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Sample records for bacterial dna viruses

  1. Characterization of DNA conformation inside bacterial viruses

    NASA Astrophysics Data System (ADS)

    Petrov, Anton S.; Locker, C. Rebecca; Harvey, Stephen C.

    2009-08-01

    In this study we develop a formalism to describe the organization of DNA inside bacteriophage capsids during genome packaging. We have previously shown that DNA inside bacteriophage phi29 (ϕ29) is organized into folded toroids [A. S. Petrov and S. C. Harvey, Structure 15, 21 (2007)], whereas epsilon15 (ɛ15) reveals the coaxial organization of the genetic material [A. S. Petrov, K. Lim-Hing, and S. C. Harvey, Structure 15, 807 (2007)]. We now show that each system undergoes two consecutive transitions. The first transition corresponds to the formation of global conformations and is analogous to a disorder-order conformational transition. The second transition is characterized by a significant loss of DNA mobility at the local level leading to glasslike dynamic behavior. Packing genetic material inside bacteriophages can be used as a general model to study the behavior of semiflexible chains inside confined spaces, and the proposed formalism developed here can be used to study other systems of linear polymer chains confined to closed spaces.

  2. Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates.

    PubMed

    Borenstein, Ronen; Frenkel, Niza

    2009-11-10

    Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constraints. We introduced the 160-kb human herpes virus 6A (HHV-6A) genome into BACs by digesting the viral DNA replicative intermediates with the Sfil enzyme that cleaves the viral genome in a single site. The recombinant BACs contained also the puromycin selection gene, GFP, and LoxP sites flanking the BAC sequences. The HHV-6A-BAC vectors were retained stably in puromycin selected 293T cells. In the presence of irradiated helper virus, supplying most likely proteins enhancing gene expression they expressed early and late genes in SupT1 T cells. The method is especially attractive for viruses that replicate inefficiently and for viruses propagated in suspension cells. We have used the fact that the BAC cloning "freezes" the viral DNA replication intermediates to analyze their structure. The results revealed that HHV-6A-BACs contained a single direct repeat (DR) rather than a DR-DR sequence, predicted to arise by circularization of parental genomes with a DR at each terminus. HHV-6A DNA molecules prepared from the infected cells also contained DNA molecules with a single DR. Such forms were not previously described for HHV-6 DNA.

  3. A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes.

    PubMed

    Zhou, Fuchun; Li, Qiuhua; Gao, Shou-Jiang

    2009-01-01

    Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses.

  4. Study of the fine structure of adeno-associated virus DNA with bacterial restriction endonucleases.

    PubMed Central

    Berns, K I; Kort, J; Fife, K H; Grogan, E W; Spear, I

    1975-01-01

    A physical map of the adeno-associated virus type 2 genome has been constructed on the basis of the five fragments produced by the restriction endonucleases HindII + III from Hemophilus influenzae. There are three endo R-HindII cleavage sites and one endo R-HindIII site. Evidence has been obtained to support the existence of two nucleotide sequence permutations in adeno-associated virus DNA, the start points of which have been estimated to be separated by 1% of the genome. The three cleavage fragments produced by endo R-Eco RI have been ordered and oriented with respect to the endo R-HindII + III cleavage map. Images PMID:1159899

  5. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease.

    PubMed

    Kennedy, Edward M; Bassit, Leda C; Mueller, Henrik; Kornepati, Anand V R; Bogerd, Hal P; Nie, Ting; Chatterjee, Payel; Javanbakht, Hassan; Schinazi, Raymond F; Cullen, Bryan R

    2015-02-01

    Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome.

    PubMed

    Usme-Ciro, Jose A; Lopera, Jaime A; Enjuanes, Luis; Almazán, Fernando; Gallego-Gomez, Juan C

    2014-02-13

    Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Tumorigenic DNA viruses

    SciTech Connect

    Klein, G.

    1989-01-01

    The eighth volume of Advances in Viral Oncology focuses on the three major DNA virus groups with a postulated or proven tumorigenic potential: papillomaviruses, animal hepatitis viruses, and the Epstein-Bar virus. In the opening chapters, the contributors analyze the evidence that papillomaviruses and animal hepatitis viruses are involved in tumorigenesis and describe the mechanisms that trigger virus-host cell interactions. A detailed section on the Epstein-Barr virus (EBV) - comprising more than half the book - examines the transcription and mRNA processing patterns of the virus genome; the mechanisms by which EBV infects lymphoid and epithelial cells; the immunological aspects of the virus; the actions of EBV in hosts with Acquired Immune Deficiency Syndrome; and the involvement of EBV in the etiology of Burkitt's lymphoma.

  8. Isolation and Characterization of Bacterial DNA.

    ERIC Educational Resources Information Center

    Wilson, W. David; Davidson, Michael W.

    1979-01-01

    An inexpensive DNA preparation is presented which starts with commercially available frozen packed bacterial cells. Methods for analyzing the DNA are also presented, along with physical chemical experiments which can be done using the purified DNA. (BB)

  9. Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

    SciTech Connect

    Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu; Lin, Zihan; Liang, Lingfei; Lynn, Anna Y.; Varnado, Brittany; Weiss, Thomas M.; Tang, Liang; Schuch, Raymond

    2016-02-16

    The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.

  10. Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

    DOE PAGES

    Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu; ...

    2016-02-16

    The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

  11. Collective decisions among bacterial viruses

    NASA Astrophysics Data System (ADS)

    Joh, Richard; Mileyko, Yuriy; Voit, Eberhard; Weitz, Joshua

    2010-03-01

    For many temperate bacteriophages, the decision of whether to kill hosts or enter a latent state depends on the multiplicity of infection. In this talk, I present a quantitative model of gene regulatory dynamics to describe how phages make collective decisions within host cells. Unlike most previous studies, the copy number of viral genomes is treated as a variable. In the absence of feedback loops, viral mRNA transcription is expected to be proportional to the viral copy number. However, when there are nonlinear feedback loops in viral gene regulation, our model shows that gene expression patterns are sensitive to changes in viral copy number and there can be a domain of copy number where the system becomes bistable. Hence, the viral copy number is a key control parameter determining host cell fates. This suggests that bacterial viruses can respond adaptively to changes in population dynamics, and can make alternative decisions as a bet-hedging strategy. Finally, I present a stochastic version of viral gene regulation and discuss speed-accuracy trade-offs in the context of cell fate determination by viruses.

  12. Nuclear entry of DNA viruses

    PubMed Central

    Fay, Nikta; Panté, Nelly

    2015-01-01

    DNA viruses undertake their replication within the cell nucleus, and therefore they must first deliver their genome into the nucleus of their host cells. Thus, trafficking across the nuclear envelope is at the basis of DNA virus infections. Nuclear transport of molecules with diameters up to 39 nm is a tightly regulated process that occurs through the nuclear pore complex (NPC). Due to the enormous diversity of virus size and structure, each virus has developed its own strategy for entering the nucleus of their host cells, with no two strategies alike. For example, baculoviruses target their DNA-containing capsid to the NPC and subsequently enter the nucleus intact, while the hepatitis B virus capsid crosses the NPC but disassembles at the nuclear side of the NPC. For other viruses such as herpes simplex virus and adenovirus, although both dock at the NPC, they have each developed a distinct mechanism for the subsequent delivery of their genome into the nucleus. Remarkably, other DNA viruses, such as parvoviruses and human papillomaviruses, access the nucleus through an NPC-independent mechanism. This review discusses our current understanding of the mechanisms used by DNA viruses to deliver their genome into the nucleus, and further presents the experimental evidence for such mechanisms. PMID:26029198

  13. A novel virus-like particle based on hepatitis B core antigen and substrate-binding domain of bacterial molecular chaperone DnaK.

