micronutrients in the human diet and is extensively consumed around the world. Virus is among the major constraints on tomato production. Categorizing virus species that are capable of infecting tomato and understanding their diversity and evolution are challenging due to difficulties in detecting such fast-evolving biological entities. Here, we report the landscape of the tomato virome in China, the leading country in tomato production. We identified dozens of viruses present in tomato, including both well-documented and completely new viruses. Some newly emerged viruses in tomato were found to spread fast, and therefore, prompt attention is needed to control them. Moreover, we show that the virus genomes exhibit considerable degree of polymorphisms and recombination, and the virus-derived small interfering RNA (vsiRNA) sequences indicate distinct vsiRNA biogenesis mechanisms for different viruses. The Chinese tomato virome that we developed provides valuable information to facilitate the management of tomato viral diseases. Copyright © 2017 American Society for Microbiology.

Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing

PubMed Central

Xu, Chenxi; Taylor, Angela; Jiao, Chen; Xu, Yimin; Cai, Xiaofeng; Wang, Xiaoli; Ge, Chenhui; Pan, Guanghui; Wang, Quanxi

2017-01-01

ABSTRACT Tomato is a major vegetable crop that has tremendous popularity. However, viral disease is still a major factor limiting tomato production. Here, we report the tomato virome identified through sequencing small RNAs of 170 field-grown samples collected in China. A total of 22 viruses were identified, including both well-documented and newly detected viruses. The tomato viral community is dominated by a few species, and they exhibit polymorphisms and recombination in the genomes with cold spots and hot spots. Most samples were coinfected by multiple viruses, and the majority of identified viruses are positive-sense single-stranded RNA viruses. Evolutionary analysis of one of the most dominant tomato viruses, Tomato yellow leaf curl virus (TYLCV), predicts its origin and the time back to its most recent common ancestor. The broadly sampled data have enabled us to identify several unreported viruses in tomato, including a completely new virus, which has a genome of ∼13.4 kb and groups with aphid-transmitted viruses in the genus Cytorhabdovirus. Although both DNA and RNA viruses can trigger the biogenesis of virus-derived small interfering RNAs (vsiRNAs), we show that features such as length distribution, paired distance, and base selection bias of vsiRNA sequences reflect different plant Dicer-like proteins and Argonautes involved in vsiRNA biogenesis. Collectively, this study offers insights into host-virus interaction in tomato and provides valuable information to facilitate the management of viral diseases. IMPORTANCE Tomato is an important source of micronutrients in the human diet and is extensively consumed around the world. Virus is among the major constraints on tomato production. Categorizing virus species that are capable of infecting tomato and understanding their diversity and evolution are challenging due to difficulties in detecting such fast-evolving biological entities. Here, we report the landscape of the tomato virome in China, the leading country in tomato production. We identified dozens of viruses present in tomato, including both well-documented and completely new viruses. Some newly emerged viruses in tomato were found to spread fast, and therefore, prompt attention is needed to control them. Moreover, we show that the virus genomes exhibit considerable degree of polymorphisms and recombination, and the virus-derived small interfering RNA (vsiRNA) sequences indicate distinct vsiRNA biogenesis mechanisms for different viruses. The Chinese tomato virome that we developed provides valuable information to facilitate the management of tomato viral diseases. PMID:28331089

The number of genes encoding repeat domain-containing proteins positively correlates with genome size in amoebal giant viruses

PubMed Central

Shukla, Avi; Chatterjee, Anirvan

2018-01-01

Abstract Curiously, in viruses, the virion volume appears to be predominantly driven by genome length rather than the number of proteins it encodes or geometric constraints. With their large genome and giant particle size, amoebal viruses (AVs) are ideally suited to study the relationship between genome and virion size and explore the role of genome plasticity in their evolutionary success. Different genomic regions of AVs exhibit distinct genealogies. Although the vertically transferred core genes and their functions are universally conserved across the nucleocytoplasmic large DNA virus (NCLDV) families and are essential for their replication, the horizontally acquired genes are variable across families and are lineage-specific. When compared with other giant virus families, we observed a near–linear increase in the number of genes encoding repeat domain-containing proteins (RDCPs) with the increase in the genome size of AVs. From what is known about the functions of RDCPs in bacteria and eukaryotes and their prevalence in the AV genomes, we envisage important roles for RDCPs in the life cycle of AVs, their genome expansion, and plasticity. This observation also supports the evolution of AVs from a smaller viral ancestor by the acquisition of diverse gene families from the environment including RDCPs that might have helped in host adaption. PMID:29308275

Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

PubMed

Hancock, Meaghan H; Cliffe, Anna R; Knipe, David M; Smiley, James R

2010-02-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

Herpes Simplex Virus VP16, but Not ICP0, Is Required To Reduce Histone Occupancy and Enhance Histone Acetylation on Viral Genomes in U2OS Osteosarcoma Cells▿ †

PubMed Central

Hancock, Meaghan H.; Cliffe, Anna R.; Knipe, David M.; Smiley, James R.

2010-01-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure. PMID:19939931

Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection.

PubMed

Keele, Brandon F; Giorgi, Elena E; Salazar-Gonzalez, Jesus F; Decker, Julie M; Pham, Kimmy T; Salazar, Maria G; Sun, Chuanxi; Grayson, Truman; Wang, Shuyi; Li, Hui; Wei, Xiping; Jiang, Chunlai; Kirchherr, Jennifer L; Gao, Feng; Anderson, Jeffery A; Ping, Li-Hua; Swanstrom, Ronald; Tomaras, Georgia D; Blattner, William A; Goepfert, Paul A; Kilby, J Michael; Saag, Michael S; Delwart, Eric L; Busch, Michael P; Cohen, Myron S; Montefiori, David C; Haynes, Barton F; Gaschen, Brian; Athreya, Gayathri S; Lee, Ha Y; Wood, Natasha; Seoighe, Cathal; Perelson, Alan S; Bhattacharya, Tanmoy; Korber, Bette T; Hahn, Beatrice H; Shaw, George M

2008-05-27

The precise identification of the HIV-1 envelope glycoprotein (Env) responsible for productive clinical infection could be instrumental in elucidating the molecular basis of HIV-1 transmission and in designing effective vaccines. Here, we developed a mathematical model of random viral evolution and, together with phylogenetic tree construction, used it to analyze 3,449 complete env sequences derived by single genome amplification from 102 subjects with acute HIV-1 (clade B) infection. Viral env genes evolving from individual transmitted or founder viruses generally exhibited a Poisson distribution of mutations and star-like phylogeny, which coalesced to an inferred consensus sequence at or near the estimated time of virus transmission. Overall, 78 of 102 subjects had evidence of productive clinical infection by a single virus, and 24 others had evidence of productive clinical infection by a minimum of two to five viruses. Phenotypic analysis of transmitted or early founder Envs revealed a consistent pattern of CCR5 dependence, masking of coreceptor binding regions, and equivalent or modestly enhanced resistance to the fusion inhibitor T1249 and broadly neutralizing antibodies compared with Envs from chronically infected subjects. Low multiplicity infection and limited viral evolution preceding peak viremia suggest a finite window of potential vulnerability of HIV-1 to vaccine-elicited immune responses, although phenotypic properties of transmitted Envs pose a formidable defense.

Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection

PubMed Central

Keele, Brandon F.; Giorgi, Elena E.; Salazar-Gonzalez, Jesus F.; Decker, Julie M.; Pham, Kimmy T.; Salazar, Maria G.; Sun, Chuanxi; Grayson, Truman; Wang, Shuyi; Li, Hui; Wei, Xiping; Jiang, Chunlai; Kirchherr, Jennifer L.; Gao, Feng; Anderson, Jeffery A.; Ping, Li-Hua; Swanstrom, Ronald; Tomaras, Georgia D.; Blattner, William A.; Goepfert, Paul A.; Kilby, J. Michael; Saag, Michael S.; Delwart, Eric L.; Busch, Michael P.; Cohen, Myron S.; Montefiori, David C.; Haynes, Barton F.; Gaschen, Brian; Athreya, Gayathri S.; Lee, Ha Y.; Wood, Natasha; Seoighe, Cathal; Perelson, Alan S.; Bhattacharya, Tanmoy; Korber, Bette T.; Hahn, Beatrice H.; Shaw, George M.

2008-01-01

The precise identification of the HIV-1 envelope glycoprotein (Env) responsible for productive clinical infection could be instrumental in elucidating the molecular basis of HIV-1 transmission and in designing effective vaccines. Here, we developed a mathematical model of random viral evolution and, together with phylogenetic tree construction, used it to analyze 3,449 complete env sequences derived by single genome amplification from 102 subjects with acute HIV-1 (clade B) infection. Viral env genes evolving from individual transmitted or founder viruses generally exhibited a Poisson distribution of mutations and star-like phylogeny, which coalesced to an inferred consensus sequence at or near the estimated time of virus transmission. Overall, 78 of 102 subjects had evidence of productive clinical infection by a single virus, and 24 others had evidence of productive clinical infection by a minimum of two to five viruses. Phenotypic analysis of transmitted or early founder Envs revealed a consistent pattern of CCR5 dependence, masking of coreceptor binding regions, and equivalent or modestly enhanced resistance to the fusion inhibitor T1249 and broadly neutralizing antibodies compared with Envs from chronically infected subjects. Low multiplicity infection and limited viral evolution preceding peak viremia suggest a finite window of potential vulnerability of HIV-1 to vaccine-elicited immune responses, although phenotypic properties of transmitted Envs pose a formidable defense. PMID:18490657

Fulminant hepatitis failure in adults and children from a Public Hospital in Rio de Janeiro, Brazil.

PubMed

Santos, Damião Carlos Moraes dos; Martinho, José Manoel da Silva Gomes; Pacheco-Moreira, Lucio Filgueiras; Araújo, Cristina Carvalho Viana de; Oliveira, Barbara Cristina Euzebio Pereira Dias de; Lago, Barbara Vieira; Pinto, Marcelo Alves; Paula, Vanessa Salete de

2009-10-01

Fulminant hepatic failure (FHF) is characterized by massive hepatocellular injury, whose physiopathology is still unclear. Hepatitis B (HBV) is probably the most common viral cause of FHF, while hepatitis A (HAV) virus seem occurs less frequently. However, the host and viral factors that determine the outcome of these infections are poorly understood. In the present study, viral load and genotyping determining regions of HAV and HBV genomes were sequenced. Eight FHF patients and one patient with severe acute hepatitis (SAH) were included. Liver and blood samples were collected during liver transplantation or necropsy procedures. HAV-RNA and HBV-DNA were extracted from serum, biopsy and paraffin liver. Nucleotide sequencing of HAV-RNA was performed from VP1/2A and HBV-DNA from PreS/S region. The amplified samples were quantified by Real-Time PCR. The cases of HAV infection were due to subgenotype IA. The cases of HBV infection were due to genotype A2 and D4. The case of HAV/HBV coinfection was infected by genotype IA and D3. Hepatitis A and B infection were associated with genotypes most prevalent in Brazil. In hepatitis A infection the mean of period evolution was 13 days. In hepatitis B, FHF patients infected by genotype D have a shorter period of evolution than FHF patients infected by genotype A (mean 15 v. 53 days). There was no association with genotype-determining region with the severity of hepatitis, however nucleotide differences and high viral load could be observed among FHF.

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

PubMed

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Evolution of feline immunodeficiency virus in Felidae: implications for human health and wildlife ecology.

PubMed

Pecon-Slattery, Jill; Troyer, Jennifer L; Johnson, Warren E; O'Brien, Stephen J

2008-05-15

Genetic analyses of feline immunodeficiency viruses provide significant insights on the worldwide distribution and evolutionary history of this emerging pathogen. Large-scale screening of over 3000 samples from all species of Felidae indicates that at least some individuals from most species possess antibodies that cross react to FIV. Phylogenetic analyses of genetic variation in the pol-RT gene demonstrate that FIV lineages are species-specific and suggest that there has been a prolonged period of viral-host co-evolution. The clinical effects of FIV specific to species other than domestic cat are controversial. Comparative genomic analyses of all full-length FIV genomes confirmed that FIV is host specific. Recently sequenced lion subtype E is marginally more similar to Pallas cat FIV though env is more similar to that of domestic cat FIV, indicating a possible recombination between two divergent strains in the wild. Here we review global patterns of FIV seroprevalence and endemnicity, assess genetic differences within and between species-specific FIV strains, and interpret these with patterns of felid speciation to propose an ancestral origin of FIV in Africa followed by interspecies transmission and global dissemination to Eurasia and the Americas. Continued comparative genomic analyses of full-length FIV from all seropositive animals, along with whole genome sequence of host species, will greatly advance our understanding of the role of recombination, selection and adaptation in retroviral emergence.

Evolution of feline immunodeficiency virus in Felidae: Implications for human health and wildlife ecology

PubMed Central

Pecon-Slattery, Jill; Troyer, Jennifer L.; Johnson, Warren E.; O’Brien, Stephen J.

2008-01-01

Genetic analyses of feline immunodeficiency viruses provide significant insights on the worldwide distribution and evolutionary history of this emerging pathogen. Large-scale screening of over 3000 samples from all species of Felidae indicates that at least some individuals from most species possess antibodies that cross react to FIV. Phylogenetic analyses of genetic variation in the pol-RT gene demonstrate that FIV lineages are species-specific and suggest that there has been a prolonged period of viral-host co-evolution. The clinical effects of FIV specific to species other than domestic cat are controversial. Comparative genomic analyses of all full-length FIV genomes confirmed that FIV is host specific. Recently sequenced lion subtype E is marginally more similar to Pallas cat FIV though env is more similar to that of domestic cat FIV, indicating a possible recombination between two divergent strains in the wild. Here we review global patterns of FIV seroprevalence and endemnicity, assess genetic differences within and between species-specific FIV strains, and interpret these with patterns of felid speciation to propose an ancestral origin of FIV in Africa followed by interspecies transmission and global dissemination to Eurasia and the Americas. Continued comparative genomic analyses of full-length FIV from all seropositive animals, along with whole genome sequence of host species, will greatly advance our understanding of the role of recombination, selection and adaptation in retroviral emergence. PMID:18359092

Patterns of amino acid conservation in human and animal immunodeficiency viruses.

PubMed

Voitenko, Olga S; Dhroso, Andi; Feldmann, Anna; Korkin, Dmitry; Kalinina, Olga V

2016-09-01

Due to their high genomic variability, RNA viruses and retroviruses present a unique opportunity for detailed study of molecular evolution. Lentiviruses, with HIV being a notable example, are one of the best studied viral groups: hundreds of thousands of sequences are available together with experimentally resolved three-dimensional structures for most viral proteins. In this work, we use these data to study specific patterns of evolution of the viral proteins, and their relationship to protein interactions and immunogenicity. We propose a method for identification of two types of surface residues clusters with abnormal conservation: extremely conserved and extremely variable clusters. We identify them on the surface of proteins from HIV and other animal immunodeficiency viruses. Both types of clusters are overrepresented on the interaction interfaces of viral proteins with other proteins, nucleic acids or low molecular-weight ligands, both in the viral particle and between the virus and its host. In the immunodeficiency viruses, the interaction interfaces are not more conserved than the corresponding proteins on an average, and we show that extremely conserved clusters coincide with protein-protein interaction hotspots, predicted as the residues with the largest energetic contribution to the interaction. Extremely variable clusters have been identified here for the first time. In the HIV-1 envelope protein gp120, they overlap with known antigenic sites. These antigenic sites also contain many residues from extremely conserved clusters, hence representing a unique interacting interface enriched both in extremely conserved and in extremely variable clusters of residues. This observation may have important implication for antiretroviral vaccine development. A Python package is available at https://bioinf.mpi-inf.mpg.de/publications/viral-ppi-pred/ voitenko@mpi-inf.mpg.de or kalinina@mpi-inf.mpg.de Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

West Nile virus population genetics and evolution

PubMed Central

Pesko, Kendra N.; Ebel, Gregory D.

2015-01-01

West Nile virus (WNV) (Flaviviridae: Flavivirus) is transmitted from mosquitoes to birds, but can cause fatal encephalitis in infected humans. Since its introduction into North America in New York in 1999, it has spread throughout the western hemisphere. Multiple outbreaks have also occurred in Europe over the last 20 years. This review highlights recent efforts to understand how host pressures impact viral population genetics, genotypic and phenotypic changes which have occurred in the WNV genome as it adapts to this novel environment, and molecular epidemiology of WNV worldwide. Future research directions are also discussed. PMID:22226703

Epigenetic Alterations in Epstein-Barr Virus-Associated Diseases.

PubMed

Niller, Hans Helmut; Banati, Ferenc; Salamon, Daniel; Minarovits, Janos

2016-01-01

Latent Epstein-Bar virus genomes undergo epigenetic modifications which are dependent on the respective tissue type and cellular phenotype. These define distinct viral epigenotypes corresponding with latent viral gene expression profiles. Viral Latent Membrane Proteins 1 and 2A can induce cellular DNA methyltransferases, thereby influencing the methylation status of the viral and cellular genomes. Therefore, not only the viral genomes carry epigenetic modifications, but also the cellular genomes adopt major epigenetic alterations upon EBV infection. The distinct cellular epigenotypes of EBV-infected cells differ from the epigenotypes of their normal counterparts. In Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC) significant changes in the host cell methylome with a strong tendency towards CpG island hypermethylation are observed. Hypermethylated genes unique for EBVaGC suggest the existence of an EBV-specific "epigenetic signature". Contrary to the primary malignancies carrying latent EBV genomes, lymphoblastoid cells (LCs) established by EBV infection of peripheral B cells in vitro are characterized by a massive genome-wide demethylation and a significant decrease and redistribution of heterochromatic histone marks. Establishing complete epigenomes of the diverse EBV-associated malignancies shall clarify their similarities and differences and further clarify the contribution of EBV to the pathogenesis, especially for the epithelial malignancies, NPC and EBVaGC.

New parvovirus in child with unexplained diarrhea, Tunisia.

PubMed

Phan, Tung G; Sdiri-Loulizi, Khira; Aouni, Mahjoub; Ambert-Balay, Katia; Pothier, Pierre; Deng, Xutao; Delwart, Eric

2014-11-01

A divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a Tunisian child with unexplained diarrhea. Tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1, respectively, of the closest genome, Kilham rat parvovirus, indicating presence of a new human viral species in the Protoparvovirus genus.

New Parvovirus in Child with Unexplained Diarrhea, Tunisia

PubMed Central

Phan, Tung G.; Sdiri-Loulizi, Khira; Aouni, Mahjoub; Ambert-Balay, Katia; Pothier, Pierre; Deng, Xutao

2014-01-01

A divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a Tunisian child with unexplained diarrhea. Tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1, respectively, of the closest genome, Kilham rat parvovirus, indicating presence of a new human viral species in the Protoparvovirus genus. PMID:25340816

K-bZIP Mediated SUMO-2/3 Specific Modification on the KSHV Genome Negatively Regulates Lytic Gene Expression and Viral Reactivation

PubMed Central

Yang, Wan-Shan; Hsu, Hung-Wei; Campbell, Mel; Cheng, Chia-Yang; Chang, Pei-Ching

2015-01-01

SUMOylation is associated with epigenetic regulation of chromatin structure and transcription. Epigenetic modifications of herpesviral genomes accompany the transcriptional switch of latent and lytic genes during the virus life cycle. Here, we report a genome-wide comparison of SUMO paralog modification on the KSHV genome. Using chromatin immunoprecipitation in conjunction with high-throughput sequencing, our study revealed highly distinct landscape changes of SUMO paralog genomic modifications associated with KSHV reactivation. A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed. Interestingly, SUMO-2/3 enrichment was inversely correlated with H3K9me3 mark after reactivation, indicating that SUMO-2/3 may be responsible for regulating the expression of viral genes located in low heterochromatin regions during viral reactivation. RNA-sequencing analysis showed that the SUMO-2/3 enrichment pattern positively correlated with KSHV gene expression profiles. Activation of KSHV lytic genes located in regions with high SUMO-2/3 enrichment was enhanced by SUMO-2/3 knockdown. These findings suggest that SUMO-2/3 viral chromatin modification contributes to the diminution of viral gene expression during reactivation. Our previous study identified a SUMO-2/3-specific viral E3 ligase, K-bZIP, suggesting a potential role of this enzyme in regulating SUMO-2/3 enrichment and viral gene repression. Consistent with this prediction, higher K-bZIP binding on SUMO-2/3 enrichment region during reactivation was observed. Moreover, a K-bZIP SUMO E3 ligase dead mutant, K-bZIP-L75A, in the viral context, showed no SUMO-2/3 enrichment on viral chromatin and higher expression of viral genes located in SUMO-2/3 enriched regions during reactivation. Importantly, virus production significantly increased in both SUMO-2/3 knockdown and KSHV K-bZIP-L75A mutant cells. These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation. As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy. PMID:26197391

Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

PubMed Central

Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel

2017-01-01

ABSTRACT Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells. IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression. PMID:28331082

Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination

PubMed Central

Chao, Mei; Wang, Tzu-Chi; Lin, Chia-Chi; Yung-Liang Wang, Robert; Lin, Wen-Bin; Lee, Shang-En; Cheng, Ying-Yu; Yeh, Chau-Ting; Iang, Shan-Bei

2017-01-01

The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens (the only viral-encoded protein) and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription. PMID:28977829

Novel cis-acting element within the capsid-coding region enhances flavivirus viral-RNA replication by regulating genome cyclization.

PubMed

Liu, Zhong-Yu; Li, Xiao-Feng; Jiang, Tao; Deng, Yong-Qiang; Zhao, Hui; Wang, Hong-Jiang; Ye, Qing; Zhu, Shun-Ya; Qiu, Yang; Zhou, Xi; Qin, E-De; Qin, Cheng-Feng

2013-06-01

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5' cyclization sequence (5'CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5'CS, and the presence of DCS-PK facilitates the formation of 5'-3' RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.

Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

PubMed Central

Liu, Zhong-Yu; Li, Xiao-Feng; Jiang, Tao; Deng, Yong-Qiang; Zhao, Hui; Wang, Hong-Jiang; Ye, Qing; Zhu, Shun-Ya; Qiu, Yang; Zhou, Xi; Qin, E-De

2013-01-01

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution. PMID:23576500

Fitness cost of reassortment in human influenza.

PubMed

Villa, Mara; Lässig, Michael

2017-11-01

Reassortment, which is the exchange of genome sequence between viruses co-infecting a host cell, plays an important role in the evolution of segmented viruses. In the human influenza virus, reassortment happens most frequently between co-existing variants within the same lineage. This process breaks genetic linkage and fitness correlations between viral genome segments, but the resulting net effect on viral fitness has remained unclear. In this paper, we determine rate and average selective effect of reassortment processes in the human influenza lineage A/H3N2. For the surface proteins hemagglutinin and neuraminidase, reassortant variants with a mean distance of at least 3 nucleotides to their parent strains get established at a rate of about 10-2 in units of the neutral point mutation rate. Our inference is based on a new method to map reassortment events from joint genealogies of multiple genome segments, which is tested by extensive simulations. We show that intra-lineage reassortment processes are, on average, under substantial negative selection that increases in strength with increasing sequence distance between the parent strains. The deleterious effects of reassortment manifest themselves in two ways: there are fewer reassortment events than expected from a null model of neutral reassortment, and reassortant strains have fewer descendants than their non-reassortant counterparts. Our results suggest that influenza evolves under ubiquitous epistasis across proteins, which produces fitness barriers against reassortment even between co-circulating strains within one lineage.

A Genome-Wide Association Study for the Incidence of Persistent Bovine Viral Diarrhea Virus Infection in Cattle.

USDA-ARS?s Scientific Manuscript database

Bovine Viral Diarrhea Virus (BVDV) is a diverse group of viruses causing disease in ruminants. The objective was to determine genomic regions harboring single nucleotide polymorphisms (SNP) associated with presence or absence of persistent BVDV infections. A genome wide association approach based on...

A Drosophila Toolkit for the Visualization and Quantification of Viral Replication Launched from Transgenic Genomes

PubMed Central

Wernet, Mathias F.; Klovstad, Martha; Clandinin, Thomas R.

2014-01-01

Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile Drosophila toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication in vivo. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our in vivo system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development. PMID:25386852

DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication.

PubMed

Bristol, Molly L; Wang, Xu; Smith, Nathan W; Son, Minkyeong P; Evans, Michael R; Morgan, Iain M

2016-06-22

Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

Comprehensive viral enrichment enables sensitive respiratory virus genomic identification and analysis by next generation sequencing.

PubMed

O'Flaherty, Brigid M; Li, Yan; Tao, Ying; Paden, Clinton R; Queen, Krista; Zhang, Jing; Dinwiddie, Darrell L; Gross, Stephen M; Schroth, Gary P; Tong, Suxiang

2018-06-01

Next generation sequencing (NGS) technologies have revolutionized the genomics field and are becoming more commonplace for identification of human infectious diseases. However, due to the low abundance of viral nucleic acids (NAs) in relation to host, viral identification using direct NGS technologies often lacks sufficient sensitivity. Here, we describe an approach based on two complementary enrichment strategies that significantly improves the sensitivity of NGS-based virus identification. To start, we developed two sets of DNA probes to enrich virus NAs associated with respiratory diseases. The first set of probes spans the genomes, allowing for identification of known viruses and full genome sequencing, while the second set targets regions conserved among viral families or genera, providing the ability to detect both known and potentially novel members of those virus groups. Efficiency of enrichment was assessed by NGS testing reference virus and clinical samples with known infection. We show significant improvement in viral identification using enriched NGS compared to unenriched NGS. Without enrichment, we observed an average of 0.3% targeted viral reads per sample. However, after enrichment, 50%-99% of the reads per sample were the targeted viral reads for both the reference isolates and clinical specimens using both probe sets. Importantly, dramatic improvements on genome coverage were also observed following virus-specific probe enrichment. The methods described here provide improved sensitivity for virus identification by NGS, allowing for a more comprehensive analysis of disease etiology. © 2018 O'Flaherty et al.; Published by Cold Spring Harbor Laboratory Press.

Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment

PubMed Central

Cohen, Camille; Streichenberger, Nathalie; Texier, Pascale; Takissian, Julie; Rousseau, Antoine; Poccardi, Nolwenn; Welsch, Jérémy; Corpet, Armelle; Schaeffer, Laurent; Labetoulle, Marc; Lomonte, Patrick

2016-01-01

Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely “multiple-acute”). Viral genomes in the “multiple-acute” pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the “multiple-latency” pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual. PMID:27618691

Within-Host Variations of Human Papillomavirus Reveal APOBEC Signature Mutagenesis in the Viral Genome.

PubMed

Hirose, Yusuke; Onuki, Mamiko; Tenjimbayashi, Yuri; Mori, Seiichiro; Ishii, Yoshiyuki; Takeuchi, Takamasa; Tasaka, Nobutaka; Satoh, Toyomi; Morisada, Tohru; Iwata, Takashi; Miyamoto, Shingo; Matsumoto, Koji; Sekizawa, Akihiko; Kukimoto, Iwao

2018-06-15

Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied by the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here, we explored within-host genetic diversity of HPV by performing deep-sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52, and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) and were deep sequenced. After constructing a reference viral genome sequence for each specimen, nucleotide positions showing changes with >0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, n = 56; CIN2/3, n = 68; ICC, n = 27), with various numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the trinucleotide context encompassing substituted bases revealed that TpCpN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions. IMPORTANCE HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep-sequencing analyses, we show for the first time a comprehensive snapshot of the within-host genetic diversity of high-risk HPVs during cervical carcinogenesis. Quasispecies harboring minor nucleotide variations in viral whole-genome sequences were extensively observed across different grades of CIN and cervical cancer. Among the within-host variations, C-to-T transitions, a characteristic change mediated by cellular APOBEC cytosine deaminases, were predominantly detected throughout the whole viral genome, most strikingly in low-grade CIN lesions. The results strongly suggest that within-host variations of the HPV genome are primarily generated through the interaction with host cell DNA-editing enzymes and that such within-host variability is an evolutionary source of the genetic diversity of HPVs. Copyright © 2018 American Society for Microbiology.

Driving forces behind the evolution of the Aleutian mink disease parvovirus in the context of intensive farming.

PubMed

Canuti, Marta; O'Leary, Kimberly E; Hunter, Bruce D; Spearman, Grant; Ojkic, Davor; Whitney, Hugh G; Lang, Andrew S

2016-01-01

Aleutian mink disease virus (AMDV) causes plasmacytosis, an immune complex-associated syndrome that affects wild and farmed mink. The virus can also infect other small mammals (e.g., ferrets, skunks, ermines, and raccoons), but the disease in these hosts has been studied less. In 2007, a mink plasmacytosis outbreak began on the Island of Newfoundland, and the virus has been endemic in farms since then. In this study, we evaluated the molecular epidemiology of AMDV in farmed and wild animals of Newfoundland since before the beginning of the outbreak and investigated the epidemic in a global context by studying AMDV worldwide, thereby examining its diffusion and phylogeography. Furthermore, AMDV evolution was examined in the context of intensive farming, where host population dynamics strongly influence viral evolution. Partial NS1 sequences and several complete genomes were obtained from Newfoundland viruses and analyzed along with numerous sequences from other locations worldwide that were either obtained as part of this study or from public databases. We observed very high viral diversity within Newfoundland and within single farms, where high rates of co-infection, recombinant viruses and polymorphisms were observed within single infected individuals. Worldwide, we documented a partial geographic distribution of strains, where viruses from different countries co-exist within clades but form country-specific subclades. Finally, we observed the occurrence of recombination and the predominance of negative selection pressure on AMDV proteins. A surprisingly low number of immunoepitopic sites were under diversifying pressure, possibly because AMDV gains no benefit by escaping the immune response as viral entry into target cells is mediated through interactions with antibodies, which therefore contribute to cell infection. In conclusion, the high prevalence of AMDV in farms facilitates the establishment of co-infections that can favor the occurrence of recombination and enhance viral diversity. Viruses are then exchanged between different farms and countries and can be introduced into the wild, with the rapidly evolving viruses producing many parallel lineages.

Engineering Molecular Immunity Against Plant Viruses.

PubMed

Zaidi, Syed Shan-E-Ali; Tashkandi, Manal; Mahfouz, Magdy M

2017-01-01

Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections. © 2017 Elsevier Inc. All rights reserved.

Redefining the genetics of Murine Gammaherpesvirus 68 via transcriptome-based annotation

PubMed Central

Johnson, L. Steven; Willert, Erin K.; Virgin, Herbert W.

2010-01-01

Summary Viral genetic studies often focus on large open reading frames (ORFs) identified during genome annotation (ORF-based annotation). Here we provide a tool and software set for defining gene expression by murine gammaherpesvirus 68 (γHV68) nucleotide-by-nucleotide across the 119,450 basepair (bp) genome. These tools allowed us to determine that viral RNA expression was significantly more complex than predicted from ORF-based annotation, including over 73,000 nucleotides of unexpected transcription within 30 expressed genomic regions (EGRs). Approximately 90% of this RNA expression was antisense to genomic regions containing known large ORFs. We verified the existence of novel transcripts in three EGRs using standard methods to validate the approach and determined which parts of the transcriptome depend on protein or viral DNA synthesis. This redefines the genetic map of γHV68, indicates that herpesviruses contain significantly more genetic complexity than predicted from ORF-based genome annotations, and provides new tools and approaches for viral genetic studies. PMID:20542255

Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.

1988-01-01

By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alterationmore » of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).« less

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

PubMed

Gokhale, Nandan S; McIntyre, Alexa B R; McFadden, Michael J; Roder, Allison E; Kennedy, Edward M; Gandara, Jorge A; Hopcraft, Sharon E; Quicke, Kendra M; Vazquez, Christine; Willer, Jason; Ilkayeva, Olga R; Law, Brittany A; Holley, Christopher L; Garcia-Blanco, Mariano A; Evans, Matthew J; Suthar, Mehul S; Bradrick, Shelton S; Mason, Christopher E; Horner, Stacy M

2016-11-09

The RNA modification N6-methyladenosine (m 6 A) post-transcriptionally regulates RNA function. The cellular machinery that controls m 6 A includes methyltransferases and demethylases that add or remove this modification, as well as m 6 A-binding YTHDF proteins that promote the translation or degradation of m 6 A-modified mRNA. We demonstrate that m 6 A modulates infection by hepatitis C virus (HCV). Depletion of m 6 A methyltransferases or an m 6 A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m 6 A sites across the HCV genome and determined that inactivating m 6 A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m 6 A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m 6 A. Altogether, this work identifies m 6 A as a conserved regulatory mark across Flaviviridae genomes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Detecting exact breakpoints of deletions with diversity in hepatitis B viral genomic DNA from next-generation sequencing data.

PubMed

Cheng, Ji-Hong; Liu, Wen-Chun; Chang, Ting-Tsung; Hsieh, Sun-Yuan; Tseng, Vincent S

2017-10-01

Many studies have suggested that deletions of Hepatitis B Viral (HBV) are associated with the development of progressive liver diseases, even ultimately resulting in hepatocellular carcinoma (HCC). Among the methods for detecting deletions from next-generation sequencing (NGS) data, few methods considered the characteristics of virus, such as high evolution rates and high divergence among the different HBV genomes. Sequencing high divergence HBV genome sequences using the NGS technology outputs millions of reads. Thus, detecting exact breakpoints of deletions from these big and complex data incurs very high computational cost. We proposed a novel analytical method named VirDelect (Virus Deletion Detect), which uses split read alignment base to detect exact breakpoint and diversity variable to consider high divergence in single-end reads data, such that the computational cost can be reduced without losing accuracy. We use four simulated reads datasets and two real pair-end reads datasets of HBV genome sequence to verify VirDelect accuracy by score functions. The experimental results show that VirDelect outperforms the state-of-the-art method Pindel in terms of accuracy score for all simulated datasets and VirDelect had only two base errors even in real datasets. VirDelect is also shown to deliver high accuracy in analyzing the single-end read data as well as pair-end data. VirDelect can serve as an effective and efficient bioinformatics tool for physiologists with high accuracy and efficient performance and applicable to further analysis with characteristics similar to HBV on genome length and high divergence. The software program of VirDelect can be downloaded at https://sourceforge.net/projects/virdelect/. Copyright © 2017. Published by Elsevier Inc.

Signaling Lymphocytic Activation Molecule Family Receptor Homologs in New World Monkey Cytomegaloviruses.

PubMed

Pérez-Carmona, Natàlia; Farré, Domènec; Martínez-Vicente, Pablo; Terhorst, Cox; Engel, Pablo; Angulo, Ana

2015-11-01

Throughout evolution, large DNA viruses have been usurping genes from their hosts to equip themselves with proteins that restrain host immune defenses. Signaling lymphocytic activation molecule (SLAM) family (SLAMF) receptors are involved in the regulation of both innate and adaptive immunity, which occurs upon engagement with their ligands via homotypic or heterotypic interactions. Here we report a total of seven SLAMF genes encoded by the genomes of two cytomegalovirus (CMV) species, squirrel monkey CMV (SMCMV) and owl monkey CMV (OMCMV), that infect New World monkeys. Our results indicate that host genes were captured by retrotranscription at different stages of the CMV-host coevolution. The most recent acquisition led to S1 in SMCMV. S1 is a SLAMF6 homolog with an amino acid sequence identity of 97% to SLAMF6 in its ligand-binding N-terminal Ig domain. We demonstrate that S1 is a cell surface glycoprotein capable of binding to host SLAMF6. Furthermore, the OMCMV genome encodes A33, an LY9 (SLAMF3) homolog, and A43, a CD48 (SLAMF2) homolog, two soluble glycoproteins which recognize their respective cellular counterreceptors and thus are likely to be viral SLAMF decoy receptors. In addition, distinct copies of further divergent CD48 homologs were found to be encoded by both CMV genomes. Remarkably, all these molecules display a number of unique features, including cytoplasmic tails lacking characteristic SLAMF signaling motifs. Taken together, our findings indicate a novel immune evasion mechanism in which incorporation of host SLAMF receptors that retain their ligand-binding properties enables viruses to interfere with SLAMF functions and to supply themselves with convenient structural molds for expanding their immunomodulatory repertoires. The way in which viruses shape their genomes under the continual selective pressure exerted by the host immune system is central for their survival. Here, we report that New World monkey cytomegaloviruses have broadly captured and duplicated immune cell receptors of the signaling lymphocyte activation molecule (SLAM) family during host-virus coevolution. Notably, we demonstrate that several of these viral SLAMs exhibit exceptional preservation of their N-terminal immunoglobulin domains, which results in maintenance of their ligand-binding capacities. At the same time, these molecules present distinctive structural properties which include soluble forms and the absence of typical SLAM signaling motifs in their cytoplasmic domains, likely reflecting the evolutionary adaptation undergone to efficiently interfere with host SLAM family activities. The observation that the genomes of other large DNA viruses might bear SLAM family homologs further underscores the importance of these molecules as a novel class of immune regulators and as convenient scaffolds for viral evolution. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The not so universal tree of life or the place of viruses in the living world

PubMed Central

Brüssow, Harald

2009-01-01

Darwin provided a great unifying theory for biology; its visual expression is the universal tree of life. The tree concept is challenged by the occurrence of horizontal gene transfer and—as summarized in this review—by the omission of viruses. Microbial ecologists have demonstrated that viruses are the most numerous biological entities on earth, outnumbering cells by a factor of 10. Viral genomics have revealed an unexpected size and distinctness of the viral DNA sequence space. Comparative genomics has shown elements of vertical evolution in some groups of viruses. Furthermore, structural biology has demonstrated links between viruses infecting the three domains of life pointing to a very ancient origin of viruses. However, presently viruses do not find a place on the universal tree of life, which is thus only a tree of cellular life. In view of the polythetic nature of current life definitions, viruses cannot be dismissed as non-living material. On earth we have therefore at least two large DNA sequence spaces, one represented by capsid-encoding viruses and another by ribosome-encoding cells. Despite their probable distinct evolutionary origin, both spheres were and are connected by intensive two-way gene transfers. PMID:19571246

Continuous Influx of Genetic Material from Host to Virus Populations

PubMed Central

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane

2016-01-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors. PMID:26829124

Continuous Influx of Genetic Material from Host to Virus Populations.

PubMed

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane; Cordaux, Richard; Herniou, Elisabeth A

2016-02-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors.

In-vitro and in-vivo phenotype of type Asia 1 foot-and-mouth disease viruses utilizing two non-RGD receptor recognition sites

PubMed Central

2011-01-01

Background Foot-and-mouth disease virus (FMDV) uses a highly conserved Arg-Gly-Asp (RGD) triplet for attachment to host cells and this motif is believed to be essential for virus viability. Previous sequence analyses of the 1D-encoding region of an FMDV field isolate (Asia1/JS/CHA/05) and its two derivatives indicated that two viruses, which contained an Arg-Asp-Asp (RDD) or an Arg-Ser-Asp (RSD) triplet instead of the RGD integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. To examine the influence of single amino acid substitutions in the receptor binding site of the RDD-containing FMD viral genome on virus viability and the ability of non-RGD FMDVs to cause disease in susceptible animals, we constructed an RDD-containing FMDV full-length cDNA clone and derived mutant molecules with RGD or RSD receptor recognition motifs. Following transfection of BSR cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals. Results Amino acid sequence analysis of the 1D-coding region of different derivatives derived from the Asia1/JS/CHA/05 field isolate revealed that the RDD mutants became dominant or achieved population equilibrium with coexistence of the RGD and RSD subpopulations at an early phase of type Asia1 FMDV quasispecies evolution. Furthermore, the RDD and RSD sequences remained genetically stable for at least 20 passages. Using reverse genetics, the RDD-, RSD-, and RGD-containing FMD viruses were rescued from full-length cDNA clones, and single amino acid substitution in RDD-containing FMD viral genome did not affect virus viability. The genetically engineered viruses replicated stably in BHK-21 cells and had similar growth properties to the parental virus. The RDD parental virus and two non-RGD recombinant viruses were virulent to pigs and bovines that developed typical clinical disease and viremia. Conclusions FMDV quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition site. The RDD-containing FMD viral genome can accommodate substitutions in the receptor binding site without additional changes in the capsid. The viruses expressing non-RGD receptor binding sites can replicate stably in vitro and produce typical FMD clinical disease in susceptible animals. PMID:21711567

Immunity: Insect Immune Memory Goes Viral.

PubMed

Ligoxygakis, Petros

2017-11-20

Adaptive memory in insect immunity has been controversial. In this issue, Andino and co-workers propose that acquisition of viral sequences in the host genome gives rise to anti-sense, anti-viral piRNAs. Such sequences can be regarded as both a genomic archive of past infections and as an armour of potential heritable memory. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly.

PubMed

Huang, Zhihong; Pan, Mengjia; Zhu, Silei; Zhang, Hao; Wu, Wenbi; Yuan, Meijin; Yang, Kai

2017-03-01

Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83 -encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis -acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis -acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis -acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process. IMPORTANCE Virus nucleocapsid assembly usually requires specific cis -acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis -acting elements in the viral genome. Copyright © 2017 American Society for Microbiology.

Potential Links between Hepadnavirus and Bornavirus Sequences in the Host Genome and Cancer.

PubMed

Honda, Tomoyuki

2017-01-01

Various viruses leave their sequences in the host genomes during infection. Such events occur mainly in retrovirus infection but also sometimes in DNA and non-retroviral RNA virus infections. If viral sequences are integrated into the genomes of germ line cells, the sequences can become inherited as endogenous viral elements (EVEs). The integration events of viral sequences may have oncogenic potential. Because proviral integrations of some retroviruses and/or reactivation of endogenous retroviruses are closely linked to cancers, viral insertions related to non-retroviral viruses also possibly contribute to cancer development. This article focuses on genomic viral sequences derived from two non-retroviral viruses, whose endogenization is already reported, and discusses their possible contributions to cancer. Viral insertions of hepatitis B virus play roles in the development of hepatocellular carcinoma. Endogenous bornavirus-like elements, the only non-retroviral RNA virus-related EVEs found in the human genome, may also be involved in cancer formation. In addition, the possible contribution of the interactions between viruses and retrotransposons, which seem to be a major driving force for generating EVEs related to non-retroviral RNA viruses, to cancers will be discussed. Future studies regarding the possible links described here may open a new avenue for the development of novel therapeutics for tumor virus-related cancers and/or provide novel insights into EVE functions.

Mapping EBNA-1 Domains Involved in Binding to Metaphase Chromosomes

PubMed Central

Marechal, Vincent; Dehee, Axelle; Chikhi-Brachet, Roxane; Piolot, Tristan; Coppey-Moisan, Maité; Nicolas, Jean-Claude

1999-01-01

The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids. PMID:10196336

Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains.

PubMed

Kanda, Teru; Furuse, Yuki; Oshitani, Hitoshi; Kiyono, Tohru

2016-05-01

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death. Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Viral Predation and Host Immunity Structure Microbial Communities in a Terrestrial Deep Subsurface, Hydraulically Fractured Shale System

NASA Astrophysics Data System (ADS)

Daly, R. A.; Mouser, P. J.; Trexler, R.; Wrighton, K. C.

2014-12-01

Despite a growing appreciation for the ecological role of viruses in marine and gut systems, little is known about their role in the terrestrial deep (> 2000 m) subsurface. We used assembly-based metagenomics to examine the viral component in fluids from hydraulically fractured Marcellus shale gas wells. Here we reconstructed microbial and viral genomes from samples collected 7, 82, and 328 days post fracturing. Viruses accounted for 4.14%, 0.92% and 0.59% of the sample reads that mapped to the assembly. We identified 6 complete, circularized viral genomes and an additional 92 viral contigs > 5 kb with a maximum contig size of 73.6 kb. A BLAST comparison to NCBI viral genomes revealed that 85% of viral contigs had significant hits to the viral order Caudovirales, with 43% of sequences belonging to the family Siphoviridae, 38% to Myoviridae, and 12% to Podoviridae. Enrichment of Caudovirales viruses was supported by a large number of predicted proteins characteristic of tailed viruses including terminases (TerL), tape measure, tail formation, and baseplate related proteins. The viral contigs included evidence of lytic and temperate lifestyles, with the 7 day sample having the greatest number of detected lytic viruses. Notably in this sample, the most abundant virus was lytic and its inferred host, a member of the Vibrionaceae, was not detected at later time points. Analyses of CRISPR sequences (a viral and foreign DNA immune system in bacteria and archaea), linked 18 viral contigs to hosts. CRISPR linkages increased through time and all bacterial and archaeal genomes recovered in the final time point had genes for CRISPR-mediated viral defense. The majority of CRISPR sequences linked phage genomes to several Halanaerobium strains, which are the dominant and persisting members of the community inferred to be responsible for carbon and sulfur cycling in these shales. Network analysis revealed that several viruses were present in the 82 and 328 day samples; this viral persistence is consistent with concomitant temporal stability in geochemistry and microbial community composition. Our findings suggest that after a disturbance (hydraulic fracturing) viral predation and host immunity is an important controller of microbial community structure, metabolism, and thus biogeochemical cycling in the deep subsurface.

Chromosomally Integrated Human Herpesvirus 6: Models of Viral Genome Release from the Telomere and Impacts on Human Health.

PubMed

Wood, Michael L; Royle, Nicola J

2017-07-12

Human herpesvirus 6A and 6B, alongside some other herpesviruses, have the striking capacity to integrate into telomeres, the terminal repeated regions of chromosomes. The chromosomally integrated forms, ciHHV-6A and ciHHV-6B, are proposed to be a state of latency and it has been shown that they can both be inherited if integration occurs in the germ line. The first step in full viral reactivation must be the release of the integrated viral genome from the telomere and here we propose various models of this release involving transcription of the viral genome, replication fork collapse, and t-circle mediated release. In this review, we also discuss the relationship between ciHHV-6 and the telomere carrying the insertion, particularly how the presence and subsequent partial or complete release of the ciHHV-6 genome may affect telomere dynamics and the risk of disease.

Samba virus: a novel mimivirus from a giant rain forest, the Brazilian Amazon.

PubMed

Campos, Rafael K; Boratto, Paulo V; Assis, Felipe L; Aguiar, Eric R G R; Silva, Lorena C F; Albarnaz, Jonas D; Dornas, Fabio P; Trindade, Giliane S; Ferreira, Paulo P; Marques, João T; Robert, Catherine; Raoult, Didier; Kroon, Erna G; La Scola, Bernard; Abrahão, Jônatas S

2014-05-14

The identification of novel giant viruses from the nucleocytoplasmic large DNA viruses group and their virophages has increased in the last decade and has helped to shed light on viral evolution. This study describe the discovery, isolation and characterization of Samba virus (SMBV), a novel giant virus belonging to the Mimivirus genus, which was isolated from the Negro River in the Brazilian Amazon. We also report the isolation of an SMBV-associated virophage named Rio Negro (RNV), which is the first Mimivirus virophage to be isolated in the Americas. Based on a phylogenetic analysis, SMBV belongs to group A of the putative Megavirales order, possibly a new virus related to Acanthamoeba polyphaga mimivirus (APMV). SMBV is the largest virus isolated in Brazil, with an average particle diameter about 574 nm. The SMBV genome contains 938 ORFs, of which nine are ORFans. The 1,213.6 kb SMBV genome is one of the largest genome of any group A Mimivirus described to date. Electron microscopy showed RNV particle accumulation near SMBV and APMV factories resulting in the production of defective SMBV and APMV particles and decreasing the infectivity of these two viruses by several logs. This discovery expands our knowledge of Mimiviridae evolution and ecology.

Viral Evolution Core | FNLCR Staging

Cancer.gov

Brandon F. Keele, Ph.D. PI/Senior Principal Investigator, Retroviral Evolution Section Head, Viral Evolution Core Leidos Biomedical Research, Inc. Frederick National Laboratory for Cancer Research Frederick, MD 21702-1201 Tel: 301-846-173

Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting Paramecium bursaria chlorella virus-1

PubMed Central

Shimoni, Eyal; Dadosh, Tali; Rechav, Katya; Unger, Tamar

2017-01-01

A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle. PMID:28850602

Experimental Approaches to Study Genome Packaging of Influenza A Viruses.

PubMed

Isel, Catherine; Munier, Sandie; Naffakh, Nadia

2016-08-09

The genome of influenza A viruses (IAV) consists of eight single-stranded negative sense viral RNAs (vRNAs) encapsidated into viral ribonucleoproteins (vRNPs). It is now well established that genome packaging (i.e., the incorporation of a set of eight distinct vRNPs into budding viral particles), follows a specific pathway guided by segment-specific cis-acting packaging signals on each vRNA. However, the precise nature and function of the packaging signals, and the mechanisms underlying the assembly of vRNPs into sub-bundles in the cytoplasm and their selective packaging at the viral budding site, remain largely unknown. Here, we review the diverse and complementary methods currently being used to elucidate these aspects of the viral cycle. They range from conventional and competitive reverse genetics, single molecule imaging of vRNPs by fluorescence in situ hybridization (FISH) and high-resolution electron microscopy and tomography of budding viral particles, to solely in vitro approaches to investigate vRNA-vRNA interactions at the molecular level.

Evasion of superinfection exclusion and elimination of primary viral RNA by an adapted strain of hepatitis C virus.

PubMed

Webster, Brian; Ott, Melanie; Greene, Warner C

2013-12-01

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequence-barcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/UC) tract of the 3' untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo.

DNA methylation at hepatitis B viral integrants is associated with methylation at flanking human genomic sequences

PubMed Central

Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Oikawa, Ritsuko; Toyota, Minoru; Yamamoto, Masakazu; Kokudo, Norihiro; Tanaka, Shinji; Arii, Shigeki; Yotsuyanagi, Hiroshi; Koike, Kazuhiko; Itoh, Fumio

2015-01-01

Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)–related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis. PMID:25653310

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

PubMed Central

Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth

2017-01-01

ABSTRACT Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets. PMID:28978704

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

PubMed

Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth; Dutch, Rebecca Ellis

2017-12-15

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets. Copyright © 2017 American Society for Microbiology.

[Genome organization and life cycle of the hepatitis c virus].

PubMed

Kalinina, O V; Dmitriev, A V

2015-01-01

The review summarizes the current data about the hepatitis C viral genome and polyprotein organization. The functional role of the structural and non-structural viral proteins including their interaction with cellular regulatory proteins and cell structural elements is discussed. Specific peculiarities of the life cycle of the hepatitis C virus important for the understanding of the viral hepatitis C pathogenesis are summarized.

Viral entry pathways: the example of common cold viruses.

PubMed

Blaas, Dieter

2016-05-01

For infection, viruses deliver their genomes into the host cell. These nucleic acids are usually tightly packed within the viral capsid, which, in turn, is often further enveloped within a lipid membrane. Both protect them against the hostile environment. Proteins and/or lipids on the viral particle promote attachment to the cell surface and internalization. They are likewise often involved in release of the genome inside the cell for its use as a blueprint for production of new viruses. In the following, I shall cursorily discuss the early more general steps of viral infection that include receptor recognition, uptake into the cell, and uncoating of the viral genome. The later sections will concentrate on human rhinoviruses, the main cause of the common cold, with respect to the above processes. Much of what is known on the underlying mechanisms has been worked out by Renate Fuchs at the Medical University of Vienna.