    PubMed

    Wang, Xue Jun; Gu, Kai; Xiong, Qi Yan; Shen, Liang; Cao, Rong Yue; Li, Ming Hui; Li, Tai Ming; Wu, Jie; Liu, Jing Jing

    2009-12-09

    Hepatitis B virus core (HBc) protein has been proved to be an attractive carrier for foreign epitopes, and can display green fluorescent protein (GFP) on its surface. The structure of substrate-binding domain of DnaK [DnaK (394-504 aa), DnaK SBD] is similar to GFP, we therefore reasoned that DnaK SBD might also be tolerated. Electron microscopic observations suggested that the chimeric proteins containing the truncated HBc (HBcDelta) and DnaK SBD could self-assemble into virus-like particle (VLP). Then the accessibility of DnaK SBD and the adjuvanticity of VLP HBcDelta-SBD were demonstrated by two recombinant peptide vaccines against gonadotropin-releasing hormone (GnRH), GhM and GhMNR. The latter carries in addition the peptide motif NRLLLTG which is known to bind to DnaK and DnaK SBD. The combination of VLP HBcDelta-SBD and GhMNR elicited stronger humoral responses and caused further testicular atrophy than the combinations of VLP HBcDelta and GhMNR or VLP HBcDelta-SBD and GhM in Balb/c mice. These findings indicate VLP HBcDelta-SBD might serve as an excellent carrier for GhMNR and some other peptide vaccines.

  14. I am what I eat and I eat what I am: acquisition of bacterial genes by giant viruses.

    PubMed

    Filée, Jonathan; Siguier, Patricia; Chandler, Mick

    2007-01-01

    Giant viruses are nucleocytoplasmic large DNA viruses (NCLDVs) that infect algae (phycodnaviruses) and amoebae (Mimivirus). We report an unexpected abundance in these giant viruses of islands of bacterial-type genes, including apparently intact prokaryotic mobile genetic elements, and hypothesize that NCLDV genomes undergo successive accretions of bacterial genes. The viruses could acquire bacterial genes within their bacteria-feeding eukaryotic hosts, and we suggest that such acquisition is driven by the intimate coupling of recombination and replication in NCLDVs.

  15. DNA Virus Replication Compartments

    PubMed Central

    Schmid, Melanie; Speiseder, Thomas; Dobner, Thomas

    2014-01-01

    Viruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication. PMID:24257611

  16. Bacterial identification and subtyping using DNA microarray and DNA sequencing.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

    2012-01-01

    The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

  17. Oral bacterial DNA findings in pericardial fluid

    PubMed Central

    Louhelainen, Anne-Mari; Aho, Joonas; Tuomisto, Sari; Aittoniemi, Janne; Vuento, Risto; Karhunen, Pekka J.; Pessi, Tanja

    2014-01-01

    Background We recently reported that large amounts of oral bacterial DNA can be found in thrombus aspirates of myocardial infarction patients. Some case reports describe bacterial findings in pericardial fluid, mostly done with conventional culturing and a few with PCR; in purulent pericarditis, nevertheless, bacterial PCR has not been used as a diagnostic method before. Objective To find out whether bacterial DNA can be measured in the pericardial fluid and if it correlates with pathologic–anatomic findings linked to cardiovascular diseases. Methods Twenty-two pericardial aspirates were collected aseptically prior to forensic autopsy at Tampere University Hospital during 2009–2010. Of the autopsies, 10 (45.5%) were free of coronary artery disease (CAD), 7 (31.8%) had mild and 5 (22.7%) had severe CAD. Bacterial DNA amounts were determined using real-time quantitative PCR with specific primers and probes for all bacterial strains associated with endodontic disease (Streptococcus mitis group, Streptococcus anginosus group, Staphylococcus aureus/Staphylococcus epidermidis, Prevotella intermedia, Parvimonas micra) and periodontal disease (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatus, and Dialister pneumosintes). Results Of 22 cases, 14 (63.6%) were positive for endodontic and 8 (36.4%) for periodontal-disease-associated bacteria. Only one case was positive for bacterial culturing. There was a statistically significant association between the relative amount of bacterial DNA in the pericardial fluid and the severity of CAD (p=0.035). Conclusions Oral bacterial DNA was detectable in pericardial fluid and an association between the severity of CAD and the total amount of bacterial DNA in pericardial fluid was found, suggesting that this kind of measurement might be useful for clinical purposes. PMID:25412607

  18. Translocation of DNA across bacterial membranes.

    PubMed Central

    Dreiseikelmann, B

    1994-01-01

    DNA translocation across bacterial membranes occurs during the biological processes of infection by bacteriophages, conjugative DNA transfer of plasmids, T-DNA transfer, and genetic transformation. The mechanism of DNA translocation in these systems is not fully understood, but during the last few years extensive data about genes and gene products involved in the translocation processes have accumulated. One reason for the increasing interest in this topic is the discussion about horizontal gene transfer and transkingdom sex. Analyses of genes and gene products involved in DNA transfer suggest that DNA is transferred through a protein channel spanning the bacterial envelope. No common model exists for DNA translocation during phage infection. Perhaps various mechanisms are necessary as a result of the different morphologies of bacteriophages. The DNA translocation processes during conjugation, T-DNA transfer, and transformation are more consistent and may even be compared to the excretion of some proteins. On the basis of analogies and homologies between the proteins involved in DNA translocation and protein secretion, a common basic model for these processes is presented. PMID:7968916

  19. Collective decision making in bacterial viruses.

    PubMed

    Weitz, Joshua S; Mileyko, Yuriy; Joh, Richard I; Voit, Eberhard O

    2008-09-15

    For many bacterial viruses, the choice of whether to kill host cells or enter a latent state depends on the multiplicity of coinfection. Here, we present a mathematical theory of how bacterial viruses can make collective decisions concerning the fate of infected cells. We base our theory on mechanistic models of gene regulatory dynamics. Unlike most previous work, we treat the copy number of viral genes as variable. Increasing the viral copy number increases the rate of transcription of viral mRNAs. When viral regulation of cell fate includes nonlinear feedback loops, very small changes in transcriptional rates can lead to dramatic changes in steady-state gene expression. Hence, we prove that deterministic decisions can be reached, e.g., lysis or latency, depending on the cellular multiplicity of infection within a broad class of gene regulatory models of viral decision-making. Comparisons of a parameterized version of the model with molecular studies of the decision structure in the temperate bacteriophage lambda are consistent with our conclusions. Because the model is general, it suggests that bacterial viruses can respond adaptively to changes in population dynamics, and that features of collective decision-making in viruses are evolvable life history traits.

  20. Comparing Bacterial DNA Microarray Fingerprints

    SciTech Connect

    Willse, Alan R.; Chandler, Darrell P.; White, Amanda M.; Protic, Miroslava; Daly, Don S.; Wunschel, Sharon C.

    2005-08-15

    Detecting subtle genetic differences between microorganisms is an important problem in molecular epidemiology and microbial forensics. In a typical investigation, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial strains, where the patterns of DNA fragment sizes are proxies for a microbe's genotype. The limited genomic sample captured on a gel is often insufficient to discriminate nearly identical strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if the number of probes on the microarray is sufficiently large, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate the statistical fingerprinting problem for 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

  1. Bacterial Virus Ontology; Coordinating across Databases.

    PubMed

    Hulo, Chantal; Masson, Patrick; Toussaint, Ariane; Osumi-Sutherland, David; de Castro, Edouard; Auchincloss, Andrea H; Poux, Sylvain; Bougueleret, Lydie; Xenarios, Ioannis; Le Mercier, Philippe

    2017-05-23

    Bacterial viruses, also called bacteriophages, display a great genetic diversity and utilize unique processes for infecting and reproducing within a host cell. All these processes were investigated and indexed in the ViralZone knowledge base. To facilitate standardizing data, a simple ontology of viral life-cycle terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address unique viral replication cycle processes, and existing terminology was modified and adapted. Classically, the viral life-cycle is described by schematic pictures. Using this ontology, it can be represented by a combination of successive events: entry, latency, transcription/replication, host-virus interactions and virus release. Each of these parts is broken down into discrete steps. For example enterobacteria phage lambda entry is broken down in: viral attachment to host adhesion receptor, viral attachment to host entry receptor, viral genome ejection and viral genome circularization. To demonstrate the utility of a standard ontology for virus biology, this work was completed by annotating virus data in the ViralZone, UniProtKB and Gene Ontology databases.

  2. Bacterial Virus Ontology; Coordinating across Databases

    PubMed Central

    Hulo, Chantal; Masson, Patrick; Toussaint, Ariane; Osumi-Sutherland, David; de Castro, Edouard; Auchincloss, Andrea H.; Poux, Sylvain; Bougueleret, Lydie; Xenarios, Ioannis; Le Mercier, Philippe

    2017-01-01

    Bacterial viruses, also called bacteriophages, display a great genetic diversity and utilize unique processes for infecting and reproducing within a host cell. All these processes were investigated and indexed in the ViralZone knowledge base. To facilitate standardizing data, a simple ontology of viral life-cycle terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address unique viral replication cycle processes, and existing terminology was modified and adapted. Classically, the viral life-cycle is described by schematic pictures. Using this ontology, it can be represented by a combination of successive events: entry, latency, transcription/replication, host–virus interactions and virus release. Each of these parts is broken down into discrete steps. For example enterobacteria phage lambda entry is broken down in: viral attachment to host adhesion receptor, viral attachment to host entry receptor, viral genome ejection and viral genome circularization. To demonstrate the utility of a standard ontology for virus biology, this work was completed by annotating virus data in the ViralZone, UniProtKB and Gene Ontology databases. PMID:28545254

  3. DNA vaccines for targeting bacterial infections

    PubMed Central

    Ingolotti, Mariana; Kawalekar, Omkar; Shedlock, Devon J; Muthumani, Karuppiah; Weiner, David B

    2010-01-01

    DNA vaccination has been of great interest since its discovery in the 1990s due to its ability to elicit both humoral and cellular immune responses. DNA vaccines consist of a DNA plasmid containing a transgene that encodes the sequence of a target protein from a pathogen under the control of a eukaryotic promoter. This revolutionary technology has proven to be effective in animal models and four DNA vaccine products have recently been approved for veterinary use. Although few DNA vaccines against bacterial infections have been tested, the results are encouraging. Because of their versatility, safety and simplicity a wider range of organisms can be targeted by these vaccines, which shows their potential advantages to public health. This article describes the mechanism of action of DNA vaccines and their potential use for targeting bacterial infections. In addition, it provides an updated summary of the methods used to enhance immunogenicity from codon optimization and adjuvants to delivery techniques including electroporation and use of nanoparticles. PMID:20624048

  4. Recombination in Eukaryotic Single Stranded DNA Viruses

    PubMed Central

    Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-01-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

  5. Recombination in eukaryotic single stranded DNA viruses.

    PubMed

    Martin, Darren P; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-09-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

  6. Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

    USDA-ARS?s Scientific Manuscript database

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek’s disease virus (MDV) is a highly oncogenic herpe...

  7. Bacterial Respiratory Infections Complicating Human Immunodeficiency Virus.

    PubMed

    Feldman, Charles; Anderson, Ronald

    2016-04-01

    Opportunistic bacterial and fungal infections of the lower respiratory tract, most commonly those caused by Streptococcus pneumoniae (the pneumococcus), Mycobacterium tuberculosis, and Pneumocystis jirovecii, remain the major causes of mortality in those infected with human immunodeficiency virus (HIV). Bacterial respiratory pathogens most prevalent in those infected with HIV, other than M. tuberculosis, represent the primary focus of the current review with particular emphasis on the pneumococcus, the leading cause of mortality due to HIV infection in the developed world. Additional themes include (1) risk factors; (2) the predisposing effects of HIV-mediated suppression on pulmonary host defenses, possibly intensified by smoking; (3) clinical and laboratory diagnosis, encompassing assessment of disease severity and outcome; and (4) antibiotic therapy. The final section addresses current recommendations with respect to pneumococcal immunization in the context of HIV infection, including an overview of the rationale underpinning the current "prime-boost" immunization strategy based on sequential administration of pneumococcal conjugate vaccine 13 and pneumococcal polysaccharide vaccine 23.

  8. Structure of large dsDNA viruses

    PubMed Central

    Klose, Thomas; Rossmann, Michael G.

    2015-01-01

    Nucleocytoplasmic large dsDNA viruses (NCLDVs) encompass an ever-increasing group of large eukaryotic viruses, infecting a wide variety of organisms. The set of core genes shared by all these viruses includes a major capsid protein with a double jelly-roll fold forming an icosahedral capsid, which surrounds a double layer membrane that contains the viral genome. Furthermore, some of these viruses, such as the members of the Mimiviridae and Phycodnaviridae have a unique vertex that is used during infection to transport DNA into the host. PMID:25003382

  9. Virulent Marek's Disease Virus Generated from Infectious Bacterial Artificial Chromosome Clones with Complete DNA Sequence and Implication of Viral Genetic Homogeniety in Pathogenesis

    USDA-ARS?s Scientific Manuscript database

    Genetic homogeneity of a test population is essential to precisely associate a viral genome sequence and its phenotype at the nucleotide level. However, homogeneity is not easy to achieve for Marek’s disease virus (MDV) due to its strictly cell-associated replication. To address this problem, two vi...

  10. Detection of pseudorabies virus by DNA hybridization

    SciTech Connect

    McFarlane, R.G.

    1985-01-01

    A DNA hybridization technique was developed in order to detect the presence of pseudorabies virus (PRV) deoxyribonucleic acid (DNA) in swine tissue. Seven, /sup 32/P-nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam H1) restriction fragments of PRV-DNA had been inserted. Under optimal hybridization conditions, the minimum detection level of PRV-DNA was 10/sup -11/ g, which is equivalent to 1 copy of the PRV genome/80 host cells. PRV-DNA was detected in the DNA extracted from the tissues of 10 out of 11 swine previously shown to harbor infective virus. Furthermore, PRV-DNA was present in all four seropositive swine that had recovered from pseudorabies, where no infective virus or viral products were detected at necropsy. The PRV-DNA was present in either the anterior cerebral cortex in 2 swine, or the medulla oblongata and trigeminal ganglion in 1 swine. This perhaps indicates the portal of entry of the virus into the central nervous system. This DNA hybridization assay, which utilizes restriction fragments, may be useful for studying the dynamics and molecular biologic properties of the latency of pseudorabies virus in swine.

  11. Synthesis of Sindbis virus complementary DNA by avian myeloblastosis virus RNA-directed DNA polymerase.

    PubMed

    Yuferov, V; Grandgenett, D P; Bondurant, M; Riggin, C; Tigges, M

    1978-07-24

    Sindbis virus 42 S RNA was efficiently transcribed into complementary DNA (CDNA) by avian myeloblastosis virus alphabeta DNA polymerase using oligo- (dT) or single-stranded calf thymus DNA as primers. Both of the Sindbis virus cDNA products were able to protect 60% of 125I-labeled Sindbis virus RNA, at near equal weight ratios, from RNAase A and T1 digestion. Using hybridization kinetics, the Crt 1/2 value for hybridization of the calf thymus-primed cDNA product with excess Sindbis RNA was determined to be 1.8 9 10-2 mol . s . 1-1. Thes data demonstrate that the Sindbis virus cDNA products are relatively uniform representations of Sindbis virus RNA sequences.

  12. Resistance of Aerosolized Bacterial Viruses to Four Germicidal Products

    PubMed Central

    Turgeon, Nathalie; Michel, Kevin; Ha, Thi-Lan; Robine, Enric; Moineau, Sylvain; Duchaine, Caroline

    2016-01-01

    Viral diseases can spread through a variety of routes including aerosols. Yet, limited data are available on the efficacy of aerosolized chemicals to reduce viral loads in the air. Bacteriophages (phages) are often used as surrogates for hazardous viruses in aerosol studies because they are inexpensive, easy to handle, and safe for laboratory workers. Moreover, several of these bacterial viruses display physical characteristics similar to pathogenic human and animal viruses, like morphological size, type of nucleic acids, capsid morphology, and the presence of an envelope. In this study, the efficacy of four chemicals was evaluated on four airborne phages at two different relative humidity levels. Non-tailed bacteriophages MS2 (single-stranded RNA), ϕ6 (double-stranded RNA, enveloped), PR772 (double-stranded DNA), and ϕX174 (single-stranded DNA) were first aerosolized in a 55L rotative environmental chamber at 19°C with 25% and 50% relative humidity. Then, hydrogen peroxide, Eugenol (phenylpropene used in commercial perfumes and flavorings), Mist® (automobile disinfectant containing Triethylene glycol), and Pledge® (multisurface disinfectant containing Isopropanol, n-Alkyl Dimethyl Benzyl Amonium Chlorides, and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride) were nebulized with the phages using a separate nebulizer. Aerosols were maintained in suspension during 10 minutes, 1 hour, and 2 hours. Viral aerosols were sampled using an SKC BioSampler and samples were analyzed using qPCR and plaque assays. The resistance levels of the four phages varied depending on the relative humidity (RH) and germicidal products tested. Phage MS2 was the most stable airborne virus under the environmental conditions tested while phage PR772 was the least stable. Pledge® and Eugenol reduced the infectivity of all airborne phages tested. At 25% RH, Pledge® and Eugenol were more effective at reducing infectivity of RNA phages ϕ6 and MS2. At 50% RH, Pledge® was the most effective