Phage-Mediated Gene Therapy.

PubMed

Hosseinidoust, Zeinab

2017-01-01

Bacteriophages (bacterial viruses) have long been under investigation as vectors for gene therapy. Similar to other viral vectors, the phage coat proteins have evolved over millions of years to protect the viral genome from degradation post injection, offering protection for the valuable therapeutic sequence. However, what sets phage apart from other viral gene delivery vectors is their safety for human use and the relative ease by which foreign molecules can be expressed on the phage outer surface, enabling highly targeted gene delivery. The latter property also makes phage a popular choice for gene therapy target discovery through directed evolution. Although promising, phage-mediated gene therapy faces several outstanding challenges, the most notable being lower gene delivery efficiency compared to animal viruses, vector stability, and nondesirable immune stimulation. This review presents a critical review of promises and challenges of employing phage as gene delivery vehicles as well as an introduction to the concept of phage-based microbiome therapy as the new frontier and perhaps the most promising application of phage-based gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

PubMed Central

Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Jianguo

2016-01-01

ABSTRACT Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5′ untranslated region (5′UTR) and a polyadenylated 3′UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3Dpol protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3Dpol, resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5′UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5′UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication. PMID:27875274

Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration.

PubMed

Thierry, Sylvain; Munir, Soundasse; Thierry, Eloïse; Subra, Frédéric; Leh, Hervé; Zamborlini, Alessia; Saenz, Dyana; Levy, David N; Lesbats, Paul; Saïb, Ali; Parissi, Vincent; Poeschla, Eric; Deprez, Eric; Delelis, Olivier

2015-03-12

Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication. Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.

Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia

PubMed Central

Oshima, Koichi; Khiabanian, Hossein; da Silva-Almeida, Ana C.; Tzoneva, Gannie; Abate, Francesco; Ambesi-Impiombato, Alberto; Sanchez-Martin, Marta; Carpenter, Zachary; Penson, Alex; Perez-Garcia, Arianne; Eckert, Cornelia; Nicolas, Concepción; Balbin, Milagros; Sulis, Maria Luisa; Kato, Motohiro; Koh, Katsuyoshi; Paganin, Maddalena; Basso, Giuseppe; Gastier-Foster, Julie M.; Devidas, Meenakshi; Loh, Mignon L.; Kirschner-Schwabe, Renate; Palomero, Teresa; Rabadan, Raul; Ferrando, Adolfo A.

2016-01-01

Although multiagent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die because of chemorefractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape at relapse in pediatric ALL cases. These analyses identified numerous relapse-associated mutated genes intertwined in chemotherapy resistance-related protein complexes. In this context, RAS-MAPK pathway-activating mutations in the neuroblastoma RAS viral oncogene homolog (NRAS), kirsten rat sarcoma viral oncogene homolog (KRAS), and protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) genes were present in 24 of 55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, whereas in others RAS mutant clones present at diagnosis were replaced by RAS wild-type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts. These results highlight the central role of chemotherapy-driven selection as a central mechanism of leukemia clonal evolution in relapsed ALL, and demonstrate a previously unrecognized dual role of RAS mutations as drivers of both sensitivity and resistance to chemotherapy. PMID:27655895

Population structure of the dengue viruses, Aragua, Venezuela, 2006–2007. Insights into dengue evolution under hyperendemic transmission

PubMed Central

Rodriguez-Roche, Rosmari; Villegas, Elci; Cook, Shelley; Poh Kim, Pauline A.W.; Hinojosa, Yoandri; Rosario, Delfina; Villalobos, Iris; Bendezu, Herminia; Hibberd, Martin L.; Guzman, Maria G.

2012-01-01

During the past three decades there has been a notable increase in dengue disease severity in Venezuela. Nevertheless, the population structure of the viruses being transmitted in this country is not well understood. Here, we present a molecular epidemiological study on dengue viruses (DENV) circulating in Aragua State, Venezuela during 2006–2007. Twenty-one DENV full-length genomes representing all of the four serotypes were amplified and sequenced directly from the serum samples. Notably, only DENV-2 was associated with severe disease. Phylogenetic trees constructed using Bayesian methods indicated that only one genotype was circulating for each serotype. However, extensive viral genetic diversity was found in DENV isolated from the same area during the same period, indicating significant in situ evolution since the introduction of these genotypes. Collectively, the results suggest that the non-structural (NS) proteins may play an important role in DENV evolution, particularly NS1, NS2A and NS4B proteins. The phylogenetic data provide evidence to suggest that multiple introductions of DENV have occurred from the Latin American region into Venezuela and vice versa. The implications of the significant viral genetic diversity generated during hyperendemic transmission, particularly in NS protein are discussed and considered in the context of future development and use of human monoclonal antibodies as antivirals and tetravalent vaccines. PMID:22197765

The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores

PubMed Central

Huffman, Jamie B.; Daniel, Gina R.; Falck-Pedersen, Erik; Huet, Alexis

2017-01-01

ABSTRACT The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids. IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release. PMID:28490590

Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins

NASA Astrophysics Data System (ADS)

Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

2016-05-01

A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks.

Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko

In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres,more » and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.« less

Experimentally guided models reveal replication principles that shape the mutation distribution of RNA viruses

PubMed Central

Schulte, Michael B; Draghi, Jeremy A; Plotkin, Joshua B; Andino, Raul

2015-01-01

Life history theory posits that the sequence and timing of events in an organism's lifespan are fine-tuned by evolution to maximize the production of viable offspring. In a virus, a life history strategy is largely manifested in its replication mode. Here, we develop a stochastic mathematical model to infer the replication mode shaping the structure and mutation distribution of a poliovirus population in an intact single infected cell. We measure production of RNA and poliovirus particles through the infection cycle, and use these data to infer the parameters of our model. We find that on average the viral progeny produced from each cell are approximately five generations removed from the infecting virus. Multiple generations within a single cell infection provide opportunities for significant accumulation of mutations per viral genome and for intracellular selection. DOI: http://dx.doi.org/10.7554/eLife.03753.001 PMID:25635405

Transposases are the most abundant, most ubiquitous genes in nature.

PubMed

Aziz, Ramy K; Breitbart, Mya; Edwards, Robert A

2010-07-01

Genes, like organisms, struggle for existence, and the most successful genes persist and widely disseminate in nature. The unbiased determination of the most successful genes requires access to sequence data from a wide range of phylogenetic taxa and ecosystems, which has finally become achievable thanks to the deluge of genomic and metagenomic sequences. Here, we analyzed 10 million protein-encoding genes and gene tags in sequenced bacterial, archaeal, eukaryotic and viral genomes and metagenomes, and our analysis demonstrates that genes encoding transposases are the most prevalent genes in nature. The finding that these genes, classically considered as selfish genes, outnumber essential or housekeeping genes suggests that they offer selective advantage to the genomes and ecosystems they inhabit, a hypothesis in agreement with an emerging body of literature. Their mobile nature not only promotes dissemination of transposable elements within and between genomes but also leads to mutations and rearrangements that can accelerate biological diversification and--consequently--evolution. By securing their own replication and dissemination, transposases guarantee to thrive so long as nucleic acid-based life forms exist.

The First Isolation and Whole Genome Sequencing of Murray Valley Encephalitis Virus from Cerebrospinal Fluid of a Patient with Encephalitis.

PubMed

Russell, Jessica S; Caly, Leon; Kostecki, Renata; McGuinness, Sarah L; Carter, Glen; Bulach, Dieter; Seemann, Torsten; Stinear, Tim P; Baird, Rob; Catton, Mike; Druce, Julian

2018-06-11

Murray Valley Encephalitis virus (MVEV) is a mosquito-borne Flavivirus. Clinical presentation is rare but severe, with a case fatality rate of 15⁻30%. Here we report a case of MVEV from the cerebrospinal fluid (CSF) of a patient in the Northern Territory in Australia. Initial diagnosis was performed using both MVEV-specific real-time, and Pan- Flavivirus conventional, Polymerase Chain Reaction (PCR), with confirmation by Sanger sequencing. Subsequent isolation, the first from CSF, was conducted in Vero cells and the observed cytopathic effect was confirmed by increasing viral titre in the real-time PCR. Isolation allowed for full genome sequencing using the Scriptseq V2 RNASeq library preparation kit. A consensus genome for VIDRL-MVE was generated and phylogenetic analysis identified it as Genotype 2. This is the first reported isolation, and full genome sequencing of MVEV from CSF. It is also the first time Genotype 2 has been identified in humans. As such, this case has significant implications for public health surveillance, epidemiology, and the understanding of MVEV evolution.

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis.

PubMed

DiGiuseppe, Stephen; Luszczek, Wioleta; Keiffer, Timothy R; Bienkowska-Haba, Malgorzata; Guion, Lucile G M; Sapp, Martin J

2016-05-31

During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral genome dissociates from the TGN and associates with microtubules after the onset of mitosis. Deposition onto mitotic chromosomes is L2-mediated. Using differential staining of an incoming viral genome by small molecular dyes in selectively permeabilized cells, nuclease protection, and flotation assays, we found that HPV resides in a membrane-bound vesicle until mitosis is completed and the nuclear envelope has reformed. As a result, expression of the incoming viral genome is delayed. Taken together, these data provide evidence that HPV has evolved a unique strategy for delivering the viral genome to the nucleus of dividing cells. Furthermore, it is unlikely that nuclear vesicles are unique to HPV, and thus we may have uncovered a hitherto unrecognized cellular pathway that may be of interest for future cell biological studies.

Emergence of host-adapted Salmonella Enteritidis through rapid evolution in an immunocompromised host.

PubMed

Klemm, Elizabeth J; Gkrania-Klotsas, Effrossyni; Hadfield, James; Forbester, Jessica L; Harris, Simon R; Hale, Christine; Heath, Jennifer N; Wileman, Thomas; Clare, Simon; Kane, Leanne; Goulding, David; Otto, Thomas D; Kay, Sally; Doffinger, Rainer; Cooke, Fiona J; Carmichael, Andrew; Lever, Andrew Ml; Parkhill, Julian; MacLennan, Calman A; Kumararatne, Dinakantha; Dougan, Gordon; Kingsley, Robert A

2016-03-01

Host adaptation is a key factor contributing to the emergence of new bacterial, viral and parasitic pathogens. Many pathogens are considered promiscuous because they cause disease across a range of host species, while others are host-adapted, infecting particular hosts 1 . Host adaptation can potentially progress to host restriction where the pathogen is strictly limited to a single host species and is frequently associated with more severe symptoms. Host-adapted and host-restricted bacterial clades evolve from within a broader host-promiscuous species and sometimes target different niches within their specialist hosts, such as adapting from a mucosal to a systemic lifestyle. Genome degradation, marked by gene inactivation and deletion, is a key feature of host adaptation, although the triggers initiating genome degradation are not well understood. Here, we show that a chronic systemic non-typhoidal Salmonella infection in an immunocompromised human patient resulted in genome degradation targeting genes that are expendable for a systemic lifestyle. We present a genome-based investigation of a recurrent blood-borne Salmonella enterica serotype Enteritidis ( S . Enteritidis) infection covering 15 years in an interleukin (IL)-12 β-1 receptor-deficient individual that developed into an asymptomatic chronic infection. The infecting S. Enteritidis harbored a mutation in the mismatch repair gene mutS that accelerated the genomic mutation rate. Phylogenetic analysis and phenotyping of multiple patient isolates provides evidence for a remarkable level of within-host evolution that parallels genome changes present in successful host-restricted bacterial pathogens but never before observed on this timescale. Our analysis identifies common pathways of host adaptation and demonstrates the role that immunocompromised individuals can play in this process.

The Molecular Biology of Frog Virus 3 and other Iridoviruses Infecting Cold-Blooded Vertebrates

PubMed Central

Chinchar, V. Gregory; Yu, Kwang H.; Jancovich, James K.

2011-01-01

Frog virus 3 (FV3) is the best characterized member of the family Iridoviridae. FV3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. Although the broad outlines of FV3 replication have been elucidated, the precise roles of most viral proteins remain unknown. Current studies using knock down (KD) mediated by antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA), knock out (KO) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. In addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. In this review, we summarize current studies using not only FV3, but also other iridoviruses infecting ectotherms. As described below, general principles ascertained using FV3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. Collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease. PMID:22069524

Improved bacteriophage genome data is necessary for integrating viral and bacterial ecology.

PubMed

Bibby, Kyle

2014-02-01

The recent rise in "omics"-enabled approaches has lead to improved understanding in many areas of microbial ecology. However, despite the importance that viruses play in a broad microbial ecology context, viral ecology remains largely not integrated into high-throughput microbial ecology studies. A fundamental hindrance to the integration of viral ecology into omics-enabled microbial ecology studies is the lack of suitable reference bacteriophage genomes in reference databases-currently, only 0.001% of bacteriophage diversity is represented in genome sequence databases. This commentary serves to highlight this issue and to promote bacteriophage genome sequencing as a valuable scientific undertaking to both better understand bacteriophage diversity and move towards a more holistic view of microbial ecology.

Molecular Mechanisms of Foot-and-Mouth Disease Virus Targeting the Host Antiviral Response.

PubMed

Rodríguez Pulido, Miguel; Sáiz, Margarita

2017-01-01

Foot-and-mouth disease virus (FMDV) is the causative agent of an acute vesicular disease affecting pigs, cattle and other domestic, and wild animals worldwide. The aim of the host interferon (IFN) response is to limit viral replication and spread. Detection of the viral genome and products by specialized cellular sensors initiates a signaling cascade that leads to a rapid antiviral response involving the secretion of type I- and type III-IFNs and other antiviral cytokines with antiproliferative and immunomodulatory functions. During co-evolution with their hosts, viruses have acquired strategies to actively counteract host antiviral responses and the balance between innate response and viral antagonism may determine the outcome of disease and pathogenesis. FMDV proteases Lpro and 3C have been found to antagonize the host IFN response by a repertoire of mechanisms. Moreover, the putative role of other viral proteins in IFN antagonism is being recently unveiled, uncovering sophisticated immune evasion strategies different to those reported to date for other members of the Picornaviridae family. Here, we review the interplay between antiviral responses induced by FMDV infection and viral countermeasures to block them. Research on strategies used by viruses to modulate immunity will provide insights into the function of host pathways involved in defense against pathogens and will also lead to development of new therapeutic strategies to fight virus infections.

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

PubMed Central

Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Sola, Isabel

2013-01-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. PMID:23966403

Wild-Type Measles Viruses with Non-Standard Genome Lengths

PubMed Central

Bankamp, Bettina; Liu, Chunyu; Rivailler, Pierre; Bera, Jayati; Shrivastava, Susmita; Kirkness, Ewen F.; Bellini, William J.; Rota, Paul A.

2014-01-01

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible. PMID:24748123

Reverse Genetics for Mammalian Orthoreovirus.

PubMed

Stuart, Johnasha D; Phillips, Matthew B; Boehme, Karl W

2017-01-01

Reverse genetics allows introduction of specific alterations into a viral genome. Studies performed with mutant viruses generated using reverse genetics approaches have contributed immeasurably to our understanding of viral replication and pathogenesis, and also have led to development of novel vaccines and virus-based vectors. Here, we describe the reverse genetics system that allows for production and recovery of mammalian orthoreovirus, a double-stranded (ds) RNA virus, from plasmids that encode the viral genome.

Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P2.

PubMed

Kakisaka, Michinori; Yamada, Kazunori; Yamaji-Hasegawa, Akiko; Kobayashi, Toshihide; Aida, Yoko

2016-09-01

To be incorporated into progeny virions, the viral genome must be transported to the inner leaflet of the plasma membrane (PM) and accumulate there. Some viruses utilize lipid components to assemble at the PM. For example, simian virus 40 (SV40) targets the ganglioside GM1 and human immunodeficiency virus type 1 (HIV-1) utilizes phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. Recent studies clearly indicate that Rab11-mediated recycling endosomes are required for influenza A virus (IAV) trafficking of vRNPs to the PM but it remains unclear how IAV vRNP localized or accumulate underneath the PM for viral genome incorporation into progeny virions. In this study, we found that the second intrinsically disordered region (IDR2) of NP regulates two binding steps involved in viral genome packaging. First, IDR2 facilitates NP oligomer binding to viral RNA to form vRNP. Secondly, vRNP assemble by interacting with PI(4,5)P2 at the PM via IDR2. These findings suggest that PI(4,5)P2 functions as the determinant of vRNP accumulation at the PM. Copyright © 2016 Elsevier Inc. All rights reserved.

Genomic diversity of Bombyx mori nucleopolyhedrovirus strains.

PubMed

Xu, Yi-Peng; Cheng, Ruo-Lin; Xi, Yu; Zhang, Chuan-Xi

2013-07-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that selectively infects the domestic silkworm. In this study, six BmNPV strains were compared at the whole genome level. We found that the number of bro genes and the composition of the homologous regions (hrs) are the two primary areas of divergence within these genomes. When we compared the ORFs of these BmNPV variants, we noticed a high degree of sequence divergence in the ORFs that are not baculovirus core genes. This result is consistent with the results derived from phylogenetic trees and evolutionary pressure analyses of these ORFs, indicating that ORFs that are not core genes likely play important roles in the evolution of BmNPV strains. The evolutionary relationships of these BmNPV strains might be explained by their geographic origins or those of their hosts. In addition, the total number of hr palindromes seems to affect viral DNA replication in Bm5 cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Revisiting the genome packaging in viruses with lessons from the "Giants".

PubMed

Chelikani, Venkata; Ranjan, Tushar; Kondabagil, Kiran

2014-10-01

Genome encapsidation is an essential step in the life cycle of viruses. Viruses either use some of the most powerful ATP-dependent motors to compel the genetic material into the preformed capsid or make use of the positively charged proteins to bind and condense the negatively charged genome in an energy-independent manner. While the former is a hallmark of large DNA viruses, the latter is commonly seen in small DNA and RNA viruses. Discoveries of many complex giant viruses such as mimivirus, megavirus, pandoravirus, etc., belonging to the nucleo-cytoplasmic large DNA virus (NCLDV) superfamily have changed the perception of genome packaging in viruses. From what little we have understood so far, it seems that the genome packaging mechanism in NCLDVs has nothing in common with other well-characterized viral packaging systems such as the portal-terminase system or the energy-independent system. Recent findings suggest that in giant viruses, the genome segregation and packaging processes are more intricately coupled than those of other viral systems. Interestingly, giant viral packaging systems also seem to possess features that are analogous to bacterial and archaeal chromosome segregation. Although there is a lot of diversity in terms of host range, type of genome, and genome size among viruses, they all seem to use three major types of independent innovations to accomplish genome encapsidation. Here, we have made an attempt to comprehensively review all the known viral genome packaging systems, including the one that is operative in giant viruses, by proposing a simple and expanded classification system that divides the viral packaging systems into three large groups (types I-III) on the basis of the mechanism employed and the relatedness of the major packaging proteins. Known variants within each group have been further classified into subgroups to reflect their unique adaptations. Copyright © 2014 Elsevier Inc. All rights reserved.

The Effect of Vaccination on the Evolution and Population Dynamics of Avian Paramyxovirus-1

PubMed Central

Hudson, Peter J.; Poss, Mary

2010-01-01

Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19th century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins. PMID:20421950

Comparative Genomic Analysis MERS CoV Isolated from Humans and Camels with Special Reference to Virus Encoded Helicase.

PubMed

Alnazawi, Mohamed; Altaher, Abdallah; Kandeel, Mahmoud

2017-01-01

Middle East Respiratory Syndrome Coronavirus (MERS CoV) is a new emerging viral disease characterized by high fatality rate. Understanding MERS CoV genetic aspects and codon usage pattern is important to understand MERS CoV survival, adaptation, evolution, resistance to innate immunity, and help in finding the unique aspects of the virus for future drug discovery experiments. In this work, we provide comprehensive analysis of 238 MERS CoV full genomes comprised of human (hMERS) and camel (cMERS) isolates of the virus. MERS CoV genome shaping seems to be under compositional and mutational bias, as revealed by preference of A/T over G/C nucleotides, preferred codons, nucleotides at the third position of codons (NT3s), relative synonymous codon usage, hydropathicity (Gravy), and aromaticity (Aromo) indices. Effective number of codons (ENc) analysis reveals a general slight codon usage bias. Codon adaptation index reveals incomplete adaptation to host environment. MERS CoV showed high ability to resist the innate immune response by showing lower CpG frequencies. Neutrality evolution analysis revealed a more significant role of mutation pressure in cMERS over hMERS. Correspondence analysis revealed that MERS CoV genomes have three genetic clusters, which were distinct in their codon usage, host, and geographic distribution. Additionally, virtual screening and binding experiments were able to identify three new virus-encoded helicase binding compounds. These compounds can be used for further optimization of inhibitors.

Noumeavirus replication relies on a transient remote control of the host nucleus

PubMed Central

Fabre, Elisabeth; Jeudy, Sandra; Santini, Sébastien; Legendre, Matthieu; Trauchessec, Mathieu; Couté, Yohann; Claverie, Jean-Michel; Abergel, Chantal

2017-01-01

Acanthamoeba are infected by a remarkable diversity of large dsDNA viruses, the infectious cycles of which have been characterized using genomics, transcriptomics and electron microscopy. Given their gene content and the persistence of the host nucleus throughout their infectious cycle, the Marseilleviridae were initially assumed to fully replicate in the cytoplasm. Unexpectedly, we find that their virions do not incorporate the virus-encoded transcription machinery, making their replication nucleus-dependent. However, instead of delivering their DNA to the nucleus, the Marseilleviridae initiate their replication by transiently recruiting the nuclear transcription machinery to their cytoplasmic viral factory. The nucleus recovers its integrity after becoming leaky at an early stage. This work highlights the importance of virion proteomic analyses to complement genome sequencing in the elucidation of the replication scheme and evolution of large dsDNA viruses. PMID:28429720

Introduction, Evolution, and Dissemination of Influenza A Viruses in Exhibition Swine in the United States during 2009 to 2013

PubMed Central

Nelson, Martha I.; Stucker, Karla M.; Schobel, Seth A.; Dugan, Vivien G.; Nelson, Sarah W.; Sreevatsan, Srinand; Killian, Mary L.; Nolting, Jacqueline M.; Wentworth, David E.

2016-01-01

ABSTRACT The swine-human interface created at agricultural fairs, along with the generation of and maintenance of influenza A virus diversity in exhibition swine, presents an ongoing threat to public health. Nucleotide sequences of influenza A virus isolates collected from exhibition swine in Ohio (n = 262) and Indiana (n = 103) during 2009 to 2013 were used to investigate viral evolution and movement within this niche sector of the swine industry. Phylogenetic and Bayesian analyses were employed to identify introductions of influenza A virus to exhibition swine and study viral population dynamics. In 2013 alone, we identified 10 independent introductions of influenza A virus into Ohio and/or Indiana exhibition swine. Frequently, viruses from the same introduction were identified at multiple fairs within the region, providing evidence of rapid and widespread viral movement within the exhibition swine populations of the two states. While pigs moving from fair to fair to fair is possible in some locations, the concurrent detection of nearly identical strains at several fairs indicates that a common viral source was more likely. Importantly, we detected an association between the high number of human variant H3N2 (H3N2v) virus infections in 2012 and the widespread circulation of influenza A viruses of the same genotype in exhibition swine in Ohio fairs sampled that year. The extent of viral diversity observed in exhibition swine and the rapidity with which it disseminated across long distances indicate that novel strains of influenza A virus will continue to emerge and spread within exhibition swine populations, presenting an ongoing threat to humans. IMPORTANCE Understanding the underlying population dynamics of influenza A viruses in commercial and exhibition swine is central to assessing the risk for human infections with variant viruses, including H3N2v. We used viral genomic sequences from isolates collected from exhibition swine during 2009 to 2013 to understand how the peak of H3N2v cases in 2012 relates to long-term trends in the population dynamics of pandemic viruses recently introduced into commercial and exhibition swine in the United States. The results of our spatial analysis underscore the key role of rapid viral dispersal in spreading multiple genetic lineages throughout a multistate network of agricultural fairs, providing opportunities for divergent lineages to coinfect, reassort, and generate new viral genotypes. The higher genetic diversity of genotypes cocirculating in exhibition swine since 2013 could facilitate the evolution of new reassortants, potentially with even greater ability to cause severe infections in humans or cause human-to-human transmission, highlighting the need for continued vigilance. PMID:27681134

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

PubMed

Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François-Loïc

2017-03-01

Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Cis-acting RNA elements in the Hepatitis C virus RNA genome

PubMed Central

Sagan, Selena M.; Chahal, Jasmin; Sarnow, Peter

2017-01-01

Hepatitis C virus (HCV) infection is a rapidly increasing global health problem with an estimated 170 million people infected worldwide. HCV is a hepatotropic, positive-sense RNA virus of the family Flaviviridae. As a positive-sense RNA virus, the HCV genome itself must serve as a template for translation, replication and packaging. The viral RNA must therefore be a dynamic structure that is able to readily accommodate structural changes to expose different regions of the genome to viral and cellular proteins to carry out the HCV life cycle. The ∼9600 nucleotide viral genome contains a single long open reading frame flanked by 5′ and 3′ non-coding regions that contain cis-acting RNA elements important for viral translation, replication and stability. Additional cis-acting RNA elements have also been identified in the coding sequences as well as in the 3′ end of the negative-strand replicative intermediate. Herein, we provide an overview of the importance of these cis-acting RNA elements in the HCV life cycle. PMID:25576644

Fitness cost of reassortment in human influenza

PubMed Central

Lässig, Michael

2017-01-01

Reassortment, which is the exchange of genome sequence between viruses co-infecting a host cell, plays an important role in the evolution of segmented viruses. In the human influenza virus, reassortment happens most frequently between co-existing variants within the same lineage. This process breaks genetic linkage and fitness correlations between viral genome segments, but the resulting net effect on viral fitness has remained unclear. In this paper, we determine rate and average selective effect of reassortment processes in the human influenza lineage A/H3N2. For the surface proteins hemagglutinin and neuraminidase, reassortant variants with a mean distance of at least 3 nucleotides to their parent strains get established at a rate of about 10−2 in units of the neutral point mutation rate. Our inference is based on a new method to map reassortment events from joint genealogies of multiple genome segments, which is tested by extensive simulations. We show that intra-lineage reassortment processes are, on average, under substantial negative selection that increases in strength with increasing sequence distance between the parent strains. The deleterious effects of reassortment manifest themselves in two ways: there are fewer reassortment events than expected from a null model of neutral reassortment, and reassortant strains have fewer descendants than their non-reassortant counterparts. Our results suggest that influenza evolves under ubiquitous epistasis across proteins, which produces fitness barriers against reassortment even between co-circulating strains within one lineage. PMID:29112968

Molecular Genetic Analysis of Orf Virus: A Poxvirus That Has Adapted to Skin

PubMed Central

Fleming, Stephen B.; Wise, Lyn M.; Mercer, Andrew A.