  13. Resistance of Aerosolized Bacterial Viruses to Four Germicidal Products.

    PubMed

    Turgeon, Nathalie; Michel, Kevin; Ha, Thi-Lan; Robine, Enric; Moineau, Sylvain; Duchaine, Caroline

    2016-01-01

    Viral diseases can spread through a variety of routes including aerosols. Yet, limited data are available on the efficacy of aerosolized chemicals to reduce viral loads in the air. Bacteriophages (phages) are often used as surrogates for hazardous viruses in aerosol studies because they are inexpensive, easy to handle, and safe for laboratory workers. Moreover, several of these bacterial viruses display physical characteristics similar to pathogenic human and animal viruses, like morphological size, type of nucleic acids, capsid morphology, and the presence of an envelope. In this study, the efficacy of four chemicals was evaluated on four airborne phages at two different relative humidity levels. Non-tailed bacteriophages MS2 (single-stranded RNA), ϕ6 (double-stranded RNA, enveloped), PR772 (double-stranded DNA), and ϕX174 (single-stranded DNA) were first aerosolized in a 55L rotative environmental chamber at 19°C with 25% and 50% relative humidity. Then, hydrogen peroxide, Eugenol (phenylpropene used in commercial perfumes and flavorings), Mist® (automobile disinfectant containing Triethylene glycol), and Pledge® (multisurface disinfectant containing Isopropanol, n-Alkyl Dimethyl Benzyl Amonium Chlorides, and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride) were nebulized with the phages using a separate nebulizer. Aerosols were maintained in suspension during 10 minutes, 1 hour, and 2 hours. Viral aerosols were sampled using an SKC BioSampler and samples were analyzed using qPCR and plaque assays. The resistance levels of the four phages varied depending on the relative humidity (RH) and germicidal products tested. Phage MS2 was the most stable airborne virus under the environmental conditions tested while phage PR772 was the least stable. Pledge® and Eugenol reduced the infectivity of all airborne phages tested. At 25% RH, Pledge® and Eugenol were more effective at reducing infectivity of RNA phages ϕ6 and MS2. At 50% RH, Pledge® was the most effective

  14. Temperate bacterial viruses as double-edged swords in bacterial warfare.

    PubMed

    Gama, João Alves; Reis, Ana Maria; Domingues, Iolanda; Mendes-Soares, Helena; Matos, Ana Margarida; Dionisio, Francisco

    2013-01-01

    It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a "replicating toxin". However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails.

  15. Temperate Bacterial Viruses as Double-Edged Swords in Bacterial Warfare

    PubMed Central

    Gama, João Alves; Reis, Ana Maria; Domingues, Iolanda; Mendes-Soares, Helena; Matos, Ana Margarida; Dionisio, Francisco

    2013-01-01

    It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a “replicating toxin”. However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails. PMID:23536852

  16. Structural Organization of DNA in Chlorella Viruses

    PubMed Central

    Wulfmeyer, Timo; Polzer, Christian; Hiepler, Gregor; Hamacher, Kay; Shoeman, Robert; Dunigan, David D.; Van Etten, James L.; Lolicato, Marco; Moroni, Anna; Thiel, Gerhard; Meckel, Tobias

    2012-01-01

    Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes. PMID:22359540

  17. Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses.

    PubMed

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

    2015-12-29

    Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

  18. Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

    PubMed Central

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

    2015-01-01

    Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase. PMID:26780115

  19. 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.

  20. 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

    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

  1. Mobile elements and viral integrations prompt considerations for bacterial DNA integration as a novel carcinogen

    PubMed Central

    Robinson, Kelly M.; Hotopp, Julie C. Dunning

    2014-01-01

    Insertional mutagenesis has been repeatedly demonstrated in cancer genomes and has a role in oncogenesis. Mobile genetic elements can induce cancer development by random insertion into cancer related genes or by inducing translocations. L1s are typically implicated in cancers of an epithelial cell origin, while Alu elements have been implicated in leukemia as well as epithelial cell cancers. Likewise, viral infections have a significant role in cancer development predominantly through integration into the human genome and mutating or deregulating cancer related genes. Human papilloma virus is the best-known example of viral integrations contributing to carcinogenesis. However, hepatitis B virus, Epstein-Barr virus, and Merkel cell polyomavirus also integrate into the human genome and disrupt cancer related genes. Thus far, the role of microbes in cancer has primarily been attributed to mutations induced through chronic inflammation or toxins, as is the case with Helicobacter pylori and enterotoxigenic Bacteroides fragilis. We hypothesize that like mobile elements and viral DNA, bacterial and parasitic DNA may also integrate into the human somatic genome and be oncogenic. Until recently it was believed that bacterial DNA could not integrate into the human genome, but new evidence demonstrates that bacterial insertional mutagenesis may occur in cancer cells. Although this work does not show causation between bacterial insertions and cancer, it prompts more research in this area. Promising new sequencing technologies may reduce the risk of artifactual chimeric sequences, thus diminishing some of the challenges of identifying novel insertions in the somatic human genome. PMID:24956175

  2. Secondary bacterial infections in influenza virus infection pathogenesis.

    PubMed

    Smith, Amber M; McCullers, Jonathan A

    2014-01-01

    Influenza is often complicated by bacterial pathogens that colonize the nasopharynx and invade the middle ear and/or lung epithelium. Incidence and pathogenicity of influenza-bacterial coinfections are multifactorial processes that involve various pathogenic virulence factors and host responses with distinct site- and strain-specific differences. Animal models and kinetic models have improved our understanding of how influenza viruses interact with their bacterial co-pathogens and the accompanying immune responses. Data from these models indicate that considerable alterations in epithelial surfaces and aberrant immune responses lead to severe inflammation, a key driver of bacterial acquisition and infection severity following influenza. However, further experimental and analytical studies are essential to determining the full mechanistic spectrum of different viral and bacterial strains and species and to finding new ways to prevent and treat influenza-associated bacterial coinfections. Here, we review recent advances regarding transmission and disease potential of influenza-associated bacterial infections and discuss the current gaps in knowledge.

  3. DNA Repair Is Associated with Information Content in Bacteria, Archaea, and DNA Viruses.

    PubMed

    Acosta, Sharlene; Carela, Miguelina; Garcia-Gonzalez, Aurian; Gines, Mariela; Vicens, Luis; Cruet, Ricardo; Massey, Steven E

    2015-01-01

    The concept of a "proteomic constraint" proposes that DNA repair capacity is positively correlated with the information content of a genome, which can be approximated to the size of the proteome (P). This in turn implies that DNA repair genes are more likely to be present in genomes with larger values of P. This stands in contrast to the common assumption that informational genes have a core function and so are evenly distributed across organisms. We examined the presence/absence of 18 DNA repair genes in bacterial genomes. A positive relationship between gene presence and P was observed for 17 genes in the total dataset, and 16 genes when only nonintracellular bacteria were examined. A marked reduction of DNA repair genes was observed in intracellular bacteria, consistent with their reduced value of P. We also examined archaeal and DNA virus genomes, and show that the presence of DNA repair genes is likewise related to a larger value of P. In addition, the products of the bacterial genes mutY, vsr, and ndk, involved in the correction of GC/AT mutations, are strongly associated with reduced genome GC content. We therefore propose that a reduction in information content leads to a loss of DNA repair genes and indirectly to a reduction in genome GC content in bacteria by exposure to the underlying AT mutation bias. The reduction in P may also indirectly lead to the increase in substitution rates observed in intracellular bacteria via loss of DNA repair genes.

  4. Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

    SciTech Connect

    Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

    2010-03-08

    Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.