2015-01-01

Orf virus is the type species of the Parapoxvirus genus of the family Poxviridae. It induces acute pustular skin lesions in sheep and goats and is transmissible to humans. The genome is G+C rich, 138 kbp and encodes 132 genes. It shares many essential genes with vaccinia virus that are required for survival but encodes a number of unique factors that allow it to replicate in the highly specific immune environment of skin. Phylogenetic analysis suggests that both viral interleukin-10 and vascular endothelial growth factor genes have been “captured” from their host during the evolution of the parapoxviruses. Genes such as a chemokine binding protein and a protein that binds granulocyte-macrophage colony-stimulating factor and interleukin-2 appear to have evolved from a common poxvirus ancestral gene while three parapoxvirus nuclear factor (NF)-κB signalling pathway inhibitors have no homology to other known NF-κB inhibitors. A homologue of an anaphase-promoting complex subunit that is believed to manipulate the cell cycle and enhance viral DNA synthesis appears to be a specific adaptation for viral-replication in keratinocytes. The review focuses on the unique genes of orf virus, discusses their evolutionary origins and their role in allowing viral-replication in the skin epidermis. PMID:25807056

The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

PubMed

Mathew, Shomita S; Bridge, Eileen

2007-09-01

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

Yeast for virus research

PubMed Central

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

PubMed Central

Moser, Lindsey A.; Ramirez-Carvajal, Lisbeth; Puri, Vinita; Pauszek, Steven J.; Matthews, Krystal; Dilley, Kari A.; Mullan, Clancy; McGraw, Jennifer; Khayat, Michael; Beeri, Karen; Yee, Anthony; Dugan, Vivien; Heise, Mark T.; Frieman, Matthew B.; Rodriguez, Luis L.; Bernard, Kristen A.; Wentworth, David E.

2016-01-01

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. PMID:27822536

vConTACT: an iVirus tool to classify double-stranded DNA viruses that infect Archaea and Bacteria.

PubMed

Bolduc, Benjamin; Jang, Ho Bin; Doulcier, Guilhem; You, Zhi-Qiang; Roux, Simon; Sullivan, Matthew B

2017-01-01

Taxonomic classification of archaeal and bacterial viruses is challenging, yet also fundamental for developing a predictive understanding of microbial ecosystems. Recent identification of hundreds of thousands of new viral genomes and genome fragments, whose hosts remain unknown, requires a paradigm shift away from traditional classification approaches and towards the use of genomes for taxonomy. Here we revisited the use of genomes and their protein content as a means for developing a viral taxonomy for bacterial and archaeal viruses. A network-based analytic was evaluated and benchmarked against authority-accepted taxonomic assignments and found to be largely concordant. Exceptions were manually examined and found to represent areas of viral genome 'sequence space' that are under-sampled or prone to excessive genetic exchange. While both cases are poorly resolved by genome-based taxonomic approaches, the former will improve as viral sequence space is better sampled and the latter are uncommon. Finally, given the largely robust taxonomic capabilities of this approach, we sought to enable researchers to easily and systematically classify new viruses. Thus, we established a tool, vConTACT, as an app at iVirus, where it operates as a fast, highly scalable, user-friendly app within the free and powerful CyVerse cyberinfrastructure.

vConTACT: an iVirus tool to classify double-stranded DNA viruses that infect Archaea and Bacteria

PubMed Central

Doulcier, Guilhem; You, Zhi-Qiang; Roux, Simon

2017-01-01

Taxonomic classification of archaeal and bacterial viruses is challenging, yet also fundamental for developing a predictive understanding of microbial ecosystems. Recent identification of hundreds of thousands of new viral genomes and genome fragments, whose hosts remain unknown, requires a paradigm shift away from traditional classification approaches and towards the use of genomes for taxonomy. Here we revisited the use of genomes and their protein content as a means for developing a viral taxonomy for bacterial and archaeal viruses. A network-based analytic was evaluated and benchmarked against authority-accepted taxonomic assignments and found to be largely concordant. Exceptions were manually examined and found to represent areas of viral genome ‘sequence space’ that are under-sampled or prone to excessive genetic exchange. While both cases are poorly resolved by genome-based taxonomic approaches, the former will improve as viral sequence space is better sampled and the latter are uncommon. Finally, given the largely robust taxonomic capabilities of this approach, we sought to enable researchers to easily and systematically classify new viruses. Thus, we established a tool, vConTACT, as an app at iVirus, where it operates as a fast, highly scalable, user-friendly app within the free and powerful CyVerse cyberinfrastructure. PMID:28480138

vConTACT: an iVirus tool to classify double-stranded DNA viruses that infect Archaea and Bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolduc, Benjamin; Jang, Ho Bin; Doulcier, Guilhem

Taxonomic classification of archaeal and bacterial viruses is challenging, yet also fundamental for developing a predictive understanding of microbial ecosystems. Recent identification of hundreds of thousands of new viral genomes and genome fragments, whose hosts remain unknown, requires a paradigm shift away from traditional classification approaches and towards the use of genomes for taxonomy. Here we revisited the use of genomes and their protein content as a means for developing a viral taxonomy for bacterial and archaeal viruses. A network-based analytic was evaluated and benchmarked against authority-accepted taxonomic assignments and found to be largely concordant. Exceptions were manually examined andmore » found to represent areas of viral genome ‘sequence space’ that are under-sampled or prone to excessive genetic exchange. While both cases are poorly resolved by genome-based taxonomic approaches, the former will improve as viral sequence space is better sampled and the latter are uncommon. Finally, given the largely robust taxonomic capabilities of this approach, we sought to enable researchers to easily and systematically classify new viruses. Thus, we established a tool, vConTACT, as an app at iVirus, where it operates as a fast, highly scalable, user-friendly app within the free and powerful CyVerse cyberinfrastructure.« less

vConTACT: an iVirus tool to classify double-stranded DNA viruses that infect Archaea and Bacteria

DOE PAGES

Bolduc, Benjamin; Jang, Ho Bin; Doulcier, Guilhem; ...

2017-05-03

Taxonomic classification of archaeal and bacterial viruses is challenging, yet also fundamental for developing a predictive understanding of microbial ecosystems. Recent identification of hundreds of thousands of new viral genomes and genome fragments, whose hosts remain unknown, requires a paradigm shift away from traditional classification approaches and towards the use of genomes for taxonomy. Here we revisited the use of genomes and their protein content as a means for developing a viral taxonomy for bacterial and archaeal viruses. A network-based analytic was evaluated and benchmarked against authority-accepted taxonomic assignments and found to be largely concordant. Exceptions were manually examined andmore » found to represent areas of viral genome ‘sequence space’ that are under-sampled or prone to excessive genetic exchange. While both cases are poorly resolved by genome-based taxonomic approaches, the former will improve as viral sequence space is better sampled and the latter are uncommon. Finally, given the largely robust taxonomic capabilities of this approach, we sought to enable researchers to easily and systematically classify new viruses. Thus, we established a tool, vConTACT, as an app at iVirus, where it operates as a fast, highly scalable, user-friendly app within the free and powerful CyVerse cyberinfrastructure.« less

Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong Hongping; Zhang Bo; Shi Peiyong

2008-11-10

Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5'-terminal 190 nucleotides and the 3'-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5'CS/3'CSI and 5'UAR/3'UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3'-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5more » binds specifically to the 5'-terminal stem-loop (SL1) of the genomic RNA. The 5'SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5'SL1 RNA interaction facilitate NS5 binding to the 3' end of the genome for the initiation of viral minus-strand RNA synthesis.« less

Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections

PubMed Central

Schiffer, Joshua T.; Aubert, Martine; Weber, Nicholas D.; Mintzer, Esther; Stone, Daniel

2012-01-01

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches. PMID:22718830

Nucleocapsid protein-dependent assembly of the RNA packaging signal of Middle East respiratory syndrome coronavirus.

PubMed

Hsin, Wei-Chen; Chang, Chan-Hua; Chang, Chi-You; Peng, Wei-Hao; Chien, Chung-Liang; Chang, Ming-Fu; Chang, Shin C

2018-05-24

Middle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome. A culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712-19969) ME ), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712-19969) ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome. A MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712-19969) ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.

PubMed

Vashee, Sanjay; Stockwell, Timothy B; Alperovich, Nina; Denisova, Evgeniya A; Gibson, Daniel G; Cady, Kyle C; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B; Voorhies, Alexander A; Bruening, Eric; Caposio, Patrizia; Früh, Klaus

2017-01-01

Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae . Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae . Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

PubMed Central

Vashee, Sanjay; Stockwell, Timothy B.; Alperovich, Nina; Denisova, Evgeniya A.; Gibson, Daniel G.; Cady, Kyle C.; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B.; Voorhies, Alexander A.; Bruening, Eric; Caposio, Patrizia

2017-01-01

ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes. PMID:28989973

Switch from translation to RNA replication in a positive-stranded RNA virus

PubMed Central

Gamarnik, Andrea V.; Andino, Raul

1998-01-01

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication. PMID:9694795

Sequencing and annotation of mitochondrial genomes from individual parasitic helminths.

PubMed

Jex, Aaron R; Littlewood, D Timothy; Gasser, Robin B

2015-01-01

Mitochondrial (mt) genomics has significant implications in a range of fundamental areas of parasitology, including evolution, systematics, and population genetics as well as explorations of mt biochemistry, physiology, and function. Mt genomes also provide a rich source of markers to aid molecular epidemiological and ecological studies of key parasites. However, there is still a paucity of information on mt genomes for many metazoan organisms, particularly parasitic helminths, which has often related to challenges linked to sequencing from tiny amounts of material. The advent of next-generation sequencing (NGS) technologies has paved the way for low cost, high-throughput mt genomic research, but there have been obstacles, particularly in relation to post-sequencing assembly and analyses of large datasets. In this chapter, we describe protocols for the efficient amplification and sequencing of mt genomes from small portions of individual helminths, and highlight the utility of NGS platforms to expedite mt genomics. In addition, we recommend approaches for manual or semi-automated bioinformatic annotation and analyses to overcome the bioinformatic "bottleneck" to research in this area. Taken together, these approaches have demonstrated applicability to a range of parasites and provide prospects for using complete mt genomic sequence datasets for large-scale molecular systematic and epidemiological studies. In addition, these methods have broader utility and might be readily adapted to a range of other medium-sized molecular regions (i.e., 10-100 kb), including large genomic operons, and other organellar (e.g., plastid) and viral genomes.

The contrasting phylodynamics of human influenza B viruses.

PubMed

Vijaykrishna, Dhanasekaran; Holmes, Edward C; Joseph, Udayan; Fourment, Mathieu; Su, Yvonne C F; Halpin, Rebecca; Lee, Raphael T C; Deng, Yi-Mo; Gunalan, Vithiagaran; Lin, Xudong; Stockwell, Timothy B; Fedorova, Nadia B; Zhou, Bin; Spirason, Natalie; Kühnert, Denise; Bošková, Veronika; Stadler, Tanja; Costa, Anna-Maria; Dwyer, Dominic E; Huang, Q Sue; Jennings, Lance C; Rawlinson, William; Sullivan, Sheena G; Hurt, Aeron C; Maurer-Stroh, Sebastian; Wentworth, David E; Smith, Gavin J D; Barr, Ian G

2015-01-16

A complex interplay of viral, host, and ecological factors shapes the spatio-temporal incidence and evolution of human influenza viruses. Although considerable attention has been paid to influenza A viruses, a lack of equivalent data means that an integrated evolutionary and epidemiological framework has until now not been available for influenza B viruses, despite their significant disease burden. Through the analysis of over 900 full genomes from an epidemiological collection of more than 26,000 strains from Australia and New Zealand, we reveal fundamental differences in the phylodynamics of the two co-circulating lineages of influenza B virus (Victoria and Yamagata), showing that their individual dynamics are determined by a complex relationship between virus transmission, age of infection, and receptor binding preference. In sum, this work identifies new factors that are important determinants of influenza B evolution and epidemiology.

Integrated analyses using RNA-Seq data reveal viral genomes, single nucleotide variations, the phylogenetic relationship, and recombination for Apple stem grooving virus.

PubMed

Jo, Yeonhwa; Choi, Hoseong; Kim, Sang-Min; Kim, Sun-Lim; Lee, Bong Choon; Cho, Won Kyong

2016-08-09

Next-generation sequencing (NGS) provides many possibilities for plant virology research. In this study, we performed integrated analyses using plant transcriptome data for plant virus identification using Apple stem grooving virus (ASGV) as an exemplar virus. We used 15 publicly available transcriptome libraries from three different studies, two mRNA-Seq studies and a small RNA-Seq study. We de novo assembled nearly complete genomes of ASGV isolates Fuji and Cuiguan from apple and pear transcriptomes, respectively, and identified single nucleotide variations (SNVs) of ASGV within the transcriptomes. We demonstrated the application of NGS raw data to confirm viral infections in the plant transcriptomes. In addition, we compared the usability of two de novo assemblers, Trinity and Velvet, for virus identification and genome assembly. A phylogenetic tree revealed that ASGV and Citrus tatter leaf virus (CTLV) are the same virus, which was divided into two clades. Recombination analyses identified six recombination events from 21 viral genomes. Taken together, our in silico analyses using NGS data provide a successful application of plant transcriptomes to reveal extensive information associated with viral genome assembly, SNVs, phylogenetic relationships, and genetic recombination.

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

PubMed

Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

2017-06-13

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription elongation factors to viral genomes and that in the absence of ICP22 viral transcription is globally reduced late in productive infection, due to an elongation defect. This insight defines a fundamental role of ICP22 in HSV-1 infection and elucidates the involvement of cellular factors in HSV-1 transcription. Copyright © 2017 Fox et al.

Origin of porcine circovirus type 2 (PCV2) from swine affected by PCV2-associated diseases in Croatia.

PubMed

Novosel, D; Tuboly, T; Csagola, A; Lorincz, M; Cubric-Curik, V; Jungic, A; Curik, I; Segalés, J; Cortey, M; Lipej, Z

2014-04-26

Porcine circovirus type 2 (PCV2) causes some of the most significant economic losses in pig production. Several multisystemic syndromes have been attributed to PCV2 infection, which are known as PCV2-associated diseases (PCVDs). This study investigated the origin and evolution of PCV2 sequences in domestic pigs and wild boars affected by PCVDs in Croatia. Viral sequences were recovered from three wild boars diagnosed with PCV2-systemic disease (PCV2-SD), 63 fetuses positive for PCV2 DNA as determined by PCR, 14 domestic pigs affected with PCV2-SD (displaying severe interstitial nephritis) and five domestic pigs with proliferative and necrotising pneumonia. Seventeen complete PCV2 genomes were recovered. Phylogenetic and evolutionary analyses based on median-joining phylogenetic networks, amino acid alignments and principal coordinate analysis were performed using complete genomes, as well as complete and partial ORF sequences for ORF1 and ORF2. Two of the 17 PCV2 sequences belonged to PCV2a, 14 to PCV2b and one was unclustered. PCV2b was the predominant genotype in Croatia and has been linked to international trade as a route of introduction. Correlation between particular viral strains with PCVDs is lacking.

Transstadial Transmission and Long-term Association of Crimean-Congo Hemorrhagic Fever Virus in Ticks Shapes Genome Plasticity

PubMed Central

Xia, Han; Beck, Andrew S.; Gargili, Aysen; Forrester, Naomi; Barrett, Alan D. T.; Bente, Dennis A.

2016-01-01

The trade-off hypothesis, the current paradigm of arbovirus evolution, proposes that cycling between vertebrate and invertebrate hosts presents significant constraints on genetic change of arboviruses. Studying these constraints in mosquito-borne viruses has led to a new understanding of epizootics. The trade-off hypothesis is assumed to be applicable to tick-borne viruses too, although studies are lacking. Tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the family Bunyaviridae, is a major cause of severe human disease worldwide and shows an extraordinary amount of genetic diversity compared to other arboviruses, which has been linked to increased virulence and emergence in new environments. Using a transmission model for CCHFV, utilizing the main vector tick species and mice plus next generation sequencing, we detected a substantial number of consensus-level mutations in CCHFV recovered from ticks after only a single transstadial transmission, whereas none were detected in CCHFV obtained from the mammalian host. Furthermore, greater viral intra-host diversity was detected in the tick compared to the vertebrate host. Long-term association of CCHFV with its tick host for 1 year demonstrated mutations in the viral genome become fixed over time. These findings suggest that the trade-off hypothesis may not be accurate for all arboviruses. PMID:27775001

Transstadial Transmission and Long-term Association of Crimean-Congo Hemorrhagic Fever Virus in Ticks Shapes Genome Plasticity.

PubMed

Xia, Han; Beck, Andrew S; Gargili, Aysen; Forrester, Naomi; Barrett, Alan D T; Bente, Dennis A

2016-10-24

The trade-off hypothesis, the current paradigm of arbovirus evolution, proposes that cycling between vertebrate and invertebrate hosts presents significant constraints on genetic change of arboviruses. Studying these constraints in mosquito-borne viruses has led to a new understanding of epizootics. The trade-off hypothesis is assumed to be applicable to tick-borne viruses too, although studies are lacking. Tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the family Bunyaviridae, is a major cause of severe human disease worldwide and shows an extraordinary amount of genetic diversity compared to other arboviruses, which has been linked to increased virulence and emergence in new environments. Using a transmission model for CCHFV, utilizing the main vector tick species and mice plus next generation sequencing, we detected a substantial number of consensus-level mutations in CCHFV recovered from ticks after only a single transstadial transmission, whereas none were detected in CCHFV obtained from the mammalian host. Furthermore, greater viral intra-host diversity was detected in the tick compared to the vertebrate host. Long-term association of CCHFV with its tick host for 1 year demonstrated mutations in the viral genome become fixed over time. These findings suggest that the trade-off hypothesis may not be accurate for all arboviruses.

The transmission dynamics and diversity of human metapneumovirus in Peru.

PubMed

Pollett, Simon; Trovão, Nidia S; Tan, Yi; Eden, John-Sebastian; Halpin, Rebecca A; Bera, Jayati; Das, Suman R; Wentworth, David; Ocaña, Victor; Mendocilla, Silvia M; Álvarez, Carlos; Calisto, Maria E; Garcia, Josefina; Halsey, Eric; Ampuero, Julia S; Nelson, Martha I; Leguia, Mariana

2017-12-29

The transmission dynamics of human metapneumovirus (HMPV) in tropical countries remain unclear. Further understanding of the genetic diversity of the virus could aid in HMPV vaccine design and improve our understanding of respiratory virus transmission dynamics in low- and middle-income countries. We examined the evolution of HMPV in Peru through phylogenetic analysis of 61 full genome HMPV sequences collected in three ecologically diverse regions of Peru (Lima, Piura, and Iquitos) during 2008-2012, comprising the largest data set of HMPV whole genomes sequenced from any tropical country to date. We revealed extensive genetic diversity generated by frequent viral introductions, with little evidence of local persistence. While considerable viral traffic between non-Peruvian countries and Peru was observed, HMPV epidemics in Peruvian locales were more frequently epidemiologically linked with other sites within Peru. We showed that Iquitos experienced greater HMPV traffic than the similar sized city of Piura by both Bayesian and maximum likelihood methods. There is extensive HMPV genetic diversity even within smaller and relatively less connected cities of Peru and this virus is spatially fluid. Greater diversity of HMPV in Iquitos compared to Piura may relate to higher volumes of human movement, including air traffic to this location. © 2017 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda.

PubMed

Yang, Qin; Maluf, Nasib Karl; Catalano, Carlos Enrique

2008-11-28

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. "Decoration" proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to approximately 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage lambda as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage lambda capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to "idle" at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-gammaS only partially stabilizes the nucleocapsid, and a DNA is released in "quantized" steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to accommodate the packaging of large DNA substrates; (ii) the gpD decoration protein is required to stabilize the fully expanded capsid; and (iii) nucleotides regulate high-affinity DNA binding interactions that are required to maintain DNA in the packaged state.

Towards optimized methods to study viral impacts on soil microbial carbon cycling

NASA Astrophysics Data System (ADS)

Trubl, G. G.; Roux, S.; Jang, H. B.; Solonenko, N.; Sullivan, M. B.; Rich, V. I.

2016-12-01

Permafrost contains 50% of global soil carbon and is rapidly thawing. While the fate of this carbon is currently unknown, it will undoubtedly be shaped by microbes and their associated viruses, which modulate host activities via mortality and metabolic control. However, little is known about soil viruses generally and their impact on terrestrial biogeochemistry; this is partially due to the presence of inhibitory substances (e.g. humic acids) in soils that interfere with sample processing and sequence-based metagenomics surveys. To address this problem, we examined viral populations in three different peat soils along a permafrost thaw gradient. These samples yielded low viral DNA recoveries, and shallow metagenomic sequencing, but still resulted in the recovery of 40 viral genome fragments. Genome- and network-based classification suggested that these new references represented 11 viral clusters, and ecological patterns (based upon non-redundant fragment recruitment) showed that viral populations were distinct in each habitat. Although only 31% of the genes could be functionally classified, pairwise genome comparisons classified 63% of the viruses taxonomically. Additionally, comparison of the 40 viral genome fragments to 53 previously recovered fragments from the same site showed no overlap, suggesting only a small portion of the resident viral community has been sampled. A follow-up experiment was performed to remove more humics during extraction and thereby obtain better viral metagenomes. Three DNA extraction protocols were tested (CTAB, PowerSoil, and Wizard columns) and the DNA was further purified with an AMPure clean-up. The PowerSoil kit maximized DNA yield (3x CTAB and 6x Wizard), and yielded the purest DNA (based on NanoDrop 260:230 ratio). Given the important roles of viruses in biogeochemical cycles in better-studied systems, further research and humic-removal optimization on these thawing permafrost-associated viral communities is needed to clarify their involvement in carbon cycle feedbacks.

Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, C.-K.; Chen, W.-J., E-mail: wjchen@mail.cgu.edu.t; Department of Public Health and Parasitology, Chang Gung University, Kwei-San, Tao-Yuan 33332, Taiwan

2009-11-25

Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay onmore » the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.« less

Prevalence and Evolution of Core Photosystem II Genes in Marine Cyanobacterial Viruses and Their Hosts

PubMed Central

Lee, Jessica A; Thompson, Luke R; Bielawski, Joseph P

2006-01-01

Cyanophages (cyanobacterial viruses) are important agents of horizontal gene transfer among marine cyanobacteria, the numerically dominant photosynthetic organisms in the oceans. Some cyanophage genomes carry and express host-like photosynthesis genes, presumably to augment the host photosynthetic machinery during infection. To study the prevalence and evolutionary dynamics of this phenomenon, 33 cultured cyanophages of known family and host range and viral DNA from field samples were screened for the presence of two core photosystem reaction center genes, psbA and psbD. Combining this expanded dataset with published data for nine other cyanophages, we found that 88% of the phage genomes contain psbA, and 50% contain both psbA and psbD. The psbA gene was found in all myoviruses and Prochlorococcus podoviruses, but could not be amplified from Prochlorococcus siphoviruses or Synechococcus podoviruses. Nearly all of the phages that encoded both psbA and psbD had broad host ranges. We speculate that the presence or absence of psbA in a phage genome may be determined by the length of the latent period of infection. Whether it also carries psbD may reflect constraints on coupling of viral- and host-encoded PsbA–PsbD in the photosynthetic reaction center across divergent hosts. Phylogenetic clustering patterns of these genes from cultured phages suggest that whole genes have been transferred from host to phage in a discrete number of events over the course of evolution (four for psbA, and two for psbD), followed by horizontal and vertical transfer between cyanophages. Clustering patterns of psbA and psbD from Synechococcus cells were inconsistent with other molecular phylogenetic markers, suggesting genetic exchanges involving Synechococcus lineages. Signatures of intragenic recombination, detected within the cyanophage gene pool as well as between hosts and phages in both directions, support this hypothesis. The analysis of cyanophage psbA and psbD genes from field populations revealed significant sequence diversity, much of which is represented in our cultured isolates. Collectively, these findings show that photosynthesis genes are common in cyanophages and that significant genetic exchanges occur from host to phage, phage to host, and within the phage gene pool. This generates genetic diversity among the phage, which serves as a reservoir for their hosts, and in turn influences photosystem evolution. PMID:16802857

Reovirus Nonstructural Protein σNS Acts as an RNA-Stability Factor Promoting Viral Genome Replication.