  5. DNA Tumor Viruses and Cell Metabolism.

    PubMed

    Mushtaq, Muhammad; Darekar, Suhas; Kashuba, Elena

    2016-01-01

    Viruses play an important role in cancerogenesis. It is estimated that approximately 20% of all cancers are linked to infectious agents. The viral genes modulate the physiological machinery of infected cells that lead to cell transformation and development of cancer. One of the important adoptive responses by the cancer cells is their metabolic change to cope up with continuous requirement of cell survival and proliferation. In this review we will focus on how DNA viruses alter the glucose metabolism of transformed cells. Tumor DNA viruses enhance "aerobic" glycolysis upon virus-induced cell transformation, supporting rapid cell proliferation and showing the Warburg effect. Moreover, viral proteins enhance glucose uptake and controls tumor microenvironment, promoting metastasizing of the tumor cells.

  6. The Autonomous Glycosylation of Large DNA Viruses

    PubMed Central

    Piacente, Francesco; Gaglianone, Matteo; Laugieri, Maria Elena; Tonetti, Michela G.

    2015-01-01

    Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production. PMID:26690138

  7. A model capturing novel strand symmetries in bacterial DNA

    SciTech Connect

    Sobottka, Marcelo; Hart, Andrew G.

    2011-07-15

    Highlights: {yields} We propose a simple stochastic model to construct primitive DNA sequences. {yields} The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. {yields} The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. {yields} We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. {yields} We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

  8. A Hypothesis for DNA Viruses as the Origin of Eukaryotic Replication Proteins

    PubMed Central

    Villarreal, Luis P.; DeFilippis, Victor R.

    2000-01-01

    The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction. PMID:10888648

  9. Evolution of viruses by acquisition of cellular RNA or DNA nucleotide sequences and genes: an introduction.

    PubMed

    Becker, Y

    2000-01-01

    The origins of virus evolution may be traced to Archeabacteria since Inouye and Inouye (6) discovered a retroelement with a gene for reverse transcriptase in the bacterial genome and in the satellite, multiple copy single stranded DNA (msDNA) in the soil bacterium Myxococcus xanthus. It was possible (8) to define the evolution of retroelements in eukaryotic cells of plants, insects (gypsy retrovirus) and vertebrates. The replication of RNA viruses in eukaryotic cells allowed for the viral RNA genome to integrate a cellular ubiquitin mRNA, as reported for BVDV (24). Another example is the integration of 28S ribosomal RNA into the hemagglutinin gene of an influenza virus. This change in the hemagglutinin gene led to an increased pathogenicity of the influenza virus (25). In contrast to RNA viruses, DNA viruses had evolved by inserting cDNA molecules derived from mRNA transcripts of cellular genes or foreign viral RNA. It is of interest that the virus acquired cellular genes in the genomes of DNA viruses represent genes that code for proteins that inhibit cellular molecular processes related to HLA class I and II molecules. The other acquired genes are cellular genes that code for cytokines that are capable of inhibiting antigen presentation to T cells by antigen presenting cells (APC) by dendritic Langerhans cells. The acquisition of cellular genes by DNA viruses enhances their pathogenicity by inhibiting the hosts' defense systems.

  10. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase.

    PubMed

    Papillon, Julie; Ménétret, Jean-François; Batisse, Claire; Hélye, Reynald; Schultz, Patrick; Potier, Noëlle; Lamour, Valérie

    2013-09-01

    Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a 'crossover trap' that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal β-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and β-pinwheel domains. The overall conformation of the drug-induced DNA binding-cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation.

  11. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase

    PubMed Central

    Papillon, Julie; Ménétret, Jean-François; Batisse, Claire; Hélye, Reynald; Schultz, Patrick; Potier, Noëlle; Lamour, Valérie

    2013-01-01

    Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a ‘crossover trap’ that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal β-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and β-pinwheel domains. The overall conformation of the drug-induced DNA binding–cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation. PMID:23804759

  12. Presence of Bacterial Phage-Like DNA Sequences in Commercial Taq DNA Polymerase Reagents

    PubMed Central

    Newsome, Tamara; Li, Bing-Jie; Zou, Nianxiang; Lo, Shyh-Ching

    2004-01-01

    Many studies have reported the presence of bacterial DNA contamination in commercial Taq DNA polymerase reagents. This is the first report of the presence of phage-like DNA sequences in certain commercial Taq DNA polymerase reagents. Precautions are needed when using amplification reagents with exogenous DNAs. PMID:15131208

  13. Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

    PubMed Central

    Church, Geoffrey A.; Dasgupta, Anindya; Wilson, Duncan W.

    1998-01-01

    Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain tsProt.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis. PMID:9525593

  14. Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

    PubMed

    Dorn-In, Samart; Bassitta, Rupert; Schwaiger, Karin; Bauer, Johann; Hölzel, Christina S

    2015-06-01

    Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.

  15. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    PubMed

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  16. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

    PubMed Central

    Almazán, Fernando; González, José M.; Pénzes, Zoltan; Izeta, Ander; Calvo, Enrique; Plana-Durán, Juan; Enjuanes, Luis

    2000-01-01

    The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses. PMID:10805807

  17. DNA Topology and the Initiation of Virus DNA Packaging

    PubMed Central

    Oh, Choon Seok; Sippy, Jean; Charbonneau, Bridget; Crow Hutchinson, Jennifer; Mejia-Romero, Olga Esther; Barton, Michael; Patel, Priyal; Sippy, Rachel; Feiss, Michael

    2016-01-01

    During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase’s small subunit (TerS). The large terminase subunit (TerL) contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead’s portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented. PMID:27144448

  18. Superstatistical model of bacterial DNA architecture

    PubMed Central

    Bogachev, Mikhail I.; Markelov, Oleg A.; Kayumov, Airat R.; Bunde, Armin

    2017-01-01

    Understanding the physical principles that govern the complex DNA structural organization as well as its mechanical and thermodynamical properties is essential for the advancement in both life sciences and genetic engineering. Recently we have discovered that the complex DNA organization is explicitly reflected in the arrangement of nucleotides depicted by the universal power law tailed internucleotide interval distribution that is valid for complete genomes of various prokaryotic and eukaryotic organisms. Here we suggest a superstatistical model that represents a long DNA molecule by a series of consecutive ~150 bp DNA segments with the alternation of the local nucleotide composition between segments exhibiting long-range correlations. We show that the superstatistical model and the corresponding DNA generation algorithm explicitly reproduce the laws governing the empirical nucleotide arrangement properties of the DNA sequences for various global GC contents and optimal living temperatures. Finally, we discuss the relevance of our model in terms of the DNA mechanical properties. As an outlook, we focus on finding the DNA sequences that encode a given protein while simultaneously reproducing the nucleotide arrangement laws observed from empirical genomes, that may be of interest in the optimization of genetic engineering of long DNA molecules. PMID:28225058

  19. Superstatistical model of bacterial DNA architecture

    NASA Astrophysics Data System (ADS)

    Bogachev, Mikhail I.; Markelov, Oleg A.; Kayumov, Airat R.; Bunde, Armin

    2017-02-01

    Understanding the physical principles that govern the complex DNA structural organization as well as its mechanical and thermodynamical properties is essential for the advancement in both life sciences and genetic engineering. Recently we have discovered that the complex DNA organization is explicitly reflected in the arrangement of nucleotides depicted by the universal power law tailed internucleotide interval distribution that is valid for complete genomes of various prokaryotic and eukaryotic organisms. Here we suggest a superstatistical model that represents a long DNA molecule by a series of consecutive ~150 bp DNA segments with the alternation of the local nucleotide composition between segments exhibiting long-range correlations. We show that the superstatistical model and the corresponding DNA generation algorithm explicitly reproduce the laws governing the empirical nucleotide arrangement properties of the DNA sequences for various global GC contents and optimal living temperatures. Finally, we discuss the relevance of our model in terms of the DNA mechanical properties. As an outlook, we focus on finding the DNA sequences that encode a given protein while simultaneously reproducing the nucleotide arrangement laws observed from empirical genomes, that may be of interest in the optimization of genetic engineering of long DNA molecules.

  20. The immunology of influenza virus-associated bacterial pneumonia.

    PubMed

    Robinson, Keven M; Kolls, Jay K; Alcorn, John F

    2015-06-01

    Infection with influenza virus has been a significant cause of morbidity and mortality for more than a hundred years. Severe disease and increased mortality often results from bacterial super-infection of patients with influenza virus infection. Preceding influenza infection alters the host's innate and adaptive immune responses, allowing increased susceptibility to secondary bacterial pneumonia. Recent advances in the field have helped to define how influenza alters the immune response to bacteria through the dysregulation of phagocytes, antimicrobial peptides, and lymphocytes. Viral-induced interferons play a key role in altering the phenotype of the immune response. Potential genetic modifiers of disease will help to define additional immunologic mechanisms that predispose to viral, bacterial super-infection with the overarching goal of developing effective therapeutic strategies to prevent and treat disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Detection of DNA viruses in prostate cancer.