PubMed

Zamora, Paula F; Hu, Liya; Knowlton, Jonathan J; Lahr, Roni M; Moreno, Rodolfo A; Berman, Andrea J; Prasad, B V Venkataram; Dermody, Terence S

2018-05-16

Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered that σNS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the Reoviridae family assort and replicate their genomes. Copyright © 2018 American Society for Microbiology.

Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures

PubMed Central

Pride, David T; Schoenfeld, Thomas

2008-01-01

Background Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC), where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. Results From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of the Octopus and Bear Paw metagenomic contigs are predicted to belong to viruses rather than to any Bacteria or Archaea, consistent with the apparent viral origin of both metagenomes. Conclusion That BLAST searches identify no significant homologs for most metagenome contigs, while GSPC suggests their origin as archaeal viruses or bacteriophages, indicates GSPC provides a complementary approach in viral metagenomic analysis. PMID:18798991

Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures.

PubMed

Pride, David T; Schoenfeld, Thomas

2008-09-17

Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC), where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of the Octopus and Bear Paw metagenomic contigs are predicted to belong to viruses rather than to any Bacteria or Archaea, consistent with the apparent viral origin of both metagenomes. That BLAST searches identify no significant homologs for most metagenome contigs, while GSPC suggests their origin as archaeal viruses or bacteriophages, indicates GSPC provides a complementary approach in viral metagenomic analysis.

Evolution and molecular epidemiology of classical swine fever virus during a multi-annual outbreak amongst European wild boar.

PubMed

Goller, Katja V; Gabriel, Claudia; Dimna, Mireille Le; Le Potier, Marie-Frédérique; Rossi, Sophie; Staubach, Christoph; Merboth, Matthias; Beer, Martin; Blome, Sandra

2016-03-01

Classical swine fever is a viral disease of pigs that carries tremendous socio-economic impact. In outbreak situations, genetic typing is carried out for the purpose of molecular epidemiology in both domestic pigs and wild boar. These analyses are usually based on harmonized partial sequences. However, for high-resolution analyses towards the understanding of genetic variability and virus evolution, full-genome sequences are more appropriate. In this study, a unique set of representative virus strains was investigated that was collected during an outbreak in French free-ranging wild boar in the Vosges-du-Nord mountains between 2003 and 2007. Comparative sequence and evolutionary analyses of the nearly full-length sequences showed only slow evolution of classical swine fever virus strains over the years and no impact of vaccination on mutation rates. However, substitution rates varied amongst protein genes; furthermore, a spatial and temporal pattern could be observed whereby two separate clusters were formed that coincided with physical barriers.

Metagenomic Sequencing of an Echovirus 30 Genome From Cerebrospinal Fluid of a Patient With Aseptic Meningitis and Orchitis.

PubMed

Piantadosi, Anne; Mukerji, Shibani S; Chitneni, Pooja; Cho, Tracey A; Cosimi, Lisa A; Hung, Deborah T; Goldberg, Marcia B; Sabeti, Pardis C; Kuritzkes, Daniel R; Grad, Yonatan H

2017-01-01

Enteroviruses cause a wide spectrum of clinical disease. In this study, we describe the case of a young man with orchitis and aseptic meningitis who was diagnosed with enterovirus infection. Using unbiased "metagenomic" massively parallel sequencing, we assembled a near-complete viral genome, the first use of this method for full-genome viral sequencing from cerebrospinal fluid. We found that the genome belonged to the subgroup echovirus 30, which is a common cause of aseptic meningitis but has not been previously reported to cause orchitis.

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

PubMed Central

2016-01-01

SUMMARY The HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle. PMID:27357278

Current Advances on Virus Discovery and Diagnostic Role of Viral Metagenomics in Aquatic Organisms

PubMed Central

Munang'andu, Hetron M.; Mugimba, Kizito K.; Byarugaba, Denis K.; Mutoloki, Stephen; Evensen, Øystein

2017-01-01

The global expansion of the aquaculture industry has brought with it a corresponding increase of novel viruses infecting different aquatic organisms. These emerging viral pathogens have proved to be a challenge to the use of traditional cell-cultures and immunoassays for identification of new viruses especially in situations where the novel viruses are unculturable and no antibodies exist for their identification. Viral metagenomics has the potential to identify novel viruses without prior knowledge of their genomic sequence data and may provide a solution for the study of unculturable viruses. This review provides a synopsis on the contribution of viral metagenomics to the discovery of viruses infecting different aquatic organisms as well as its potential role in viral diagnostics. High throughput Next Generation sequencing (NGS) and library construction used in metagenomic projects have simplified the task of generating complete viral genomes unlike the challenge faced in traditional methods that use multiple primers targeted at different segments and VPs to generate the entire genome of a novel virus. In terms of diagnostics, studies carried out this far show that viral metagenomics has the potential to serve as a multifaceted tool able to study and identify etiological agents of single infections, co-infections, tissue tropism, profiling viral infections of different aquatic organisms, epidemiological monitoring of disease prevalence, evolutionary phylogenetic analyses, and the study of genomic diversity in quasispecies viruses. With sequencing technologies and bioinformatics analytical tools becoming cheaper and easier, we anticipate that metagenomics will soon become a routine tool for the discovery, study, and identification of novel pathogens including viruses to enable timely disease control for emerging diseases in aquaculture. PMID:28382024

Hepatitis A virus-encoded miRNAs attenuate the accumulation of viral genomic RNAs in infected cells.

PubMed

Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Hu, Yunzhang

2016-06-01

The establishment of persistent infection with hepatitis A virus (HAV) is the common result of most HAV/cell culture systems. Previous observations show that the synthesis of viral RNAs is reduced during infection. However, the underlying mechanism is poorly understood. We characterized three HAV-encoded miRNAs in our previous study. In this study, we aim to investigate the impact of these miRNAs on the accumulation of viral RNAs. The results indicated that the synthesis of viral genomic RNAs was dramatically reduced (more than 75 % reduction, P < 0.05) when transfected with one or two viral miRNA mimics. Conversely, they were significantly increased (more than 3.3-fold addition, P < 0.05) when transfected with one or two viral miRNA inhibitors. The luciferase reporter assay of miRNA targets showed that viral miRNAs were fully complementary to specific sites of the viral plus or minus strand RNA and strongly inhibited their expressions. Further data showed that the relative abundance of viral genomic RNA fragments that contain miRNA targets was also dramatically reduced (more than 80 % reduction, P < 0.05) when viral miRNAs were overexpressed with miRNA mimics. In contrast, they were significantly increased (approximately 2-fold addition, P < 0.05) when viral miRNAs were inhibited with miRNA inhibitors. In conclusion, these data suggest a possible mechanism for the reduction of viral RNA synthesis during HAV infection. Thus, we propose that it is likely that RNA virus-derived miRNA could serve as a self-mediated feedback regulator during infection.

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India.

PubMed

Bondre, Vijay P; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N

2016-11-01

Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections.

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India

PubMed Central

Bondre, Vijay P.; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N.

2016-01-01

Background & objectives: Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Methods: Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Results: Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Interpretation & conclusions: Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections. PMID:28361829

Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins.

PubMed

Batra, Jyoti; Tripathi, Shashank; Kumar, Amrita; Katz, Jacqueline M; Cox, Nancy J; Lal, Renu B; Sambhara, Suryaprakash; Lal, Sunil K

2016-01-11

A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins.

Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins

PubMed Central

Batra, Jyoti; Tripathi, Shashank; Kumar, Amrita; Katz, Jacqueline M.; Cox, Nancy J.; Lal, Renu B.; Sambhara, Suryaprakash; Lal, Sunil K.

2016-01-01

A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins. PMID:26750153

The Fecal Viral Flora of Wild Rodents

PubMed Central

Phan, Tung G.; Kapusinszky, Beatrix; Wang, Chunlin; Rose, Robert K.; Lipton, Howard L.; Delwart, Eric L.

2011-01-01

The frequent interactions of rodents with humans make them a common source of zoonotic infections. To obtain an initial unbiased measure of the viral diversity in the enteric tract of wild rodents we sequenced partially purified, randomly amplified viral RNA and DNA in the feces of 105 wild rodents (mouse, vole, and rat) collected in California and Virginia. We identified in decreasing frequency sequences related to the mammalian viruses families Circoviridae, Picobirnaviridae, Picornaviridae, Astroviridae, Parvoviridae, Papillomaviridae, Adenoviridae, and Coronaviridae. Seventeen small circular DNA genomes containing one or two replicase genes distantly related to the Circoviridae representing several potentially new viral families were characterized. In the Picornaviridae family two new candidate genera as well as a close genetic relative of the human pathogen Aichi virus were characterized. Fragments of the first mouse sapelovirus and picobirnaviruses were identified and the first murine astrovirus genome was characterized. A mouse papillomavirus genome and fragments of a novel adenovirus and adenovirus-associated virus were also sequenced. The next largest fraction of the rodent fecal virome was related to insect viruses of the Densoviridae, Iridoviridae, Polydnaviridae, Dicistroviriade, Bromoviridae, and Virgaviridae families followed by plant virus-related sequences in the Nanoviridae, Geminiviridae, Phycodnaviridae, Secoviridae, Partitiviridae, Tymoviridae, Alphaflexiviridae, and Tombusviridae families reflecting the largely insect and plant rodent diet. Phylogenetic analyses of full and partial viral genomes therefore revealed many previously unreported viral species, genera, and families. The close genetic similarities noted between some rodent and human viruses might reflect past zoonoses. This study increases our understanding of the viral diversity in wild rodents and highlights the large number of still uncharacterized viruses in mammals. PMID:21909269

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain.

PubMed

Kosutic-Gulija, Tanja; Forcic, Dubravko; Santak, Maja; Ramljak, Ana; Mateljak-Lukacevic, Sanja; Mazuran, Renata

2008-07-10

The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous. We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo. L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

PubMed Central

Dougherty, W G; Semler, B L

1993-01-01

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

Transcriptional activity across the Epstein-Barr virus genome in Raji cells during latency and after induction of an abortive lytic cycle.

PubMed

Kirchner, E A; Bornkamm, G W; Polack, A

1991-10-01

We have studied the relative rate of transcription across the Epstein-Barr virus genome in the Burkitt's lymphoma cell line Raji by nuclear run-on analysis during latency and after induction of an abortive lytic cycle with 12-0-tetradecanoylphorbol 13-acetate (TPA) and 5-iodo-2'-deoxyuridine (IUdR). During latency the entire, or almost the entire, viral genome was found to be transcriptionally active to a low or intermediate extent, with some variation in activity along the genome. The fragment with the highest transcriptional activity was EcoRI J, which contains the genes encoding the small nuclear RNAs EBER1 and -2, transcribed predominantly by RNA polymerase III. An intermediate level of transcription was observed between positions 10 and 138 (kb), with areas of slightly higher activity on the large internal repeats and the left duplicated region (DL). The remaining part of the viral genome, between position 138 and the termini, and the termini and position 10 (kb) (with the exception of the EcoRI J fragment), showed very little transcriptional activity, except for the intermediately active regions carrying the righthand oriLyt (DR) and the terminal repeats. Upon induction of the viral genome with TPA and IUdR, the viral genome was transcriptionally active at a rate at least tenfold that seen during latency. Polymerases were not equally distributed along the genome after induction; the highest density was found in regions 48 to 58 kb, 82 to 84 kb, 102 to 104 kb, 118 to 122 kb and 142 to 145 kb of the viral genome. High transcriptional activity correlated with distinct transcription units in some cases, i.e. BamHI H1LF1 (DL), BamHI MLF1, BamHI ZLF1/BamHI RLF1 and BamHI X (thymidine kinase), but not in others (BamHI H2). Besides initiation of transcription, other regulatory processes such as stabilization and processing of primary transcripts may also contribute to regulation of virus gene expression. Addition of cycloheximide completely abolished the transcriptional activation of the genome mediated by TPA and IUdR.

Genome-wide diversity and selective pressure in the human rhinovirus

PubMed Central

Kistler, Amy L; Webster, Dale R; Rouskin, Silvi; Magrini, Vince; Credle, Joel J; Schnurr, David P; Boushey, Homer A; Mardis, Elaine R; Li, Hao; DeRisi, Joseph L

2007-01-01

Background The human rhinoviruses (HRV) are one of the most common and diverse respiratory pathogens of humans. Over 100 distinct HRV serotypes are known, yet only 6 genomes are available. Due to the paucity of HRV genome sequence, little is known about the genetic diversity within HRV or the forces driving this diversity. Previous comparative genome sequence analyses indicate that recombination drives diversification in multiple genera of the picornavirus family, yet it remains unclear if this holds for HRV. Results To resolve this and gain insight into the forces driving diversification in HRV, we generated a representative set of 34 fully sequenced HRVs. Analysis of these genomes shows consistent phylogenies across the genome, conserved non-coding elements, and only limited recombination. However, spikes of genetic diversity at both the nucleotide and amino acid level are detectable within every locus of the genome. Despite this, the HRV genome as a whole is under purifying selective pressure, with islands of diversifying pressure in the VP1, VP2, and VP3 structural genes and two non-structural genes, the 3C protease and 3D polymerase. Mapping diversifying residues in these factors onto available 3-dimensional structures revealed the diversifying capsid residues partition to the external surface of the viral particle in statistically significant proximity to antigenic sites. Diversifying pressure in the pleconaril binding site is confined to a single residue known to confer drug resistance (VP1 191). In contrast, diversifying pressure in the non-structural genes is less clear, mapping both nearby and beyond characterized functional domains of these factors. Conclusion This work provides a foundation for understanding HRV genetic diversity and insight into the underlying biology driving evolution in HRV. It expands our knowledge of the genome sequence space that HRV reference serotypes occupy and how the pattern of genetic diversity across HRV genomes differs from other picornaviruses. It also reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution. PMID:17477878

Evolution, functions, and mysteries of plant ARGONAUTE proteins.

PubMed

Zhang, Han; Xia, Rui; Meyers, Blake C; Walbot, Virginia

2015-10-01

ARGONAUTE (AGO) proteins bind small RNAs (sRNAs) to form RNA-induced silencing complexes for transcriptional and post-transcriptional gene silencing. Genomes of primitive plants encode only a few AGO proteins. The Arabidopsis thaliana genome encodes ten AGO proteins, designated AGO1 to AGO10. Most early studies focused on these ten proteins and their interacting sRNAs. AGOs in other flowering plant species have duplicated and diverged from this set, presumably corresponding to new, diverged or specific functions. Among these, the grass-specific AGO18 family has been discovered and implicated as playing important roles during plant reproduction and viral defense. This review covers our current knowledge about functions and features of AGO proteins in both eudicots and monocots and compares their similarities and differences. On the basis of these features, we propose a new nomenclature for some plant AGOs. Copyright © 2015 Elsevier Ltd. All rights reserved.

A genome-wide association study for the incidence of persistent bovine viral diarrhea virus infection in cattle

USDA-ARS?s Scientific Manuscript database

Bovine Viral Diarrhea Viruses (BVDV) comprises a diverse group of viruses that causes disease in cattle. BVDV may establish both, transient and persistent infections depending on the developmental stage of the animal at exposure. The objective was to determine if genomic regions harboring single nuc...

A genome-wide association study for the incidence of persistent bovine viral diarrhea virus infection in cattle

USDA-ARS?s Scientific Manuscript database

Bovine Viral Diarrhea Viruses (BVDV) comprise a diverse group of viruses that cause disease in cattle. BVDV may establish both, transient and persistent infections depending on the developmental stage of the animal at exposure. The objective was to determine if genomic regions harboring single nucle...

Comparison of genome size and synthesis of structural proteins of Hirame Rhabdovirus, infectious hematopoietic necrosis virus, and viral hemorrhagic Septicemia virus

USGS Publications Warehouse

Nishizawa, Toyohiko; Yoshimizu, Mamoru; Winton, James R.; Kimura, Takahisa

1991-01-01

Genomic RNA was extracted from purified virions of hirame rhabdovirus (HRV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV). The full-length RNA was analyzed using formaldehyde agarose gel electrophoresis followed by ethidium bromide staining. Compared with an internal RNA size standard, all three viral genomic RNAs appeared to have identical relative mobilities and were estimated to be approximately 10.7 kilobases in length or about 3.7 megadaltons in molecular mass. Structural protein synthesis of HRV, IHNV, and VHSV was studied using cell cultures treated with actinomycin D. At 2 h intervals, proteins were labeled with 35S-methionine, extracted, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The five structural proteins of each of the three viruses appeared in the following order : nucleoprotein (N), matrix protein 1 (M1), matrix protein 2 (M2), glycoprotein (G), and polymerase (L) reflecting both the approximate relative abundance of each protein within infected cells and the gene order within the viral genome.

The Incubation Period of Primary Epstein-Barr Virus Infection: Viral Dynamics and Immunologic Events.

PubMed

Dunmire, Samantha K; Grimm, Jennifer M; Schmeling, David O; Balfour, Henry H; Hogquist, Kristin A

2015-12-01

Epstein-Barr virus (EBV) is a human herpesvirus that causes acute infectious mononucleosis and is associated with cancer and autoimmune disease. While many studies have been performed examining acute disease in adults following primary infection, little is known about the virological and immunological events during EBV's lengthy 6 week incubation period owing to the challenge of collecting samples from this stage of infection. We conducted a prospective study in college students with special emphasis on frequent screening to capture blood and oral wash samples during the incubation period. Here we describe the viral dissemination and immune response in the 6 weeks prior to onset of acute infectious mononucleosis symptoms. While virus is presumed to be present in the oral cavity from time of transmission, we did not detect viral genomes in the oral wash until one week before symptom onset, at which time viral genomes were present in high copy numbers, suggesting loss of initial viral replication control. In contrast, using a sensitive nested PCR method, we detected viral genomes at low levels in blood about 3 weeks before symptoms. However, high levels of EBV in the blood were only observed close to symptom onset-coincident with or just after increased viral detection in the oral cavity. These data imply that B cells are the major reservoir of virus in the oral cavity prior to infectious mononucleosis. The early presence of viral genomes in the blood, even at low levels, correlated with a striking decrease in the number of circulating plasmacytoid dendritic cells well before symptom onset, which remained depressed throughout convalescence. On the other hand, natural killer cells expanded only after symptom onset. Likewise, CD4+ Foxp3+ regulatory T cells decreased two fold, but only after symptom onset. We observed no substantial virus specific CD8 T cell expansion during the incubation period, although polyclonal CD8 activation was detected in concert with viral genomes increasing in the blood and oral cavity, possibly due to a systemic type I interferon response. This study provides the first description of events during the incubation period of natural EBV infection in humans and definitive data upon which to formulate theories of viral control and disease pathogenesis.

The Incubation Period of Primary Epstein-Barr Virus Infection: Viral Dynamics and Immunologic Events

PubMed Central

Dunmire, Samantha K.; Grimm, Jennifer M.; Schmeling, David O.; Balfour, Henry H.; Hogquist, Kristin A.

2015-01-01

Epstein-Barr virus (EBV) is a human herpesvirus that causes acute infectious mononucleosis and is associated with cancer and autoimmune disease. While many studies have been performed examining acute disease in adults following primary infection, little is known about the virological and immunological events during EBV’s lengthy 6 week incubation period owing to the challenge of collecting samples from this stage of infection. We conducted a prospective study in college students with special emphasis on frequent screening to capture blood and oral wash samples during the incubation period. Here we describe the viral dissemination and immune response in the 6 weeks prior to onset of acute infectious mononucleosis symptoms. While virus is presumed to be present in the oral cavity from time of transmission, we did not detect viral genomes in the oral wash until one week before symptom onset, at which time viral genomes were present in high copy numbers, suggesting loss of initial viral replication control. In contrast, using a sensitive nested PCR method, we detected viral genomes at low levels in blood about 3 weeks before symptoms. However, high levels of EBV in the blood were only observed close to symptom onset–coincident with or just after increased viral detection in the oral cavity. These data imply that B cells are the major reservoir of virus in the oral cavity prior to infectious mononucleosis. The early presence of viral genomes in the blood, even at low levels, correlated with a striking decrease in the number of circulating plasmacytoid dendritic cells well before symptom onset, which remained depressed throughout convalescence. On the other hand, natural killer cells expanded only after symptom onset. Likewise, CD4+ Foxp3+ regulatory T cells decreased two fold, but only after symptom onset. We observed no substantial virus specific CD8 T cell expansion during the incubation period, although polyclonal CD8 activation was detected in concert with viral genomes increasing in the blood and oral cavity, possibly due to a systemic type I interferon response. This study provides the first description of events during the incubation period of natural EBV infection in humans and definitive data upon which to formulate theories of viral control and disease pathogenesis. PMID:26624012

Characterization of HPV and host genome interactions in primary head and neck cancers.

PubMed

Parfenov, Michael; Pedamallu, Chandra Sekhar; Gehlenborg, Nils; Freeman, Samuel S; Danilova, Ludmila; Bristow, Christopher A; Lee, Semin; Hadjipanayis, Angela G; Ivanova, Elena V; Wilkerson, Matthew D; Protopopov, Alexei; Yang, Lixing; Seth, Sahil; Song, Xingzhi; Tang, Jiabin; Ren, Xiaojia; Zhang, Jianhua; Pantazi, Angeliki; Santoso, Netty; Xu, Andrew W; Mahadeshwar, Harshad; Wheeler, David A; Haddad, Robert I; Jung, Joonil; Ojesina, Akinyemi I; Issaeva, Natalia; Yarbrough, Wendell G; Hayes, D Neil; Grandis, Jennifer R; El-Naggar, Adel K; Meyerson, Matthew; Park, Peter J; Chin, Lynda; Seidman, J G; Hammerman, Peter S; Kucherlapati, Raju

2014-10-28

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Forces from the Portal Govern the Late-Stage DNA Transport in a Viral DNA Packaging Nanomotor.

PubMed

Jing, Peng; Burris, Benjamin; Zhang, Rong

2016-07-12

In the Phi29 bacteriophage, the DNA packaging nanomotor packs its double-stranded DNA genome into the virus capsid. At the late stage of DNA packaging, the negatively charged genome is increasingly compacted at a higher density in the capsid with a higher internal pressure. During the process, two Donnan effects, osmotic pressure and Donnan equilibrium potentials, are significantly amplified, which, in turn, affect the channel activity of the portal protein, GP10, embedded in the semipermeable capsid shell. In the research, planar lipid bilayer experiments were used to study the channel activities of the viral protein. The Donnan effect on the conformational changes of the viral protein was discovered, indicating GP10 may not be a static channel at the late stage of DNA packaging. Due to the conformational changes, GP10 may generate electrostatic forces that govern the DNA transport. For the section of the genome DNA that remains outside of the connector channel, a strong repulsive force from the viral protein would be generated against the DNA entry; however, for the section of the genome DNA within the channel, the portal protein would become a Brownian motor, which adopts the flash Brownian ratchet mechanism to pump the DNA against the increasingly built-up internal pressure (up to 20 atm) in the capsid. Therefore, the DNA transport in the nanoscale viral channel at the late stage of DNA packaging could be a consequence of Brownian movement of the genomic DNA, which would be rectified and harnessed by the forces from the interior wall of the viral channel under the influence of the Donnan effect. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome.

PubMed

Skraber, S; Gassilloud, B; Schwartzbrod, L; Gantzer, C

2004-07-01

The microbiological quality of water is currently assessed by search for fecal bacteria indicators. There is, however, a body of knowledge demonstrating that bacterial indicators are less resistant to environmental factors than human pathogenic viruses and therefore underestimate the viral risk. As river water is often used as a resource for drinking water production, it is particularly important to obtain a valid estimation of the health hazard, including specific viral risk. This work was conducted to compare the survival of infectious Poliovirus-1 used as a pathogenic virus model to the persistence of, on the one hand, thermotolerant coliforms commonly used as indicators and on the other hand, to somatic coliphages and Poliovirus-1 genome considered as potential indicators. We studied the behavior of infectious Poliovirus-1 and the three (potential) indicators of viral contamination in river water at three different temperatures (4 degrees C,18 degrees C and 25 degrees C). This experiment was performed twice with river water sampled at two different periods, once in winter and once in summer. Our results showed that the survival of thermotolerant coliforms can be 1.5-fold lower than infectious Poliovirus-1. In contrast, under all our experimental conditions, somatic coliphages and Poliovirus-1 genome persisted longer than infectious Poliovirus-1, surviving, respectively, 2-6-fold and about 2-fold longer than infectious Poliovirus-1. According to our results exclusively based on survival capacity, somatic coliphages and viral genome, unlike thermotolerant coliforms appear to be better indicators of viral contamination in river water. Moreover, the disappearance of viral genome is well-correlated to that one of infectious virus irrespective of the conditions tested.

Metavir 2: new tools for viral metagenome comparison and assembled virome analysis

PubMed Central

2014-01-01

Background Metagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome. Results To face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks. Conclusions The new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface. PMID:24646187

Dendritic transport of tick-borne flavivirus RNA by neuronal granules affects development of neurological disease.

PubMed

Hirano, Minato; Muto, Memi; Sakai, Mizuki; Kondo, Hirofumi; Kobayashi, Shintaro; Kariwa, Hiroaki; Yoshii, Kentaro

2017-09-12

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus , is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5' untranslated region of TBEV genomic RNA that act as a cis -acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease.

Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes

PubMed Central

Delwart, Eric; Li, Linlin

2011-01-01

The genomes of numerous circoviruses and distantly related circular DNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, and plants (geminivirus and nanovirus), human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting their past host range. An ancient origin for viruses with rep-encoding single stranded small circular genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular viral genomes. PMID:22155583

FIV cross-species transmission: an evolutionary prospective

PubMed Central

Troyer, Jennifer L.; VandeWoude, Sue; Pecon-Slattery, Jill; McIntosh, Carl; Franklin, Sam; Antunes, Agostinho; Johnson, Warren; O'Brien, Stephen J.

2008-01-01

Feline and primate immunodeficiency viruses (FIVs, SIVs, and HIV) are transmitted via direct contact (e.g. fighting, sexual contact, and mother-offspring transmission). This dynamic likely poses a behavioral barrier to cross-species transmission in the wild. Recently, several host intracellular anti-viral proteins that contribute to species-specificity of primate lentiviruses have been identified revealing adaptive mechanisms that further limit spread of lentiviruses between species. Consistent with these inter-species transmission barriers, phylogenetic evidence supports the prediction that FIV transmission is an exceedingly rare event between free-ranging cat species, though it has occurred occasionally in captive settings. Recently we documented that puma and bobcats in Southern California share an FIV strain, providing an opportunity to evaluate evolution of both viral strains and host intracellular restriction proteins. These studies are facilitated by the availability of the 2X cat genome sequence annotation. In addition, concurrent viral and host genetic analyses have been used to track patterns of migration of the host species and barriers to transmission of the virus within the African lion. These studies illustrate the utility of FIV as a model to discover the variables necessary for establishment and control of lentiviral infections in new species. PMID:18299153

Allexiviruses may have acquired inserted sequences between the CP and CRP genes to change the translation reinitiation strategy of CRP.

PubMed

Yoshida, Naoto; Shimura, Hanako; Masuta, Chikara

2018-06-01

Allexiviruses are economically important garlic viruses that are involved in garlic mosaic diseases. In this study, we characterized the allexivirus cysteine-rich protein (CRP) gene located just downstream of the coat protein (CP) gene in the viral genome. We determined the nucleotide sequences of the CP and CRP genes from numerous allexivirus isolates and performed a phylogenetic analysis. According to the resulting phylogenetic tree, we found that allexiviruses were clearly divided into two major groups (group I and group II) based on the sequences of the CP and CRP genes. In addition, the allexiviruses in group II had distinct sequences just before the CRP gene, while group I isolates did not. The inserted sequence between the CP and CRP genes was partially complementary to garlic 18S rRNA. Using a potato virus X vector, we showed that the CRPs affected viral accumulation and symptom induction in Nicotiana benthamiana, suggesting that the allexivirus CRP is a pathogenicity determinant. We assume that the inserted sequences before the CRP gene may have been generated during viral evolution to alter the termination-reinitiation mechanism for coupled translation of CP and CRP.

Sequential Bottlenecks Drive Viral Evolution in Early Acute Hepatitis C Virus Infection

PubMed Central

McElroy, Kerensa; Gaudieri, Silvana; Pham, Son T.; Chopra, Abha; Cameron, Barbara; Maher, Lisa; Dore, Gregory J.; White, Peter A.; Lloyd, Andrew R.

2011-01-01

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection. PMID:21912520

A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production.

PubMed

Geisler, Christoph

2018-02-07

Adventitious viral contamination in cell substrates used for biologicals production is a major safety concern. A powerful new approach that can be used to identify adventitious viruses is a combination of bioinformatics tools with massively parallel sequencing technology. Typically, this involves mapping or BLASTN searching individual reads against viral nucleotide databases. Although extremely sensitive for known viruses, this approach can easily miss viruses that are too dissimilar to viruses in the database. Moreover, it is computationally intensive and requires reference cell genome databases. To avoid these drawbacks, we set out to develop an alternative approach. We reasoned that searching genome and transcriptome assemblies for adventitious viral contaminants using TBLASTN with a compact viral protein database covering extant viral diversity as the query could be fast and sensitive without a requirement for high performance computing hardware. We tested our approach on Spodoptera frugiperda Sf-RVN, a recently isolated insect cell line, to determine if it was contaminated with one or more adventitious viruses. We used Illumina reads to assemble the Sf-RVN genome and transcriptome and searched them for adventitious viral contaminants using TBLASTN with our viral protein database. We found no evidence of viral contamination, which was substantiated by the fact that our searches otherwise identified diverse sequences encoding virus-like proteins. These sequences included Maverick, R1 LINE, and errantivirus transposons, all of which are common in insect genomes. We also identified previously described as well as novel endogenous viral elements similar to ORFs encoded by diverse insect viruses. Our results demonstrate TBLASTN searching massively parallel sequencing (MPS) assemblies with a compact, manually curated viral protein database is more sensitive for adventitious virus detection than BLASTN, as we identified various sequences that encoded virus-like proteins, but had no similarity to viral sequences at the nucleotide level. Moreover, searches were fast without requiring high performance computing hardware. Our study also documents the enhanced biosafety profile of Sf-RVN as compared to other Sf cell lines, and supports the notion that Sf-RVN is highly suitable for the production of safe biologicals.

Myocarditis in children and detection of viruses in myocardial tissue: implications for immunosuppressive therapy.

PubMed

Camargo, Paulo Roberto; Okay, Thelma Suely; Yamamoto, Lídia; Del Negro, Gilda Maria Bárbaro; Lopes, Antonio Augusto

2011-04-14

There is scarce information on the potential benefits of immunosuppression in children with myocarditis and viral genomes in myocardium. We investigated the occurrence of myocarditis in children with a preliminary diagnosis of dilated cardiomyopathy, the frequency of cardiotropic viruses in the myocardium, and the response to immunosuppression. Thirty patients (nine months to 12 years) with left ventricular ejection fraction of 22.8 ± 4.1% were subjected to right cardiac catheterization and endomyocardial biopsy. Specimens were analyzed for the presence of inflammatory elements (Dallas criteria) and viral genome (polymerase chain reaction). Patients with active myocarditis received immunosuppressants (azatioprine and prednisone) and were re-catheterized nine months later. A historical control group of nine patients with myocarditis who did not receive immunosuppressants was included. Active myocarditis was diagnosed in ten patients (five with viral genomes detected). Immunosuppression resulted in a significant increase in left ventricular ejection fraction from 25.2 ± 2.8% to 45.7 ± 8.6% (versus 20.0 ± 4.0% to 22.0 ± 9.0% in historical controls, p<0.01) and cardiac index from 3.28 ± 0.51 L/min/m(2) to 4.40 ± 0.49 L/min/m(2) (versus 3.50 ± 0.40 L/min/m(2) to 3.70 ± 0.50 L/min/m(2) in controls, p<0.01), regardless of the presence of viral genomes (p=0.98 and p=0.22, respectively for the two variables). No relevant clinical events were observed. Non-inflammatory cardiomyopathy was diagnosed in 20 patients (seven with viral genomes). While on conventional therapy, there were four deaths and three assignments to transplantation, and no improvement of left ventricular ejection fraction in the remaining ones (22.5 ± 3.6% to 27.5 ± 10.6%). Children with chronic myocarditis seem to benefit from immunosuppressive therapy, regardless of the presence of viral genome in the myocardium. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

The Complete Genomic Sequence of Pepper Yellow Leaf Curl Virus (PYLCV) and Its Implications for Our Understanding of Evolution Dynamics in the Genus Polerovirus

PubMed Central

Dombrovsky, Aviv; Glanz, Eyal; Lachman, Oded; Sela, Noa; Doron-Faigenboim, Adi; Antignus, Yehezkel

2013-01-01

We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range. PMID:23936244

The complete genomic sequence of pepper yellow leaf curl virus (PYLCV) and its implications for our understanding of evolution dynamics in the genus polerovirus.

PubMed

Dombrovsky, Aviv; Glanz, Eyal; Lachman, Oded; Sela, Noa; Doron-Faigenboim, Adi; Antignus, Yehezkel

2013-01-01

We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range.

Residual Viremia in Treated HIV+ Individuals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conway, Jessica M.; Perelson, Alan S.

Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads. However, some residual virus remains, below the level of detection, in HIV-infected patients on ART. Furthermore, the source of this viremia is an area of debate: does it derive primarily from activation of infected cells in the latent reservoir, or from ongoing viral replication? Our observations seem to be contradictory: there is evidence of short term evolution, implying that there must be ongoing viral replication, and viral strains should thus evolve. The phylogenetic analyses, and rare emergent drug resistance, suggest no long-term viral evolution, implying that virus derived frommore » activated latent cells must dominate. We use simple deterministic and stochastic models to gain insight into residual viremia dynamics in HIV-infected patients. Our modeling relies on two underlying assumptions for patients on suppressive ART: that latent cell activation drives viral dynamics and that the reproductive ratio of treated infection is less than 1. Nonetheless, the contribution of viral replication to residual viremia in patients on ART may be non-negligible. However, even if the portion of viremia attributable to viral replication is significant, our model predicts (1) that latent reservoir re-seeding remains negligible, and (2) some short-term viral evolution is permitted, but long-term evolution can still be limited: stochastic analysis of our model shows that de novo emergence of drug resistance is rare. Thus, our simple models reconcile the seemingly contradictory observations on residual viremia and, with relatively few parameters, recapitulates HIV viral dynamics observed in patients on suppressive therapy.« less

Residual Viremia in Treated HIV+ Individuals

DOE PAGES

Conway, Jessica M.; Perelson, Alan S.

2016-01-06

Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads. However, some residual virus remains, below the level of detection, in HIV-infected patients on ART. Furthermore, the source of this viremia is an area of debate: does it derive primarily from activation of infected cells in the latent reservoir, or from ongoing viral replication? Our observations seem to be contradictory: there is evidence of short term evolution, implying that there must be ongoing viral replication, and viral strains should thus evolve. The phylogenetic analyses, and rare emergent drug resistance, suggest no long-term viral evolution, implying that virus derived frommore » activated latent cells must dominate. We use simple deterministic and stochastic models to gain insight into residual viremia dynamics in HIV-infected patients. Our modeling relies on two underlying assumptions for patients on suppressive ART: that latent cell activation drives viral dynamics and that the reproductive ratio of treated infection is less than 1. Nonetheless, the contribution of viral replication to residual viremia in patients on ART may be non-negligible. However, even if the portion of viremia attributable to viral replication is significant, our model predicts (1) that latent reservoir re-seeding remains negligible, and (2) some short-term viral evolution is permitted, but long-term evolution can still be limited: stochastic analysis of our model shows that de novo emergence of drug resistance is rare. Thus, our simple models reconcile the seemingly contradictory observations on residual viremia and, with relatively few parameters, recapitulates HIV viral dynamics observed in patients on suppressive therapy.« less

Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus.

PubMed Central

Majima, K; Kobara, R; Maeda, S

1993-01-01

Homologous regions (hrs) (hr1,hr2-left,hr2-right,hr3,hr4-left,hr 4-right, and hr5) similar to those found in the Autographa californica nuclear polyhedrosis virus (AcNPV) genome were found in the Bombyx mori NPV (BmNPV) genome. The BmNPV hrs contained two to eight repeats of a homologous nucleotide sequence which were on average about 75 bp long. All of these homologous sequence repeats contained a 26-bp-long palindrome motif with an EcoRI or EcoRI-like site at its core. The consensus sequence of the BmNPV hrs showed 95% conservation with respect to those found in AcNPV. Nucleotide sequence analysis indicated that hr2-left and hr2-right of BmNPV evolved from an ancestor similar to hr2 of AcNPV by inversion, cleavage, and ligation. The polarities of the BmNPV and AcNPV hrs were conserved except for that of hr4-left. Within hr4-right of BmNPV, four repeats of a previously underscribed palindrome motif were found. Bmhr5D, a BmNPV mutant which lacked hr5, replicated at a rate similar to that of wild-type BmNPV in BmN cells and silkworm larvae, indicating that hr5 was not essential for viral replication. After ten passages of Bmhr5D in BmN cells, no detectable changes in its genome were observed by restriction endonuclease analysis. The evolution and divergence of the BmNPV genome are also discussed. Images PMID:8230471

Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses.

PubMed

Roux, Simon; Brum, Jennifer R; Dutilh, Bas E; Sunagawa, Shinichi; Duhaime, Melissa B; Loy, Alexander; Poulos, Bonnie T; Solonenko, Natalie; Lara, Elena; Poulain, Julie; Pesant, Stéphane; Kandels-Lewis, Stefanie; Dimier, Céline; Picheral, Marc; Searson, Sarah; Cruaud, Corinne; Alberti, Adriana; Duarte, Carlos M; Gasol, Josep M; Vaqué, Dolors; Bork, Peer; Acinas, Silvia G; Wincker, Patrick; Sullivan, Matthew B

2016-09-29

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting 'global ocean virome' dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses

NASA Astrophysics Data System (ADS)

2016-09-01

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting ‘global ocean virome’ dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Genomic differentiation among wild cyanophages despite widespread horizontal gene transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gregory, Ann C.; Solonenko, Sergei A.; Ignacio-Espinoza, J. Cesar

Genetic recombination is a driving force in genome evolution. Among viruses it has a dual role. For genomes with higher fitness, it maintains genome integrity in the face of high mutation rates. Conversely, for genomes with lower fitness, it provides immediate access to sequence space that cannot be reached by mutation alone. Understanding how recombination impacts the cohesion and dissolution of individual whole genomes within viral sequence space is poorly understood across double-stranded DNA bacteriophages (a.k.a phages) due to the challenges of obtaining appropriately scaled genomic datasets. Here in this study we explore the role of recombination in both maintainingmore » and differentiating whole genomes of 142 wild double-stranded DNA marine cyanophages. Phylogenomic analysis across the 51 core genes revealed ten lineages, six of which were well represented. These phylogenomic lineages represent discrete genotypic populations based on comparisons of intra- and inter- lineage shared gene content, genome-wide average nucleotide identity, as well as detected gaps in the distribution of pairwise differences between genomes. McDonald-Kreitman selection tests identified putative niche-differentiating genes under positive selection that differed across the six well-represented genotypic populations and that may have driven initial divergence. Concurrent with patterns of recombination of discrete populations, recombination analyses of both genic and intergenic regions largely revealed decreased genetic exchange across individual genomes between relative to within populations. Lastly, these findings suggest that discrete double-stranded DNA marine cyanophage populations occur in nature and are maintained by patterns of recombination akin to those observed in bacteria, archaea and in sexual eukaryotes.« less

Genomic differentiation among wild cyanophages despite widespread horizontal gene transfer

DOE PAGES

Gregory, Ann C.; Solonenko, Sergei A.; Ignacio-Espinoza, J. Cesar; ...

2016-11-16

Genetic recombination is a driving force in genome evolution. Among viruses it has a dual role. For genomes with higher fitness, it maintains genome integrity in the face of high mutation rates. Conversely, for genomes with lower fitness, it provides immediate access to sequence space that cannot be reached by mutation alone. Understanding how recombination impacts the cohesion and dissolution of individual whole genomes within viral sequence space is poorly understood across double-stranded DNA bacteriophages (a.k.a phages) due to the challenges of obtaining appropriately scaled genomic datasets. Here in this study we explore the role of recombination in both maintainingmore » and differentiating whole genomes of 142 wild double-stranded DNA marine cyanophages. Phylogenomic analysis across the 51 core genes revealed ten lineages, six of which were well represented. These phylogenomic lineages represent discrete genotypic populations based on comparisons of intra- and inter- lineage shared gene content, genome-wide average nucleotide identity, as well as detected gaps in the distribution of pairwise differences between genomes. McDonald-Kreitman selection tests identified putative niche-differentiating genes under positive selection that differed across the six well-represented genotypic populations and that may have driven initial divergence. Concurrent with patterns of recombination of discrete populations, recombination analyses of both genic and intergenic regions largely revealed decreased genetic exchange across individual genomes between relative to within populations. Lastly, these findings suggest that discrete double-stranded DNA marine cyanophage populations occur in nature and are maintained by patterns of recombination akin to those observed in bacteria, archaea and in sexual eukaryotes.« less

Transmission between Archaic and Modern Human Ancestors during the Evolution of the Oncogenic Human Papillomavirus 16.

PubMed

Pimenoff, Ville N; de Oliveira, Cristina Mendes; Bravo, Ignacio G

2017-01-01

Every human suffers through life a number of papillomaviruses (PVs) infections, most of them asymptomatic. A notable exception are persistent infections by Human papillomavirus 16 (HPV16), the most oncogenic infectious agent for humans and responsible for most infection-driven anogenital cancers. Oncogenic potential is not homogeneous among HPV16 lineages, and genetic variation within HPV16 exhibits some geographic structure. However, an in-depth analysis of the HPV16 evolutionary history was still wanting. We have analyzed extant HPV16 diversity and compared the evolutionary and phylogeographical patterns of humans and of HPV16. We show that codivergence with modern humans explains at most 30% of the present viral geographical distribution. The most explanatory scenario suggests that ancestral HPV16 already infected ancestral human populations and that viral lineages co-diverged with the hosts in parallel with the split between archaic Neanderthal-Denisovans and ancestral modern human populations, generating the ancestral HPV16A and HPV16BCD viral lineages, respectively. We propose that after out-of-Africa migration of modern human ancestors, sexual transmission between human populations introduced HPV16A into modern human ancestor populations. We hypothesize that differential coevolution of HPV16 lineages with different but closely related ancestral human populations and subsequent host-switch events in parallel with introgression of archaic alleles into the genomes of modern human ancestors may be largely responsible for the present-day differential prevalence and association with cancers for HPV16 variants. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Evasion of Short Interfering RNA-Directed Antiviral Silencing in Musa acuminata Persistently Infected with Six Distinct Banana Streak Pararetroviruses

PubMed Central

Rajeswaran, Rajendran; Seguin, Jonathan; Chabannes, Matthieu; Duroy, Pierre-Olivier; Laboureau, Nathalie; Farinelli, Laurent; Iskra-Caruana, Marie-Line

2014-01-01

ABSTRACT Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5′-terminal guanosine was identified. In all virus-infected plants, 21- to 24-nt viral siRNAs accumulated at relatively high levels (up to 22% of the total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within open reading frame (ORF) I and II and the 5′ portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistence of pararetroviruses in banana plants. IMPORTANCE We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21- to 24-nucleotide short interfering RNAs. At the same time, the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide a possible explanation why episomal pararetroviruses can persist in plants whereas true retroviruses with an obligatory genome-integration step in their replication cycle do not exist in plants. PMID:25056897

Evasion of short interfering RNA-directed antiviral silencing in Musa acuminata persistently infected with six distinct banana streak pararetroviruses.

PubMed

Rajeswaran, Rajendran; Seguin, Jonathan; Chabannes, Matthieu; Duroy, Pierre-Olivier; Laboureau, Nathalie; Farinelli, Laurent; Iskra-Caruana, Marie-Line; Pooggin, Mikhail M

2014-10-01

Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5'-terminal guanosine was identified. In all virus-infected plants, 21- to 24-nt viral siRNAs accumulated at relatively high levels (up to 22% of the total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within open reading frame (ORF) I and II and the 5' portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistence of pararetroviruses in banana plants. We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21- to 24-nucleotide short interfering RNAs. At the same time, the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide a possible explanation why episomal pararetroviruses can persist in plants whereas true retroviruses with an obligatory genome-integration step in their replication cycle do not exist in plants. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The E1 Protein of Human Papillomavirus Type 16 Is Dispensable for Maintenance Replication of the Viral Genome

PubMed Central

Egawa, Nagayasu; Nakahara, Tomomi; Ohno, Shin-ichi; Narisawa-Saito, Mako; Yugawa, Takashi; Fujita, Masatoshi; Yamato, Kenji; Natori, Yukikazu

2012-01-01

Papillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16. PMID:22238312

Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

PubMed Central

Karttunen, Heidi; Savas, Jeffrey N.; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

2015-01-01

SUMMARY DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments and FANCI-D2 interacted with a multi-subunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, while HSV1 productive growth was impaired in monoubiquitination-defective FA patient cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for non-homologous end-joining (NHEJ). This identifies the FA-pathway as a new cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral lifecycle. PMID:24954902

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

PubMed Central

Nakagawa, K.; Lokugamage, K.G.; Makino, S.

2017-01-01

Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

PCR Amplification Strategies towards full-length HIV-1 Genome sequencing.

PubMed

Liu, Chao Chun; Ji, Hezhao

2018-06-26

The advent of next generation sequencing has enabled greater resolution of viral diversity and improved feasibility of full viral genome sequencing allowing routine HIV-1 full genome sequencing in both research and diagnostic settings. Regardless of the sequencing platform selected, successful PCR amplification of the HIV-1 genome is essential for sequencing template preparation. As such, full HIV-1 genome amplification is a crucial step in dictating the successful and reliable sequencing downstream. Here we reviewed existing PCR protocols leading to HIV-1 full genome sequencing. In addition to the discussion on basic considerations on relevant PCR design, the advantages as well as the pitfalls of published protocols were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mumps vaccine virus genome is present in throat swabs obtained from uncomplicated healthy recipients.

PubMed

Nagai, T; Nakayama, T

2001-01-08

Seven children were followed for up to 42 days post-vaccination with live mumps vaccine and 37 throat swabs were obtained serially. Viral genomic RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in the phosphoprotein (P) and hemagglutinin-neuraminidase (HN) regions. Virus isolation was also attempted. Genomic differentiation of detected mumps virus genome was performed by sequence analysis and/or restriction fragment length polymorphism (RFLP). No adverse reaction was observed in these children. Although mumps virus was not isolated from any of the samples, viral RNA was detected in four samples from three vaccine recipients, 18, 18 and 26, and 7 days after vaccination, respectively. Detected viral RNA was identified as the vaccine strain. Our data suggests that vaccine virus inoculated replicates in the parotid glands but the incidence of virus transmission from recipients to other susceptible subjects should be low.

ViralEpi v1.0: a high-throughput spectrum of viral epigenomic methylation profiles from diverse diseases.

PubMed

Khan, Mohd Shoaib; Gupta, Amit Kumar; Kumar, Manoj

2016-01-01

To develop a computational resource for viral epigenomic methylation profiles from diverse diseases. Methylation patterns of Epstein-Barr virus and hepatitis B virus genomic regions are provided as web platform developed using open source Linux-Apache-MySQL-PHP (LAMP) bundle: programming and scripting languages, that is, HTML, JavaScript and PERL. A comprehensive and integrated web resource ViralEpi v1.0 is developed providing well-organized compendium of methylation events and statistical analysis associated with several diseases. Additionally, it also facilitates 'Viral EpiGenome Browser' for user-affable browsing experience using JavaScript-based JBrowse. This web resource would be helpful for research community engaged in studying epigenetic biomarkers for appropriate prognosis and diagnosis of diseases and its various stages.

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain

PubMed Central

Kosutic-Gulija, Tanja; Forcic, Dubravko; Šantak, Maja; Ramljak, Ana; Mateljak-Lukacevic, Sanja; Mazuran, Renata

2008-01-01

Background The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous. Results We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo. Conclusion L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes. PMID:18616793

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection

PubMed Central

Alandijany, Thamir; Conn, Kristen L.; McFarlane, Steven; Orr, Anne

2018-01-01

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2’-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication. PMID:29309427

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone.

PubMed

Tong, Yi-Gang; Shi, Wei-Feng; Liu, Di; Qian, Jun; Liang, Long; Bo, Xiao-Chen; Liu, Jun; Ren, Hong-Guang; Fan, Hang; Ni, Ming; Sun, Yang; Jin, Yuan; Teng, Yue; Li, Zhen; Kargbo, David; Dafae, Foday; Kanu, Alex; Chen, Cheng-Chao; Lan, Zhi-Heng; Jiang, Hui; Luo, Yang; Lu, Hui-Jun; Zhang, Xiao-Guang; Yang, Fan; Hu, Yi; Cao, Yu-Xi; Deng, Yong-Qiang; Su, Hao-Xiang; Sun, Yu; Liu, Wen-Sen; Wang, Zhuang; Wang, Cheng-Yu; Bu, Zhao-Yang; Guo, Zhen-Dong; Zhang, Liu-Bo; Nie, Wei-Min; Bai, Chang-Qing; Sun, Chun-Hua; An, Xiao-Ping; Xu, Pei-Song; Zhang, Xiang-Li-Lan; Huang, Yong; Mi, Zhi-Qiang; Yu, Dong; Yao, Hong-Wu; Feng, Yong; Xia, Zhi-Ping; Zheng, Xue-Xing; Yang, Song-Tao; Lu, Bing; Jiang, Jia-Fu; Kargbo, Brima; He, Fu-Chu; Gao, George F; Cao, Wu-Chun

2015-08-06

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

PubMed Central

Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

2011-01-01

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

Parvovirus-Derived Endogenous Viral Elements in Two South American Rodent Genomes

PubMed Central

2014-01-01

We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the genomes of the long-tailed chinchilla (Chinchilla lanigera) and the degu (Octodon degus). The novel EVEs include dependovirus-related elements and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed in vivo, with increased expression in the liver. PMID:25078696

First complete genome sequences of genogroup V, genotype 3 porcine sapoviruses: common 5'-terminal genomic feature of sapoviruses.

PubMed

Oka, Tomoichiro; Doan, Yen Hai; Shimoike, Takashi; Haga, Kei; Takizawa, Takenori

2017-12-01

Sapoviruses (SaVs) are enteric viruses and have been detected in various mammals. They are divided into multiple genogroups and genotypes based on the entire major capsid protein (VP1) encoding region sequences. In this study, we determined the first complete genome sequences of two genogroup V, genotype 3 (GV.3) SaV strains detected from swine fecal samples, in combination with Illumina MiSeq sequencing of the libraries prepared from viral RNA and PCR products. The lengths of the viral genome (7494 nucleotides [nt] excluding polyA tail) and short 5'-untranslated region (14 nt) as well as two predicted open reading frames are similar to those of other SaVs. The amino acid differences between the two porcine SaVs are most frequent in the central region of the VP1-encoding region. A stem-loop structure which was predicted in the first 41 nt of the 5'-terminal region of GV.3 SaVs and the other available complete genome sequences of SaVs may have a critical role in viral genome replication. Our study provides complete genome sequences of rarely reported GV.3 SaV strains and highlights the common 5'-terminal genomic feature of SaVs detected from different mammalian species.