    PubMed

    Smelov, Vitaly; Bzhalava, Davit; Arroyo Mühr, Laila Sara; Eklund, Carina; Komyakov, Boris; Gorelov, Andrey; Dillner, Joakim; Hultin, Emilie

    2016-04-28

    We tested prostatic secretions from men with and without prostate cancer (13 cases and 13 matched controls) or prostatitis (18 cases and 18 matched controls) with metagenomic sequencing. A large number (>200) of viral reads was only detected among four prostate cancer cases (1 patient each positive for Merkel cell polyomavirus, JC polyomavirus and Human Papillomavirus types 89 or 40, respectively). Lower numbers of reads from a large variety of viruses were detected in all patient groups. Our knowledge of the biology of the prostate may be furthered by the fact that DNA viruses are commonly shed from the prostate and can be readily detected by metagenomic sequencing of expressed prostate secretions.

  2. Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines.

    PubMed

    Alberdi, M Pilar; Dalby, Matthew J; Rodriguez-Andres, Julio; Fazakerley, John K; Kohl, Alain; Bell-Sakyi, Lesley

    2012-06-01

    As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed 'tick-only' viruses inhabiting tick cell lines.

  3. Mechanics of DNA Packing in Viruses

    NASA Astrophysics Data System (ADS)

    Phillips, Rob

    2004-03-01

    Viruses are amongst the most beautiful and fascinating of self-assembled structures. Recently, as a result of the confluence of techniques ranging from structural biology to single molecule biophysics, it has become possible to obtain quantitative insights into the physical processes that attend the viral life cycle. In particular, the forces that build up as a result of the DNA packing process have been measured in optical tweezers experiments. The aim of this talk is to describe such experiments on the mechanics of viral DNA packing and ejection and to show how these experiments can be greeted with simple, yet predictive models.

  4. Innate Immune sensing of DNA viruses

    PubMed Central

    Rathinam, Vijay A. K.; Fitzgerald, Katherine A.

    2011-01-01

    DNA viruses are a significant contributor to human morbidity and mortality. The immune system protects against viral infections through coordinated innate and adaptive immune responses. While the antigen-specific adaptive mechanisms have been extensively studied, the critical contributions of innate immunity to anti-viral defenses have only been revealed in the very recent past. Central to these anti-viral defenses is the recognition of viral pathogens by a diverse set of germ-line encoded receptors that survey nearly all cellular compartments for the presence of pathogens. In this review, we discuss the recent advances in the innate immune sensing of DNA viruses and focus on the recognition mechanisms involved. PMID:21334037

  5. Bacterial diversity in shallow oligotrophic marine benthos and overlying waters: effects of virus infection, containment, and nutrient enrichment.

    PubMed

    Hewson, I; Vargo, G A; Fuhrman, J A

    2003-10-01

    Little is known of the factors shaping sediment bacterial communities, despite their high abundance and reports of high diversity. Two factors hypothesized to shape bacterial communities in the water column are nutrient (resource) availability and virus infection. The role these factors play in benthic bacterial diversity was assessed in oligotrophic carbonate-based sediments of Florida Bay (USA). Sediment-water mesocosm enclosures were made from 1-m diameter clear polycarbonate cylinders which were pushed into sediments to approximately 201 cm sediment depth enclosing approximately 80 L of water. Mesocosms were amended each day for 14 d with 10 microM NH4+ and 1 microM PO4(3-). In a second experiment, viruses from a benthic flocculent layer were concentrated and added back to flocculent layer samples which were collected near the mesocosm enclosures. Photosynthesis by microalgae in virus-amended incubations was monitored by pulse-amplitude modulated (PAM) fluorescence. In both experiments, bacterial diversity was estimated using automated rRNA intergenic spacer analysis (ARISA), a high-resolution fingerprinting approach. Initial sediment bacterial operational taxonomic unit (OTU) richness (236 +/- 3) was higher than in the water column (148 +/- 9), where an OTU was detectable when its amplified DNA represented >0.09% of the total amplified DNA. Effects on bacterial diversity and operational taxonomic unit (OTU) richness in nutrient-amended mesocosms may have been masked by the effects of containment, which stimulated OTU richness in the water column, but depressed OTU richness and diversity in sediments. Nutrient addition significantly elevated virus abundance and the ratio of viruses to bacteria (p < 0.05 for both) in the sediments, concomitant with elevated bacterial diversity. However, water column bacterial diversity (in unamended controls) was not affected by nutrient amendments, which may be due to rapid nutrient uptake by sediment organisms or adsorption of

  6. Cryo-electron tomography of bacterial viruses

    SciTech Connect

    Guerrero-Ferreira, Ricardo C.; Wright, Elizabeth R.

    2013-01-05

    Bacteriophage particles contain both simple and complex macromolecular assemblages and machines that enable them to regulate the infection process under diverse environmental conditions with a broad range of bacterial hosts. Recent developments in cryo-electron tomography (cryo-ET) make it possible to observe the interactions of bacteriophages with their host cells under native-state conditions at unprecedented resolution and in three-dimensions. This review describes the application of cryo-ET to studies of bacteriophage attachment, genome ejection, assembly and egress. Current topics of investigation and future directions in the field are also discussed.

  7. Two-dimensional DNA displays for comparisons of bacterial genomes

    PubMed Central

    Malloff, Chad; Dullaghan, Edie; Li, Alice; Stokes, Richard; Lam, Wan

    2003-01-01

    We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD) was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE) in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus) numerous small changes (i.e. small deletions and point mutations) unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH), was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals) represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA. PMID:14569612

  8. Horizontal transfer of DNA methylation patterns into bacterial chromosomes.

    PubMed

    Shin, Jung-Eun; Lin, Chris; Lim, Han N

    2016-05-19

    Horizontal gene transfer (HGT) is the non-inherited acquisition of novel DNA sequences. HGT is common and important in bacteria because it enables the rapid generation of new phenotypes such as antibiotic resistance. Here we show that in vivo and in vitro DNA methylation patterns can be horizontally transferred into bacterial chromosomes to program cell phenotypes. The experiments were performed using a synthetic system in Escherichia coli where different DNA methylation patterns within the cis-regulatory sequence of the agn43 gene turn on or off a fluorescent reporter (CFP). With this system we demonstrated that DNA methylation patterns not only accompany the horizontal transfer of genes into the bacterial cytoplasm but can be transferred into chromosomes by: (i) bacteriophage P1 transduction; and (ii) transformation of extracellular synthetic DNA. We also modified the experimental system by replacing CFP with the SgrS small RNA, which regulates glucose and methyl α-D-glucoside uptake, and showed that horizontally acquired DNA methylation patterns can increase or decrease cell fitness. That is, horizontally acquired DNA methylation patterns can result in the selection for and against cells that have HGT. Findings from these proof-of-concept experiments have applications in synthetic biology and potentially broad implications for bacterial adaptation and evolution. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, Susana; Arias, Carlos F.

    2014-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  10. DNA microarray for detection of gastrointestinal viruses.

    PubMed

    Martínez, Miguel A; Soto-Del Río, María de Los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y; Greninger, Alexander L; Contreras, Juan Francisco; López, Susana; Arias, Carlos F; Isa, Pavel

    2015-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  11. Rescue of measles viruses from cloned DNA.

    PubMed Central

    Radecke, F; Spielhofer, P; Schneider, H; Kaelin, K; Huber, M; Dötsch, C; Christiansen, G; Billeter, M A

    1995-01-01

    A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome. Images PMID:8846771

  12. Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

    PubMed Central

    Guo, Jin-Chao; Tang, Yan-Dong; Zhao, Kuan; Wang, Tong-Yun; Liu, Ji-Ting; Gao, Jia-Cong; Chang, Xiao-Bo; Cui, Hong-Yu; Tian, Zhi-Jun; Cai, Xue-Hui; An, Tong-Qing

    2016-01-01

    Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses. PMID:28066407

  13. Interplay between DNA tumor viruses and the host DNA damage response.

    PubMed

    McFadden, Karyn; Luftig, Micah A

    2013-01-01

    Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication.