Unifying viral genetics and human transportation data to predict the global transmission dynamics of human influenza H3N2.

PubMed

Lemey, Philippe; Rambaut, Andrew; Bedford, Trevor; Faria, Nuno; Bielejec, Filip; Baele, Guy; Russell, Colin A; Smith, Derek J; Pybus, Oliver G; Brockmann, Dirk; Suchard, Marc A

2014-02-01

Information on global human movement patterns is central to spatial epidemiological models used to predict the behavior of influenza and other infectious diseases. Yet it remains difficult to test which modes of dispersal drive pathogen spread at various geographic scales using standard epidemiological data alone. Evolutionary analyses of pathogen genome sequences increasingly provide insights into the spatial dynamics of influenza viruses, but to date they have largely neglected the wealth of information on human mobility, mainly because no statistical framework exists within which viral gene sequences and empirical data on host movement can be combined. Here, we address this problem by applying a phylogeographic approach to elucidate the global spread of human influenza subtype H3N2 and assess its ability to predict the spatial spread of human influenza A viruses worldwide. Using a framework that estimates the migration history of human influenza while simultaneously testing and quantifying a range of potential predictive variables of spatial spread, we show that the global dynamics of influenza H3N2 are driven by air passenger flows, whereas at more local scales spread is also determined by processes that correlate with geographic distance. Our analyses further confirm a central role for mainland China and Southeast Asia in maintaining a source population for global influenza diversity. By comparing model output with the known pandemic expansion of H1N1 during 2009, we demonstrate that predictions of influenza spatial spread are most accurate when data on human mobility and viral evolution are integrated. In conclusion, the global dynamics of influenza viruses are best explained by combining human mobility data with the spatial information inherent in sampled viral genomes. The integrated approach introduced here offers great potential for epidemiological surveillance through phylogeographic reconstructions and for improving predictive models of disease control.

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

PubMed

Nougairede, Antoine; De Fabritus, Lauriane; Aubry, Fabien; Gould, Ernest A; Holmes, Edward C; de Lamballerie, Xavier

2013-02-01

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

The contrasting phylodynamics of human influenza B viruses

PubMed Central

Vijaykrishna, Dhanasekaran; Holmes, Edward C; Joseph, Udayan; Fourment, Mathieu; Su, Yvonne CF; Halpin, Rebecca; Lee, Raphael TC; Deng, Yi-Mo; Gunalan, Vithiagaran; Lin, Xudong; Stockwell, Timothy B; Fedorova, Nadia B; Zhou, Bin; Spirason, Natalie; Kühnert, Denise; Bošková, Veronika; Stadler, Tanja; Costa, Anna-Maria; Dwyer, Dominic E; Huang, Q Sue; Jennings, Lance C; Rawlinson, William; Sullivan, Sheena G; Hurt, Aeron C; Maurer-Stroh, Sebastian; Wentworth, David E; Smith, Gavin JD; Barr, Ian G

2015-01-01

A complex interplay of viral, host, and ecological factors shapes the spatio-temporal incidence and evolution of human influenza viruses. Although considerable attention has been paid to influenza A viruses, a lack of equivalent data means that an integrated evolutionary and epidemiological framework has until now not been available for influenza B viruses, despite their significant disease burden. Through the analysis of over 900 full genomes from an epidemiological collection of more than 26,000 strains from Australia and New Zealand, we reveal fundamental differences in the phylodynamics of the two co-circulating lineages of influenza B virus (Victoria and Yamagata), showing that their individual dynamics are determined by a complex relationship between virus transmission, age of infection, and receptor binding preference. In sum, this work identifies new factors that are important determinants of influenza B evolution and epidemiology. DOI: http://dx.doi.org/10.7554/eLife.05055.001 PMID:25594904

Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Xueling; Zhou, Tongqing; Zhu, Jiang

2013-03-04

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution ofmore » antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.« less

Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

PubMed

Castro, Eliana F; Campos, Rodolfo H; Cavallaro, Lucía V

2014-01-01

Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

The paradox of HBV evolution as revealed from a 16th century mummy

PubMed Central

Duggan, Ana T.; Poinar, Debi; Poinar, Hendrik N.

2018-01-01

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen. PMID:29300782

Do Viruses Exchange Genes across Superkingdoms of Life?

PubMed

Malik, Shahana S; Azem-E-Zahra, Syeda; Kim, Kyung Mo; Caetano-Anollés, Gustavo; Nasir, Arshan

2017-01-01

Viruses can be classified into archaeoviruses, bacterioviruses, and eukaryoviruses according to the taxonomy of the infected host. The host-constrained perception of viruses implies preference of genetic exchange between viruses and cellular organisms of their host superkingdoms and viral origins from host cells either via escape or reduction. However, viruses frequently establish non-lytic interactions with organisms and endogenize into the genomes of bacterial endosymbionts that reside in eukaryotic cells. Such interactions create opportunities for genetic exchange between viruses and organisms of non-host superkingdoms. Here, we take an atypical approach to revisit virus-cell interactions by first identifying protein fold structures in the proteomes of archaeoviruses, bacterioviruses, and eukaryoviruses and second by tracing their spread in the proteomes of superkingdoms Archaea, Bacteria, and Eukarya. The exercise quantified protein structural homologies between viruses and organisms of their host and non-host superkingdoms and revealed likely candidates for virus-to-cell and cell-to-virus gene transfers. Unexpected lifestyle-driven genetic affiliations between bacterioviruses and Eukarya and eukaryoviruses and Bacteria were also predicted in addition to a large cohort of protein folds that were universally shared by viral and cellular proteomes and virus-specific protein folds not detected in cellular proteomes. These protein folds provide unique insights into viral origins and evolution that are generally difficult to recover with traditional sequence alignment-dependent evolutionary analyses owing to the fast mutation rates of viral gene sequences.

Patterns of Viral DNA Integration in Cells Transformed by Wild Type or DNA-Binding Protein Mutants of Adenovirus Type 5 and Effect of Chemical Carcinogens on Integration

PubMed Central

Dorsch-Häsler, Karoline; Fisher, Paul B.; Weinstein, I. Bernard; Ginsberg, Harold S.

1980-01-01

The integration pattern of viral DNA was studied in a number of cell lines transformed by wild-type adenovirus type 5 (Ad5 WT) and two mutants of the DNA-binding protein gene, H5ts125 and H5ts107. The effect of chemical carcinogens on the integration of viral DNA was also investigated. Liquid hybridization (C0t) analyses showed that rat embryo cells transformed by Ad5 WT usually contained only the left-hand end of the viral genome, whereas cell lines transformed by H5ts125 or H5ts107 at either the semipermissive (36°C) or nonpermissive (39.5°C) temperature often contained one to five copies of all or most of the entire adenovirus genome. The arrangement of the integrated adenovirus DNA sequences was determined by cleavage of transformed cell DNA with restriction endonucleases XbaI, EcoRI, or HindIII followed by transfer of separated fragments to nitrocellulose paper and hybridization according to the technique of E. M. Southern (J. Mol. Biol. 98: 503-517, 1975). It was found that the adenovirus genome is integrated as a linear sequence covalently linked to host cell DNA; that the viral DNA is integrated into different host DNA sequences in each cell line studied; that in cell lines that contain multiple copies of the Ad5 genome the viral DNA sequences can be integrated in a single set of host cell DNA sequences and not as concatemers; and that chemical carcinogens do not alter the extent or pattern of viral DNA integration. Images PMID:6246266

Human Papillomavirus Genome Integration and Head and Neck Cancer.

PubMed

Pinatti, L M; Walline, H M; Carey, T E

2018-06-01

We conducted a critical review of human papillomavirus (HPV) integration into the host genome in oral/oropharyngeal cancer, reviewed the literature for HPV-induced cancers, and obtained current data for HPV-related oral and oropharyngeal cancers. In addition, we performed studies to identify HPV integration sites and the relationship of integration to viral-host fusion transcripts and whether integration is required for HPV-associated oncogenesis. Viral integration of HPV into the host genome is not required for the viral life cycle and might not be necessary for cellular transformation, yet HPV integration is frequently reported in cervical and head and neck cancer specimens. Studies of large numbers of early cervical lesions revealed frequent viral integration into gene-poor regions of the host genome with comparatively rare integration into cellular genes, suggesting that integration is a stochastic event and that site of integration may be largely a function of chance. However, more recent studies of head and neck squamous cell carcinomas (HNSCCs) suggest that integration may represent an additional oncogenic mechanism through direct effects on cancer-related gene expression and generation of hybrid viral-host fusion transcripts. In HNSCC cell lines as well as primary tumors, integration into cancer-related genes leading to gene disruption has been reported. The studies have shown that integration-induced altered gene expression may be associated with tumor recurrence. Evidence from several studies indicates that viral integration into genic regions is accompanied by local amplification, increased expression in some cases, interruption of gene expression, and likely additional oncogenic effects. Similarly, reported examples of viral integration near microRNAs suggest that altered expression of these regulatory molecules may also contribute to oncogenesis. Future work is indicated to identify the mechanisms of these events on cancer cell behavior.

Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

PubMed

Shi, Jiandong; Sun, Jing; Wang, Bin; Wu, Meini; Zhang, Jing; Duan, Zhiqing; Wang, Haixuan; Hu, Ningzhu; Hu, Yunzhang

2014-10-01

MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs. © FASEB.

Chikungunya Virus–Vector Interactions

PubMed Central

Coffey, Lark L.; Failloux, Anna-Bella; Weaver, Scott C.

2014-01-01

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed. PMID:25421891

Global Avian Influenza Surveillance in Wild Birds: A Strategy to Capture Viral Diversity

PubMed Central

Machalaba, Catherine C.; Elwood, Sarah E.; Forcella, Simona; Smith, Kristine M.; Hamilton, Keith; Jebara, Karim B.; Swayne, David E.; Webby, Richard J.; Mumford, Elizabeth; Mazet, Jonna A.K.; Gaidet, Nicolas; Daszak, Peter

2015-01-01

Wild birds play a major role in the evolution, maintenance, and spread of avian influenza viruses. However, surveillance for these viruses in wild birds is sporadic, geographically biased, and often limited to the last outbreak virus. To identify opportunities to optimize wild bird surveillance for understanding viral diversity, we reviewed responses to a World Organisation for Animal Health–administered survey, government reports to this organization, articles on Web of Knowledge, and the Influenza Research Database. At least 119 countries conducted avian influenza virus surveillance in wild birds during 2008–2013, but coordination and standardization was lacking among surveillance efforts, and most focused on limited subsets of influenza viruses. Given high financial and public health burdens of recent avian influenza outbreaks, we call for sustained, cost-effective investments in locations with high avian influenza diversity in wild birds and efforts to promote standardized sampling, testing, and reporting methods, including full-genome sequencing and sharing of isolates with the scientific community. PMID:25811221

Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human

PubMed Central

Song, Huai-Dong; Tu, Chang-Chun; Zhang, Guo-Wei; Wang, Sheng-Yue; Zheng, Kui; Lei, Lian-Cheng; Chen, Qiu-Xia; Gao, Yu-Wei; Zhou, Hui-Qiong; Xiang, Hua; Zheng, Hua-Jun; Chern, Shur-Wern Wang; Cheng, Feng; Pan, Chun-Ming; Xuan, Hua; Chen, Sai-Juan; Luo, Hui-Ming; Zhou, Duan-Hua; Liu, Yu-Fei; He, Jian-Feng; Qin, Peng-Zhe; Li, Ling-Hui; Ren, Yu-Qi; Liang, Wen-Jia; Yu, Ye-Dong; Anderson, Larry; Wang, Ming; Xu, Rui-Heng; Wu, Xin-Wei; Zheng, Huan-Ying; Chen, Jin-Ding; Liang, Guodong; Gao, Yang; Liao, Ming; Fang, Ling; Jiang, Li-Yun; Li, Hui; Chen, Fang; Di, Biao; He, Li-Juan; Lin, Jin-Yan; Tong, Suxiang; Kong, Xiangang; Du, Lin; Hao, Pei; Tang, Hua; Bernini, Andrea; Yu, Xiao-Jing; Spiga, Ottavia; Guo, Zong-Ming; Pan, Hai-Yan; He, Wei-Zhong; Manuguerra, Jean-Claude; Fontanet, Arnaud; Danchin, Antoine; Niccolai, Neri; Li, Yi-Xue; Wu, Chung-I; Zhao, Guo-Ping

2005-01-01

The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic analysis suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geographic locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002–2003 epidemic. Major genetic variations in some critical genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003. PMID:15695582

Ultrastructure and Viral Metagenome of Bacteriophages from an Anaerobic Methane Oxidizing Methylomirabilis Bioreactor Enrichment Culture

PubMed Central

Gambelli, Lavinia; Cremers, Geert; Mesman, Rob; Guerrero, Simon; Dutilh, Bas E.; Jetten, Mike S. M.; Op den Camp, Huub J. M.; van Niftrik, Laura

2016-01-01

With its capacity for anaerobic methane oxidation and denitrification, the bacterium Methylomirabilis oxyfera plays an important role in natural ecosystems. Its unique physiology can be exploited for more sustainable wastewater treatment technologies. However, operational stability of full-scale bioreactors can experience setbacks due to, for example, bacteriophage blooms. By shaping microbial communities through mortality, horizontal gene transfer, and metabolic reprogramming, bacteriophages are important players in most ecosystems. Here, we analyzed an infected Methylomirabilis sp. bioreactor enrichment culture using (advanced) electron microscopy, viral metagenomics and bioinformatics. Electron micrographs revealed four different viral morphotypes, one of which was observed to infect Methylomirabilis cells. The infected cells contained densely packed ~55 nm icosahedral bacteriophage particles with a putative internal membrane. Various stages of virion assembly were observed. Moreover, during the bacteriophage replication, the host cytoplasmic membrane appeared extremely patchy, which suggests that the bacteriophages may use host bacterial lipids to build their own putative internal membrane. The viral metagenome contained 1.87 million base pairs of assembled viral sequences, from which five putative complete viral genomes were assembled and manually annotated. Using bioinformatics analyses, we could not identify which viral genome belonged to the Methylomirabilis- infecting bacteriophage, in part because the obtained viral genome sequences were novel and unique to this reactor system. Taken together these results show that new bacteriophages can be detected in anaerobic cultivation systems and that the effect of bacteriophages on the microbial community in these systems is a topic for further study. PMID:27877158

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification.

PubMed

Sobel Leonard, Ashley; McClain, Micah T; Smith, Gavin J D; Wentworth, David E; Halpin, Rebecca A; Lin, Xudong; Ransier, Amy; Stockwell, Timothy B; Das, Suman R; Gilbert, Anthony S; Lambkin-Williams, Robert; Ginsburg, Geoffrey S; Woods, Christopher W; Koelle, Katia

2016-12-15

Knowledge of influenza virus evolution at the point of transmission and at the intrahost level remains limited, particularly for human hosts. Here, we analyze a unique viral data set of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with cell- and egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects' viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects' nasal wash samples, the viral stock, and the influenza virus A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that (i) the viral stock contained genetic variants that originated and likely were selected for during the passaging process, (ii) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein, and (iii) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation. Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza virus sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects' viral populations differ genetically from the viral stock, with subjects' viral populations having lower representation of the amino-acid-changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid-changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections. Copyright © 2016 Sobel Leonard et al.

Genome-Wide Analysis of Evolutionary Markers of Human Influenza A(H1N1)pdm09 and A(H3N2) Viruses May Guide Selection of Vaccine Strain Candidates.

PubMed

Belanov, Sergei S; Bychkov, Dmitrii; Benner, Christian; Ripatti, Samuli; Ojala, Teija; Kankainen, Matti; Kai Lee, Hong; Wei-Tze Tang, Julian; Kainov, Denis E

2015-11-27

Here we analyzed whole-genome sequences of 3,969 influenza A(H1N1)pdm09 and 4,774 A(H3N2) strains that circulated during 2009-2015 in the world. The analysis revealed changes at 481 and 533 amino acid sites in proteins of influenza A(H1N1)pdm09 and A(H3N2) strains, respectively. Many of these changes were introduced as a result of random drift. However, there were 61 and 68 changes that were present in relatively large number of A(H1N1)pdm09 and A(H3N2) strains, respectively, that circulated during relatively long time. We named these amino acid substitutions evolutionary markers, as they seemed to contain valuable information regarding the viral evolution. Interestingly, influenza A(H1N1)pdm09 and A(H3N2) viruses acquired non-overlapping sets of evolutionary markers. We next analyzed these characteristic markers in vaccine strains recommended by the World Health Organization for the past five years. Our analysis revealed that vaccine strains carried only few evolutionary markers at antigenic sites of viral hemagglutinin (HA) and neuraminidase (NA). The absence of these markers at antigenic sites could affect the recognition of HA and NA by human antibodies generated in response to vaccinations. This could, in part, explain moderate efficacy of influenza vaccines during 2009-2014. Finally, we identified influenza A(H1N1)pdm09 and A(H3N2) strains, which contain all the evolutionary markers of influenza A strains circulated in 2015, and which could be used as vaccine candidates for the 2015/2016 season. Thus, genome-wide analysis of evolutionary markers of influenza A(H1N1)pdm09 and A(H3N2) viruses may guide selection of vaccine strain candidates. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Single-virion sequencing of lamivudine-treated HBV populations reveal population evolution dynamics and demographic history.

PubMed

Zhu, Yuan O; Aw, Pauline P K; de Sessions, Paola Florez; Hong, Shuzhen; See, Lee Xian; Hong, Lewis Z; Wilm, Andreas; Li, Chen Hao; Hue, Stephane; Lim, Seng Gee; Nagarajan, Niranjan; Burkholder, William F; Hibberd, Martin

2017-10-27

Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients - once before antiviral treatment and once after viral rebound due to resistance. With single-virion sequencing, we obtained 248-8796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently through PacBio SMRT and Illumina Pooled deep sequencing. Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance and host adaptation, allows differentiation between soft or hard selective sweeps, and may be useful in the reconstruction of intra-host viral population demographic history.

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

DOE PAGES

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; ...

2015-12-22

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus.

PubMed

Daikoku, Tohru; Kudoh, Ayumi; Fujita, Masatoshi; Sugaya, Yutaka; Isomura, Hiroki; Tsurumi, Tatsuya

2004-12-24

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.

A Membrane-Destabilizing Peptide in Capsid Protein L2 Is Required for Egress of Papillomavirus Genomes from Endosomes

PubMed Central

Kämper, Nadine; Day, Patricia M.; Nowak, Thorsten; Selinka, Hans-Christoph; Florin, Luise; Bolscher, Jan; Hilbig, Lydia; Schiller, John T.; Sapp, Martin

2006-01-01

Papillomaviruses are internalized via clathrin-dependent endocytosis. However, the mechanism by which viral genomes pass endosomal membranes has not been elucidated. In this report we show that the minor capsid protein L2 is required for egress of viral genomes from endosomes but not for initial uptake and uncoating and that a 23-amino-acid peptide at the C terminus of L2 is necessary for this function. Pseudogenomes encapsidated by L1 and L2 lacking this peptide accumulated in vesicular compartments similar to that observed with L1-only viral particles, and these mutant pseudoviruses were noninfectious. This L2 peptide displayed strong membrane-disrupting activity, induced cytolysis of bacteria and eukaryotic cells in a pH-dependent manner, and permeabilized cells after exogenous addition. Fusions between green fluorescent protein and the L2 peptide integrated into cellular membranes like the wild type but not like C-terminal mutants of L2. Our data indicate that the L2 C terminus facilitates escape of viral genomes from the endocytic compartment and that this feature is conserved among papillomaviruses. Furthermore, the characteristic of this peptide differs from the classical virus-encoded membrane-penetrating peptides. PMID:16378978

Computational approach for elucidating interactions of cross-species miRNAs and their targets in Flaviviruses.

PubMed

Shinde, Santosh P; Banerjee, Amit Kumar; Arora, Neelima; Murty, U S N; Sripathi, Venkateswara Rao; Pal-Bhadra, Manika; Bhadra, Utpal

2015-03-01

Combating viral diseases has been a challenging task since time immemorial. Available molecular approaches are limited and not much effective for this daunting task. MicroRNA based therapies have shown promise in recent times. MicroRNAs are tiny non-coding RNAs that regulate translational repression of target mRNA in highly specific manner. In this study, we have determined the target regions for human and viral microRNAs in the conserved genomic regions of selected viruses of Flaviviridae family using miRanda and performed a comparative target selectivity analysis among them. Specific target regions were determined and they were compared extensively among themselves by exploring their position to determine the vicinity. Based on the multiplicity and cooperativity analysis, interaction maps were developed manually to represent the interactions between top-ranking miRNAs and genomes of the viruses considered in this study. Self-organizing map (SOM) was used to cluster the best-ranked microRNAs based on the vital physicochemical properties. This study will provide deep insight into the interrelation of the viral and human microRNAs interactions with the selected Flaviviridae genomes and will help to identify cross-species microRNA targets on the viral genome.

Functional Role of Infective Viral Particles on Metal Reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coates, John D.

2014-04-01

A proposed strategy for the remediation of uranium (U) contaminated sites was based on the immobilization of U by reducing the oxidized soluble U, U(VI), to form a reduced insoluble end product, U(IV). Previous studies identified Geobacter sp., including G. sulfurreducens and G. metallireducens, as predominant U(VI)-reducing bacteria under acetate-oxidizing and U(VI)-reducing conditions. Examination of the finished genome sequence annotation of the canonical metal reducing species Geobacter sulfurreducens strain PCA and G. metallireduceans strain GS-15 as well as the draft genome sequence of G. uraniumreducens strain Rf4 identified phage related proteins. In addition, the completed genome for Anaeromyxobacter dehalogenans andmore » the draft genome sequence of Desulfovibrio desulfuricans strain G20, two more model metal-reducing bacteria, also revealed phage related sequences. The presence of these gene sequences indicated that Geobacter spp., Anaeromyxobacter spp., and Desulfovibrio spp. are susceptible to viral infection. Furthermore, viral populations in soils and sedimentary environments in the order of 6.4×10{sup 6}–2.7×10{sup 10} VLP’s cm{sup -3} have been observed. In some cases, viral populations exceed bacterial populations in these environments suggesting that a relationship may exist between viruses and bacteria. Our preliminary screens of samples collected from the ESR FRC indicated that viral like particles were observed in significant numbers. The objective of this study was to investigate the potential functional role viruses play in metal reduction specifically Fe(III) and U(VI) reduction, the environmental parameters affecting viral infection of metal reducing bacteria, and the subsequent effects on U transport.« less

Enhanced arbovirus surveillance with deep sequencing: Identification of novel rhabdoviruses and bunyaviruses in Australian mosquitoes.

PubMed

Coffey, Lark L; Page, Brady L; Greninger, Alexander L; Herring, Belinda L; Russell, Richard C; Doggett, Stephen L; Haniotis, John; Wang, Chunlin; Deng, Xutao; Delwart, Eric L

2014-01-05

Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥ 50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans. © 2013 Elsevier Inc. All rights reserved.

PARP1 restricts Epstein Barr Virus lytic reactivation by binding the BZLF1 promoter.

PubMed

Lupey-Green, Lena N; Moquin, Stephanie A; Martin, Kayla A; McDevitt, Shane M; Hulse, Michael; Caruso, Lisa B; Pomerantz, Richard T; Miranda, Jj L; Tempera, Italo

2017-07-01

The Epstein Barr virus (EBV) genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. Here, we demonstrate that binding of PARP1, an important cofactor of CTCF, at the BZLF1 lytic switch promoter restricts EBV reactivation. Knockdown of PARP1 in the Akata-EBV cell line significantly increases viral copy number and lytic protein expression. Interestingly, CTCF knockdown has no effect on viral reactivation, and CTCF binding across the EBV genome is largely unchanged following reactivation. Moreover, EBV reactivation attenuates PARP activity, and Zta expression alone is sufficient to decrease PARP activity. Here we demonstrate a restrictive function of PARP1 in EBV lytic reactivation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Virus Database and Online Inquiry System Based on Natural Vectors.

PubMed

Dong, Rui; Zheng, Hui; Tian, Kun; Yau, Shek-Chung; Mao, Weiguang; Yu, Wenping; Yin, Changchuan; Yu, Chenglong; He, Rong Lucy; Yang, Jie; Yau, Stephen St

2017-01-01

We construct a virus database called VirusDB (http://yaulab.math.tsinghua.edu.cn/VirusDB/) and an online inquiry system to serve people who are interested in viral classification and prediction. The database stores all viral genomes, their corresponding natural vectors, and the classification information of the single/multiple-segmented viral reference sequences downloaded from National Center for Biotechnology Information. The online inquiry system serves the purpose of computing natural vectors and their distances based on submitted genomes, providing an online interface for accessing and using the database for viral classification and prediction, and back-end processes for automatic and manual updating of database content to synchronize with GenBank. Submitted genomes data in FASTA format will be carried out and the prediction results with 5 closest neighbors and their classifications will be returned by email. Considering the one-to-one correspondence between sequence and natural vector, time efficiency, and high accuracy, natural vector is a significant advance compared with alignment methods, which makes VirusDB a useful database in further research.