  14. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    PubMed

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  15. Hyperchromicity and strand separation in bacterial DNA.

    PubMed

    FREIFELDER, D; DAVISON, P F

    1962-05-01

    Studies of the per cent of strand separation of N(14)-N(15) hybrid coli DNA heated to various temperatures in formaldehyde have shown that the process of strand separation is a function of temperature and formaldehyde concentration and is directly related to the measured hyperchromicity. No strands separate until about 75 per cent of full hyperchromicity is obtained, and even at apparently full hyperchromicity a large fraction of the strands may be held together, possibly by guanine-cytosine-rich regions.

  16. Bacterial natural transformation by highly fragmented and damaged DNA.

    PubMed

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic A A; Mayar, J Victor Moreno; Rasmussen, Simon; Dahl, Tais W; Rosing, Minik T; Poole, Anthony M; Sicheritz-Ponten, Thomas; Brunak, Søren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G; Nielsen, Rasmus; Johnsen, Pål Jarle; Nielsen, Kaare Magne; Willerslev, Eske

    2013-12-03

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥ 20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.

  17. Rescue of rinderpest virus from cloned cDNA.

    PubMed Central

    Baron, M D; Barrett, T

    1997-01-01

    Rinderpest virus is a morbillivirus and is the causative agent of a widespread and important disease of cattle. The viral genome is a single strand of RNA in the negative sense. We have constructed plasmids containing cDNA copies of the 5' and 3' termini of the virus separated by a reporter gene and have shown that antigenome-sense RNA transcripts of these model genomes can be replicated, transcribed, and packaged by helper virus, both rinderpest virus and the related measles virus. Further, these genome analogs can be replicated and transcribed by viral proteins expressed from cDNA clones by using a recombinant vaccinia virus expressing T7 RNA polymerase (MVA-T7). Using this latter system, we have rescued live rinderpest virus from a full-length cDNA copy of the genome of the RBOK vaccine strain. The recombinant virus appears to grow in tissue culture identically to the original virus. PMID:8995650

  18. Bacterial DNA replication enzymes as targets for antibacterial drug discovery.

    PubMed

    Sanyal, Gautam; Doig, Peter

    2012-04-01

    The bacterial replisome is composed of a large number of enzymes, which work in exquisite coordination to accomplish chromosomal replication. Effective inhibition inside the bacterial cell of any of the 'essential' enzymes of the DNA replication pathway should be detrimental to cell survival. This review covers DNA replication enzymes that have been shown to have a potential for delivering antibacterial compounds or drug candidates including: type II topoisomerases, a clinically validated target family, and DNA ligase, which has yielded inhibitors with in vivo efficacy. A few of the 'replisome' enzymes that are structurally and functionally well characterized and have been subjects of antibacterial discovery efforts are also discussed. Identification of several essential genes in the bacterial replication pathway raised hopes that targeting these gene products would lead to novel antibacterials. However, none of these novel, single gene targets have delivered antibacterial drug candidates into clinical trials. This lack of productivity may be due to the target properties and inhibitor identification approaches employed. For DNA primase, DNA helicase and other replisome targets, with the exception of DNA ligase, the exploitation of structure for lead generation has not been tested to the same extent that it has for DNA gyrase. Utilization of structural information should be considered to augment HTS efforts and initiate fragment-based lead generation. The complex protein-protein interactions involved in regulation of replication may explain why biochemical approaches have been less productive for some replisome targets than more independently functioning targets such as DNA ligase or DNA gyrase. © 2012 Informa UK, Ltd.

  19. Hyperchromicity and Strand Separation in Bacterial DNA

    PubMed Central

    Freifelder, David; Davison, Peter F.

    1962-01-01

    Studies of the per cent of strand separation of N14-N15 hybrid coli DNA heated to various temperatures in formaldehyde have shown that the process of strand separation is a function of temperature and formaldehyde concentration and is directly related to the measured hyperchromicity. No strands separate until about 75 per cent of full hyperchromicity is obtained, and even at apparently full hyperchromicity a large fraction of the strands may be held together, possibly by guanine-cytosine-rich regions. PMID:13894962

  20. Detection of DNA viruses in prostate cancer

    PubMed Central

    Smelov, Vitaly; Bzhalava, Davit; Arroyo Mühr, Laila Sara; Eklund, Carina; Komyakov, Boris; Gorelov, Andrey; Dillner, Joakim; Hultin, Emilie

    2016-01-01

    We tested prostatic secretions from men with and without prostate cancer (13 cases and 13 matched controls) or prostatitis (18 cases and 18 matched controls) with metagenomic sequencing. A large number (>200) of viral reads was only detected among four prostate cancer cases (1 patient each positive for Merkel cell polyomavirus, JC polyomavirus and Human Papillomavirus types 89 or 40, respectively). Lower numbers of reads from a large variety of viruses were detected in all patient groups. Our knowledge of the biology of the prostate may be furthered by the fact that DNA viruses are commonly shed from the prostate and can be readily detected by metagenomic sequencing of expressed prostate secretions. PMID:27121729

  1. Proteolysis in plasmid DNA stable maintenance in bacterial cells.

    PubMed

    Karlowicz, Anna; Wegrzyn, Katarzyna; Dubiel, Andrzej; Ropelewska, Malgorzata; Konieczny, Igor

    2016-07-01

    Plasmids, as extrachromosomal genetic elements, need to work out strategies that promote independent replication and stable maintenance in host bacterial cells. Their maintenance depends on constant formation and dissociation of nucleoprotein complexes formed on plasmid DNA. Plasmid replication initiation proteins (Rep) form specific complexes on direct repeats (iterons) localized within the plasmid replication origin. Formation of these complexes along with a strict control of Rep protein cellular concentration, quaternary structure, and activity, is essential for plasmid maintenance. Another important mechanism for maintenance of low-copy-number plasmids are the toxin-antitoxin (TA) post-segregational killing (psk) systems, which prevent plasmid loss from the bacterial cell population. In this mini review we discuss the importance of nucleoprotein complex processing by energy-dependent host proteases in plasmid DNA replication and plasmid type II toxin-antitoxin psk systems, and draw attention to the elusive role of DNA in this process.

  2. Gene Specific Impedimetric Bacterial DNA Sensor for Rheumatic Heart Disease.

    PubMed

    Singh, Swati; Kaushal, Ankur; Gupta, Sunil; Kumar, Ashok

    2017-03-01

    An impedimetric mga gene specific DNA sensor was developed by immobilization of single stranded DNA probe onto the screen printed modified gold-dendrimer nanohybrid composite electrode for early and rapid detection of S. pyogenes in human throat swab samples causing rheumatic heart disease. Electrochemical impedance response was measured after hybridization with bacterial single stranded genomic DNA (ssG-DNA) with probe. The sensor was found highly specific to S. pyogenes and can detect as low as 0.01 ng ssDNA in 6 µL sample only in 30 min. The nanohybrid sensor was also tested with non-specific pathogens and characterized by FTIR. An early detection of the pathogen S. pyogenes in human can save damage of mitral and aortic heart valves (rheumatic heart disease) by proper medical care.