Differential segregation of nodaviral coat protein and RNA into progeny virions during mixed infection with FHV and NoV

PubMed Central

Gopal, Radhika; Venter, P. Arno; Schneemann, Anette

2014-01-01

Nodaviruses are icosahedral viruses with a bipartite, positive-sense RNA genome. The two RNAs are packaged into a single virion by a poorly understood mechanism. We chose two distantly related nodaviruses, Flock House virus and Nodamura virus, to explore formation of viral reassortants as a means to further understand genome recognition and encapsidation. In mixed infections, the viruses were incompatible at the level of RNA replication and their coat proteins segregated into separate populations of progeny particles. RNA packaging, on the other hand, was indiscriminate as all four viral RNAs were detectable in each progeny population. Consistent with the trans-encapsidation phenotype, fluorescence in situ hybridization of viral RNA revealed that the genomes of the two viruses co-localized throughout the cytoplasm. Our results imply that nodaviral RNAs lack rigorously defined packaging signals and that coencapsidation of the viral RNAs does not require a pair of cognate RNA1 and RNA2. PMID:24725955

Virus-Clip: a fast and memory-efficient viral integration site detection tool at single-base resolution with annotation capability.

PubMed

Ho, Daniel W H; Sze, Karen M F; Ng, Irene O L

2015-08-28

Viral integration into the human genome upon infection is an important risk factor for various human malignancies. We developed viral integration site detection tool called Virus-Clip, which makes use of information extracted from soft-clipped sequencing reads to identify exact positions of human and virus breakpoints of integration events. With initial read alignment to virus reference genome and streamlined procedures, Virus-Clip delivers a simple, fast and memory-efficient solution to viral integration site detection. Moreover, it can also automatically annotate the integration events with the corresponding affected human genes. Virus-Clip has been verified using whole-transcriptome sequencing data and its detection was validated to have satisfactory sensitivity and specificity. Marked advancement in performance was detected, compared to existing tools. It is applicable to versatile types of data including whole-genome sequencing, whole-transcriptome sequencing, and targeted sequencing. Virus-Clip is available at http://web.hku.hk/~dwhho/Virus-Clip.zip.

Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation

PubMed Central

Betz-Stablein, B. D.; Töpfer, A.; Littlejohn, M.; Yuen, L.; Colledge, D.; Sozzi, V.; Angus, P.; Thompson, A.; Revill, P.; Beerenwinkel, N.; Warner, N.

2016-01-01

ABSTRACT Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available. PMID:27252524

Evolutionary analysis of Old World arenaviruses reveals a major adaptive contribution of the viral polymerase.

PubMed

Pontremoli, Chiara; Forni, Diego; Cagliani, Rachele; Pozzoli, Uberto; Riva, Stefania; Bravo, Ignacio G; Clerici, Mario; Sironi, Manuela

2017-10-01

The Old World (OW) arenavirus complex includes several species of rodent-borne viruses, some of which (i.e., Lassa virus, LASV and Lymphocytic choriomeningitis virus, LCMV) cause human diseases. Most LCMV and LASV infections are caused by rodent-to-human transmissions. Thus, viral evolution is largely determined by events that occur in the wildlife reservoirs. We used a set of human- and rodent-derived viral sequences to investigate the evolutionary history underlying OW arenavirus speciation, as well as the more recent selective events that accompanied LASV spread in West Africa. We show that the viral RNA polymerase (L protein) was a major positive selection target in OW arenaviruses and during LASV out-of-Nigeria migration. No evidence of selection was observed for the glycoprotein, whereas positive selection acted on the nucleoprotein (NP) during LCMV speciation. Positively selected sites in L and NP are surrounded by highly conserved residues, and the bulk of the viral genome evolves under purifying selection. Several positively selected sites are likely to modulate viral replication/transcription. In both L and NP, structural features (solvent exposed surface area) are important determinants of site-wise evolutionary rate variation. By incorporating several rodent-derived sequences, we also performed an analysis of OW arenavirus codon adaptation to the human host. Results do not support a previously hypothesized role of codon adaptation in disease severity for non-Nigerian strains. In conclusion, L and NP represent the major selection targets and possible determinants of disease presentation; these results suggest that field surveys and experimental studies should primarily focus on these proteins. © 2017 John Wiley & Sons Ltd.

Visualization of episomal and integrated Epstein-Barr virus DNA by fiber fluorescence in situ hybridization.

PubMed

Reisinger, Jürgen; Rumpler, Silvia; Lion, Thomas; Ambros, Peter F

2006-04-01

For many Epstein-Barr virus (EBV)-associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV-specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV.

Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes.

PubMed

Delwart, Eric; Li, Linlin

2012-03-01

The genomes of numerous circoviruses and distantly related circular ssDNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, plants (geminivirus and nanovirus), in human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also recently identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting the very wide past host range of rep bearing viruses. An ancient origin for viruses with Rep-encoding small circular ssDNA genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular ssDNA genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular ssDNA viral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

Adenovirus Core Protein VII Downregulates the DNA Damage Response on the Host Genome

PubMed Central

Avgousti, Daphne C.; Della Fera, Ashley N.; Otter, Clayton J.; Herrmann, Christin; Pancholi, Neha J.

2017-01-01

ABSTRACT Viral manipulation of cellular proteins allows viruses to suppress host defenses and generate infectious progeny. Due to the linear double-stranded DNA nature of the adenovirus genome, the cellular DNA damage response (DDR) is considered a barrier to successful infection. The adenovirus genome is packaged with protein VII, a virally encoded histone-like core protein that is suggested to protect incoming viral genomes from detection by the cellular DNA damage machinery. We showed that protein VII localizes to host chromatin during infection, leading us to hypothesize that protein VII may affect DNA damage responses on the cellular genome. Here we show that protein VII at cellular chromatin results in a significant decrease in accumulation of phosphorylated H2AX (γH2AX) following irradiation, indicating that protein VII inhibits DDR signaling. The oncoprotein SET was recently suggested to modulate the DDR by affecting access of repair proteins to chromatin. Since protein VII binds SET, we investigated a role for SET in DDR inhibition by protein VII. We show that knockdown of SET partially rescues the protein VII-induced decrease in γH2AX accumulation on the host genome, suggesting that SET is required for inhibition. Finally, we show that knockdown of SET also allows ATM to localize to incoming viral genomes bound by protein VII during infection with a mutant lacking early region E4. Together, our data suggest that the protein VII-SET interaction contributes to DDR evasion by adenovirus. Our results provide an additional example of a strategy used by adenovirus to abrogate the host DDR and show how viruses can modify cellular processes through manipulation of host chromatin. IMPORTANCE The DNA damage response (DDR) is a cellular network that is crucial for maintaining genome integrity. DNA viruses replicating in the nucleus challenge the resident genome and must overcome cellular responses, including the DDR. Adenoviruses are prevalent human pathogens that can cause a multitude of diseases, such as respiratory infections and conjunctivitis. Here we describe how a small adenovirus core protein that localizes to host chromatin during infection can globally downregulate the DDR. Our study focuses on key players in the damage signaling pathway and highlights how viral manipulation of chromatin may influence access of DDR proteins to the host genome. PMID:28794020

Are Evolution and the Intracellular Innate Immune System Key Determinants in HIV Transmission?

PubMed Central

Sumner, Rebecca P.; Thorne, Lucy G.; Fink, Doug L.; Khan, Hataf; Milne, Richard S.; Towers, Greg J.

2017-01-01

HIV-1 is the single most important sexually transmitted disease in humans from a global health perspective. Among human lentiviruses, HIV-1 M group has uniquely achieved pandemic levels of human-to-human transmission. The requirement to transmit between hosts likely provides the strongest selective forces on a virus, as without transmission, there can be no new infections within a host population. Our perspective is that evolution of all of the virus–host interactions, which are inherited and perpetuated from host-to-host, must be consistent with transmission. For example, CXCR4 use, which often evolves late in infection, does not favor transmission and is therefore lost when a virus transmits to a new host. Thus, transmission inevitably influences all aspects of virus biology, including interactions with the innate immune system, and dictates the biological niche in which the virus exists in the host. A viable viral niche typically does not select features that disfavor transmission. The innate immune response represents a significant selective pressure during the transmission process. In fact, all viruses must antagonize and/or evade the mechanisms of the host innate and adaptive immune systems that they encounter. We believe that viewing host–virus interactions from a transmission perspective helps us understand the mechanistic details of antiviral immunity and viral escape. This is particularly true for the innate immune system, which typically acts from the very earliest stages of the host–virus interaction, and must be bypassed to achieve successful infection. With this in mind, here we review the innate sensing of HIV, the consequent downstream signaling cascades and the viral restriction that results. The centrality of these mechanisms to host defense is illustrated by the array of countermeasures that HIV deploys to escape them, despite the coding constraint of a 10 kb genome. We consider evasion strategies in detail, in particular the role of the HIV capsid and the viral accessory proteins highlighting important unanswered questions and discussing future perspectives. PMID:29056936

Signatures of Pleiotropy, Economy and Convergent Evolution in a Domain-Resolved Map of Human–Virus Protein–Protein Interaction Networks

PubMed Central

Garamszegi, Sara; Franzosa, Eric A.; Xia, Yu

2013-01-01

A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are otherwise hidden in the traditional binary network, highlighting the power and necessity of high-resolution approaches in host-pathogen systems biology. PMID:24339775

Signatures of pleiotropy, economy and convergent evolution in a domain-resolved map of human-virus protein-protein interaction networks.

PubMed

Garamszegi, Sara; Franzosa, Eric A; Xia, Yu

2013-01-01

A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are otherwise hidden in the traditional binary network, highlighting the power and necessity of high-resolution approaches in host-pathogen systems biology.

THE PENETRATION OF REOVIRUS RNA AND INITIATION OF ITS GENETIC FUNCTION IN L-STRAIN FIBROBLASTS

PubMed Central

Silverstein, Samuel C.; Dales, Samuel

1968-01-01

Reovirus type 3 is phagocytized by L cells and rapidly sequestered inside lysosomes. Hydrolases within these organelles are capable of stripping the viral coat proteins, but they fail to degrade the double-stranded RNA genome. These observations support the view that sojourn of reovirus in lysosomes, when the lytic enzymes uncoat its genome, is an obligatory step in the sequence of infection. Although the mechanism for transferring the uncoated RNA out of lysosomes remains to be elucidated, evidence is presented suggesting that progeny genomes are bound to site(s) possessing the fine structure of viral inclusions or factories. It appears that both the synthesis of single- and double-stranded viral RNA and the morphogenesis of progeny virus particles occur in such factories. PMID:19806702

Non-coding RNAs in virology: an RNA genomics approach.

PubMed

Isaac, Christopher; Patel, Trushar R; Zovoilis, Athanasios

2018-04-01

Advances in sequencing technologies and bioinformatic analysis techniques have greatly improved our understanding of various classes of RNAs and their functions. Despite not coding for proteins, non-coding RNAs (ncRNAs) are emerging as essential biomolecules fundamental for cellular functions and cell survival. Interestingly, ncRNAs produced by viruses not only control the expression of viral genes, but also influence host cell regulation and circumvent host innate immune response. Correspondingly, ncRNAs produced by the host genome can play a key role in host-virus interactions. In this article, we will first discuss a number of types of viral and mammalian ncRNAs associated with viral infections. Subsequently, we also describe the new possibilities and opportunities that RNA genomics and next-generation sequencing technologies provide for studying ncRNAs in virology.

Giant viruses coexisted with the cellular ancestors and represent a distinct supergroup along with superkingdoms Archaea, Bacteria and Eukarya

PubMed Central

2012-01-01

Background The discovery of giant viruses with genome and physical size comparable to cellular organisms, remnants of protein translation machinery and virus-specific parasites (virophages) have raised intriguing questions about their origin. Evidence advocates for their inclusion into global phylogenomic studies and their consideration as a distinct and ancient form of life. Results Here we reconstruct phylogenies describing the evolution of proteomes and protein domain structures of cellular organisms and double-stranded DNA viruses with medium-to-very-large proteomes (giant viruses). Trees of proteomes define viruses as a ‘fourth supergroup’ along with superkingdoms Archaea, Bacteria, and Eukarya. Trees of domains indicate they have evolved via massive and primordial reductive evolutionary processes. The distribution of domain structures suggests giant viruses harbor a significant number of protein domains including those with no cellular representation. The genomic and structural diversity embedded in the viral proteomes is comparable to the cellular proteomes of organisms with parasitic lifestyles. Since viral domains are widespread among cellular species, we propose that viruses mediate gene transfer between cells and crucially enhance biodiversity. Conclusions Results call for a change in the way viruses are perceived. They likely represent a distinct form of life that either predated or coexisted with the last universal common ancestor (LUCA) and constitute a very crucial part of our planet’s biosphere. PMID:22920653

Unsuccessful therapy with adefovir and entecavir-tenofovir in a patient with chronic hepatitis B infection with previous resistance to lamivudine: a fourteen-year evolution of hepatitis B virus mutations

PubMed Central

2011-01-01

Background Complex mutants can be selected under sequential selective pressure by HBV therapy. To determine hepatitis B virus genomic evolution during antiviral therapy we characterized the HBV quasi-species in a patient who did no respond to therapy following lamivudine breakthrough for a period of 14 years. Case Presentation The polymerase and precore/core genes were amplified and sequenced at determined intervals in a period of 14 years. HBV viral load and HBeAg/Anti-HBe serological profiles as well as amino transferase levels were also measured. A mixture of lamivudine-resistant genotype A2 HBV strains harboring the rtM204V mutation coexisted in the patient following viral breakthrough to lamivudine. The L180M+M204V dominant mutant displayed strong lamivudine-resistance. As therapy was changed to adefovir, then to entecavir, and finally to entecavir-tenofovir the viral load showed fluctuations but lamivudine-resistant strains continued to be selected, with minor contributions to the HBV quasi-species composition of additional resistance-associated mutations. At the end of the 14-year follow up period, high viral loads were predominant, with viral strains harboring the lamivudine-resistance signature rtL180M+M204V. The precore/core frame A1762T and G1764A double mutation was detected before treatment and remaining in this condition during the entire follow-up. Specific entecavir and tenofovir primary resistance-associated mutations were not detected at any time. Plasma concentrations of tenofovir indicated adequate metabolism of the drug. Conclusions We report the selection of HBV mutants carrying well-defined primary resistance mutations that escaped lamivudine in a fourteen-year follow-up period. With the exception of tenofovir resistance mutations, subsequent unselected primary resistance mutations were detected as minor populations into the HBV quasispecies composition during adefovir or entecavir monotherapies. Although tenofovir is considered an appropriate therapeutic alternative for the treatment of entecavir-unresponsive patients, its use was not effective in the case reported here. PMID:21696601

Genome editing technologies to fight infectious diseases.

PubMed

Trevisan, Marta; Palù, Giorgio; Barzon, Luisa

2017-11-01

Genome editing by programmable nucleases represents a promising tool that could be exploited to develop new therapeutic strategies to fight infectious diseases. These nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) and homing endonucleases, are molecular scissors that can be targeted at predetermined loci in order to modify the genome sequence of an organism. Areas covered: By perturbing genomic DNA at predetermined loci, programmable nucleases can be used as antiviral and antimicrobial treatment. This approach includes targeting of essential viral genes or viral sequences able, once mutated, to inhibit viral replication; repurposing of CRISPR-Cas9 system for lethal self-targeting of bacteria; targeting antibiotic-resistance and virulence genes in bacteria, fungi, and parasites; engineering arthropod vectors to prevent vector-borne infections. Expert commentary: While progress has been done in demonstrating the feasibility of using genome editing as antimicrobial strategy, there are still many hurdles to overcome, such as the risk of off-target mutations, the raising of escape mutants, and the inefficiency of delivery methods, before translating results from preclinical studies into clinical applications.

Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance

PubMed Central

Zaidi, Syed Shan-e-Ali; Tashkandi, Manal; Mansoor, Shahid; Mahfouz, Magdy M.

2016-01-01

Plant viruses infect many economically important crops, including wheat, cotton, maize, cassava, and other vegetables. These viruses pose a serious threat to agriculture worldwide, as decreases in cropland area per capita may cause production to fall short of that required to feed the increasing world population. Under these circumstances, conventional strategies can fail to control rapidly evolving and emerging plant viruses. Genome-engineering strategies have recently emerged as promising tools to introduce desirable traits in many eukaryotic species, including plants. Among these genome engineering technologies, the CRISPR (clustered regularly interspaced palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) system has received special interest because of its simplicity, efficiency, and reproducibility. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. Here, we briefly describe the biology of the CRISPR/Cas9 system and plant viruses, and how different genome engineering technologies have been used to target these viruses. We further describe the main findings from recent studies of CRISPR/Cas9-mediated viral interference and discuss how these findings can be applied to improve global agriculture. We conclude by pinpointing the gaps in our knowledge and the outstanding questions regarding CRISPR/Cas9-mediated viral immunity. PMID:27877187

HHV-6A/B Integration and the Pathogenesis Associated with the Reactivation of Chromosomally Integrated HHV-6A/B.

PubMed

Collin, Vanessa; Flamand, Louis

2017-06-26

Unlike other human herpesviruses, human herpesvirus 6A and 6B (HHV-6A/B) infection can lead to integration of the viral genome in human chromosomes. When integration occurs in germinal cells, the integrated HHV-6A/B genome can be transmitted to 50% of descendants. Such individuals, carrying one copy of the HHV-6A/B genome in every cell, are referred to as having inherited chromosomally-integrated HHV-6A/B (iciHHV-6) and represent approximately 1% of the world's population. Interestingly, HHV-6A/B integrate their genomes in a specific region of the chromosomes known as telomeres. Telomeres are located at chromosomes' ends and play essential roles in chromosomal stability and the long-term proliferative potential of cells. Considering that the integrated HHV-6A/B genome is mostly intact without any gross rearrangements or deletions, integration is likely used for viral maintenance into host cells. Knowing the roles played by telomeres in cellular homeostasis, viral integration in such structure is not likely to be without consequences. At present, the mechanisms and factors involved in HHV-6A/B integration remain poorly defined. In this review, we detail the potential biological and medical impacts of HHV-6A/B integration as well as the possible chromosomal integration and viral excision processes.

Parvovirus-derived endogenous viral elements in two South American rodent genomes.

PubMed

Arriagada, Gloria; Gifford, Robert J

2014-10-01

We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the genomes of the long-tailed chinchilla (Chinchilla lanigera) and the degu (Octodon degus). The novel EVEs include dependovirus-related elements and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed in vivo, with increased expression in the liver. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

pH-Controlled Two-Step Uncoating of Influenza Virus

PubMed Central

Li, Sai; Sieben, Christian; Ludwig, Kai; Höfer, Chris T.; Chiantia, Salvatore; Herrmann, Andreas; Eghiaian, Frederic; Schaap, Iwan A.T.

2014-01-01

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5–6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection. PMID:24703306

Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

PubMed

Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

2016-06-01

Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

Template Recognition Mechanisms by Replicase Proteins Differ between Bipartite Positive-Strand Genomic RNAs of a Plant Virus▿

PubMed Central

Iwakawa, Hiro-oki; Mine, Akira; Hyodo, Kiwamu; An, Mengnan; Kaido, Masanori; Mise, Kazuyuki; Okuno, Tetsuro

2011-01-01

Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3′ untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism. PMID:20980498

The Papillomavirus E2 proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

McBride, Alison A., E-mail: amcbride@nih.gov

2013-10-15

The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. - Highlights: • Overview of E2 protein functions. • Structural domains of the papillomavirus E2 proteins. • Analysis of E2 binding sites in different genera of papillomaviruses.more » • Compilation of E2 associated proteins. • Comparison of key mutations in distinct E2 functions.« less

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

PubMed Central

Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

Limited SHIV env diversification in macaques failing oral antiretroviral pre-exposure prophylaxis.

PubMed

Zheng, Qi; Ruone, Susan; Switzer, William M; Heneine, Walid; García-Lerma, J Gerardo

2012-05-09

Pre-exposure prophylaxis (PrEP) with daily Truvada [a combination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF)] is a novel HIV prevention strategy recently found to prevent HIV transmission among men who have sex with men and heterosexual couples. Acute infection in adherent persons who fail PrEP will inevitably occur under concurrent antiretroviral therapy, thus raising questions regarding the potential impact of PrEP on early viral dynamics. We investigated viral evolution dynamics in a macaque model of PrEP consisting of repeated rectal exposures to SHIV162P3 in the presence of PrEP. Four macaques were infected during daily or intermittent PrEP with FTC or FTC/TDF, and five were untreated controls. SHIV env sequence evolution was monitored by single genome amplification with phylogenetic and sequence analysis. Mean nucleotide divergence from transmitted founder viruses calculated 17 weeks (range = 12-20) post peak viremia was significantly lower in PrEP failures than in control animals (7.2 × 10-3 compared to 1.6 × 10-2 nucleotide substitutions per site per year, respectively, p < 0.0001). Mean virus diversity was also lower in PrEP failures after 17 weeks (0.13% vs. 0.53% in controls, p < 0.0001). Our results in a macaque model of acute HIV infection suggest that infection during PrEP limits early virus evolution likely because of a direct antiviral effect of PrEP and/or reduced target cell availability. Reduced virus diversification during early infection might enhance immune control by slowing the selection of escape mutants.

Non-encapsidation Activities of the Capsid Proteins of Positive-strand RNA Viruses

PubMed Central

Ni, Peng; Kao, C. Cheng

2013-01-01

Viral capsid proteins (CPs) are characterized by their role in forming protective shells around viral genomes. However, CPs have additional and important roles in the virus infection cycles and in the cellular response to infection. These activities involve CP binding to RNAs in both sequence-specific and nonspecific manners as well as association with other proteins. This review focuses on CPs of both plant and animal-infecting viruses with positive-strand RNA genomes. We summarize the structural features of CPs and describe their modulatory roles in viral translation, RNA-dependent RNA synthesis, and host defense responses. PMID:24074574

Overlapping local and long-range RNA-RNA interactions modulate dengue virus genome cyclization and replication.

PubMed

de Borba, Luana; Villordo, Sergio M; Iglesias, Nestor G; Filomatori, Claudia V; Gebhard, Leopoldo G; Gamarnik, Andrea V

2015-03-01

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3' untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

F-Specific RNA Bacteriophages, Especially Members of Subgroup II, Should Be Reconsidered as Good Indicators of Viral Pollution of Oysters.

PubMed

Hartard, C; Leclerc, M; Rivet, R; Maul, A; Loutreul, J; Banas, S; Boudaud, N; Gantzer, C

2018-01-01

Norovirus (NoV) is the leading cause of gastroenteritis outbreaks linked to oyster consumption. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as indicators of viral contamination in oysters by focusing especially on FRNAPH subgroup II (FRNAPH-II). These viral indicators have been neglected because their behavior is sometimes different from that of NoV in shellfish, especially during the depuration processes usually performed before marketing. However, a significant bias needs to be taken into account. This bias is that, in the absence of routine culture methods, NoV is targeted by genome detection, while the presence of FRNAPH is usually investigated by isolation of infectious particles. In this study, by targeting both viruses using genome detection, a significant correlation between the presence of FRNAPH-II and that of NoV in shellfish collected from various European harvesting areas impacted by fecal pollution was observed. Moreover, during their depuration, while the long period of persistence of NoV was confirmed, a similar or even longer period of persistence of the FRNAPH-II genome, which was over 30 days, was observed. Such a striking genome persistence calls into question the relevance of molecular methods for assessing viral hazards. Targeting the same virus (i.e., FRNAPH-II) by culture and genome detection in specimens from harvesting areas as well as during depuration, we concluded that the presence of genomes in shellfish does not provide any information on the presence of the corresponding infectious particles. In view of these results, infectious FRNAPH detection should be reconsidered as a valuable indicator in oysters, and its potential for use in assessing viral hazard needs to be investigated. IMPORTANCE This work brings new data about the behavior of viruses in shellfish, as well as about the relevance of molecular methods for their detection and evaluation of the viral hazard. First, a strong correlation between the presence of F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and that of norovirus (NoV) in shellfish impacted by fecal contamination has been observed when both viruses are detected using molecular approaches. Second, when reverse transcription-PCR and culture are used to detect FRNAPH-II in shellfish, it appears that the genomes of the viruses present a longer period of persistence than infectious virus, and thus, virus genome detection fails to give information about the concomitant presence of infectious viruses. Finally, this study shows that FRNAPH persist at least as long as NoV does. These data are major arguments to reconsider the potential of FRNAPH as indicators of shellfish viral quality. Copyright © 2017 American Society for Microbiology.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

PubMed

Yoshida, Asuka; Kawabata, Ryoko; Honda, Tomoyuki; Sakai, Kouji; Ami, Yasushi; Sakaguchi, Takemasa; Irie, Takashi

2018-03-01

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-β) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-β-inducing virus. IFN-β induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells. IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-β induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity. Copyright © 2018 American Society for Microbiology.

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

PubMed Central

Zhao, Wei; Jardine, Paul J.

2015-01-01

ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. PMID:26423956

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

PubMed

Zhao, Wei; Jardine, Paul J; Grimes, Shelley

2015-12-01

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Ecology, Diversity and Comparative Genomics of Oceanic Cyanobacterial Viruses

DTIC Science & Technology

2004-06-01

microbial trophodynamics. Applied and Environmental Microbiology 56, 1400-1405. Bratbak, G. (1993). Viral mortality of the marine alga Emiliania huxleyi...inspecting regions of microbial chro- Emiliania huxleyi is a marine coccolithophorid, mosomes for the following characteristics: (1) with a world-wide...genomes are easier to under- Immun. 67, 5898-5905. Bratbatk G.. 1993. Viral mortality of the marine alga Emiliania stand-their small size makes it

Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

PubMed

Nikolaitchik, Olga A; Hu, Wei-Shau

2014-04-01

A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1 regulates its genome packaging and generate infectious viruses necessary for transmission to new hosts.