  3. Recovery of recombinant bacterial plasmids from E. coli transformed with DNA from microinjected mouse cells.

    PubMed Central

    Kretschmer, P J; Bowman, A H; Huberman, M H; Sanders-Haigh, L; Killos, L; Anderson, W F

    1981-01-01

    We have previously described the isolation of thymidine kinase positive (TK+), human beta-globin gene-containing colonies following co-microinjection of mouse TK- L cells with two recombinant pBR322 plasmids, one containing the TK gene of herpes simplex virus type I (plasmid pXl), and the second containing a human genomic DNA fragment within which is the human beta-globin gene (plasmid pRKl). DNA isolated from one such clone was used in bacterial transformation experiments with a selection for tetracycline-resistant colonies (that is, for cells containing pRKl). A total of forty-two tetracycline-resistant colonies were isolated, thirty of which contained circular pRK1 molecules identical to those originally injected. The remaining twelve colonies contained unique plasmids that were grouped into five different classes of recombinant molecules. All five of these unique recombinant classes appear to contain a common deletion endpoint occurring at a specific region of the pBR322 segment of pRKl. Four of the unique recombinant classes appear to have arisen from the deletion of a segment of a pRKl trimer or dimer molecule, while the fifth class appears to have resulted from recombination between pRKl and pXl followed by a deletion event within this recombinant. It is uncertain whether these deletions are occurring within the eukaryotic cell or upon subsequent transformation of the bacterial cell. If the latter, then the passage of the plasmid DNA through the eukaryotic cell alters a specific site of the pBR322 DNA in such a way that deletions can occur at a high frequency in this region when the plasmid DNA is introduced back into a bacterial cell. Thus, we have established a prokaryote-eukaryote-prokaryote DNA transfer and recovery system which should be useful in studies on DNA replication and the regulation of gene expression in higher eukaryotes. Images PMID:6273826

  4. 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

  5. Molecular hybridization with DNA-probes as a laboratory diagnostic test for influenza viruses.

    PubMed

    Pljusnin, A Z; Rozhkova, S A; Nolandt, O V; Bryantseva, E A; Kuznetsov, O K; Noskov, F S

    1987-01-01

    The possibilities of using DNA-copies of different influenza A virus genes cloned with recombinant bacterial plasmids for the detection of virus-specific RNA by molecular dot-hybridization were analyzed. High specificity of RNA identification has been demonstrated and it has been shown expedient to use DNA-probes with high-conservative virus genes (polymerase, nucleoprotein, or matrix) for the detection of influenza A virus subtypes (H1N1, H2N2, H3N2) and probes with corresponding hemagglutinin genes for the differentiation of the subtypes H3N2 and H1N1. The results of nasopharyngeal specimens testing proved the effectiveness of molecular dot-hybridization in epidemiological studies of influenza outbreaks, especially of mixed etiology.

  6. [Genetic transformation and fate of heterological DNA in bacterial cells].

    PubMed

    Piechowska, Mirosława

    2015-01-01

    Secretion of a metabolite enabling Streptococci to undergo genetic transformation was discovered. The metabolite combined with an optimization process were applied to increase the transformation yield about 20-fold. It was observed that large amounts of DNA exert a bactericidal effect, indicating the ability of at least 70% of cells to uptake the polymer. While studying the molecular mechanism of transformation of Bacillus subtilis it was shown that the uptaken DNA forms complexes with bacterial proteins, which hinders determination of its structure. A method was found to dissociate these complexes which enabled to determine the single-stranded structure of the uptaken DNA. Donor DNA fragments incorporated into the host DNA were of about 10 Da. Non-transforming DNA can be uptaken similarly but does not undergo incorporation into the host DNA. The selectivity of Bacillus subtilis receptors was determined towards DNA of phages containing modified bases: uracil, putrescinyl-thymine and its acetylated derivative, 5'-hydroxymethylcytosine and its glycosylated derivative and also towards double-stranded RNA of f2 phage. All these modifications were tolerated by the cellular receptors, with the exception of glycosylation and the 2'-OH group in RNA.

  7. Licensed DNA Vaccines against Infectious Hematopoietic Necrosis Virus (IHNV).

    PubMed

    Alonso, Marta; Leong, Jo-Ann C

    2013-04-01

    This article reviews some of the recent patents on DNA vaccines against fish viruses, in particular against the novirhabdovirus infectious hematopoitic necrosis virus (IHNV). Although very effective in protecting fish against IHNV, only one DNA vaccine has been approved to date for use in Canada. In Europe and in US, its commercialization is restricted due to safety concerns.

  8. Viral hemorrhagic fevers of animals caused by DNA viruses

    USDA-ARS?s Scientific Manuscript database

    Here we outline serious diseases of food and fiber animals that cause damaging economic effect on products all over the world. The only vector-borne DNA virus is included here, such as African swine fever virus, and the herpes viruses discussed have a complex epidemiology characterized by outbreak...

  9. Identification of DNA viruses by membrane filter hybridization.

    PubMed Central

    Stålhandske, P; Pettersson, U

    1982-01-01

    The use of membrane filter hybridization for the identification of DNA viruses is described. We designed and used a procedure for identification of herpes simplex virus. This method can discriminate between herpes simplex virus types 1 and 2 in a simple way. Images PMID:6279697

  10. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  11. Structures of giant icosahedral eukaryotic dsDNA viruses

    PubMed Central

    Xiao, Chuan; Rossmann, Michael G.

    2011-01-01

    In the last twenty years, numerous giant, dsDNA, icosahedral viruses have been discovered and assigned to the nucleocytoplasmic large dsDNA virus (NCLDV) clade. The major capsid proteins of these viruses consist of two consecutive jelly-roll domains, assembled into trimers, with pseudo 6-fold symmetry. The capsomers are assembled into arrays that have either p6 (as in Paramecium bursaria Chlorella virus-1) or p3 symmetry (as in Mimivirus). Most of the NCLDV viruses have a membrane that separates the nucleocapsid from the external capsid. PMID:21909343

  12. Structures of giant icosahedral eukaryotic dsDNA viruses.

    PubMed

    Xiao, Chuan; Rossmann, Michael G

    2011-08-01

    In the last twenty years, numerous giant, dsDNA, icosahedral viruses have been discovered and assigned to the nucleocytoplasmic large dsDNA virus (NCLDV) clade. The major capsid proteins of these viruses consist of two consecutive jelly-roll domains, assembled into trimers, with pseudo 6-fold symmetry. The capsomers are assembled into arrays that have either p6 (as in Paramecium bursaria Chlorella virus-1) or p3 symmetry (as in Mimivirus). Most of the NCLDV viruses have a membrane that separates the nucleocapsid from the external capsid.

  13. Amyloid-DNA composites of bacterial biofilms stimulate autoimmunity

    PubMed Central

    Gallo, Paul M.; Rapsinski, Glenn J.; Wilson, R. Paul; Oppong, Gertrude O.; Sriram, Uma; Goulian, Mark; Buttaro, Bettina; Caricchio, Roberto; Gallucci, Stefania; Tükel, Çagla

    2015-01-01

    SUMMARY Research on the human microbiome has established that commensal and pathogenic bacteria can influence obesity, cancer, and autoimmunity through mechanisms mostly unknown. We found that a component of bacterial biofilms, the amyloid protein curli, irreversibly formed fibers with bacterial DNA during biofilm formation. This interaction accelerated amyloid polymerization and created potent immunogenic complexes that activated immune cells, including dendritic cells, to produce cytokines such as Type I interferons, which are pathogenic in systemic lupus erythematosus (SLE). When given systemically, curli-DNA composites triggered immune activation and production of autoantibodies in lupus-prone and wild-type mice. We also found that the infection of lupus-prone mice with curli-producing bacteria triggered higher autoantibody titers compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity. PMID:26084027

  14. Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

    NASA Astrophysics Data System (ADS)

    Chen, K.

    2017-01-01

    With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

  15. Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.

    PubMed

    Lohman, Gregory J S; Zhang, Yinhua; Zhelkovsky, Alexander M; Cantor, Eric J; Evans, Thomas C

    2014-02-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

  16. Sensing Viruses by Mechanical Tension of DNA in Responsive Hydrogels

    NASA Astrophysics Data System (ADS)

    Shin, Jaeoh; Cherstvy, Andrey G.; Metzler, Ralf

    2014-04-01

    The rapid worldwide spread of severe viral infections, often involving novel mutations of viruses, poses major challenges to our health-care systems. This means that tools that can efficiently and specifically diagnose viruses are much needed. To be relevant for broad applications in local health-care centers, such tools should be relatively cheap and easy to use. In this paper, we discuss the biophysical potential for the macroscopic detection of viruses based on the induction of a mechanical stress in a bundle of prestretched DNA molecules upon binding of viruses to the DNA. We show that the affinity of the DNA to the charged virus surface induces a local melting of the double helix into two single-stranded DNA. This process effects a mechanical stress along the DNA chains leading to an overall contraction of the DNA. Our results suggest that when such DNA bundles are incorporated in a supporting matrix such as a responsive hydrogel, the presence of viruses may indeed lead to a significant, macroscopic mechanical deformation of the matrix. We discuss the biophysical basis for this effect and characterize the physical properties of the associated DNA melting transition. In particular, we reveal several scaling relations between the relevant physical parameters of the system. We promote this DNA-based assay as a possible tool for efficient and specific virus screening.

  17. Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology

    PubMed Central

    Grasso, Francesca; Frisan, Teresa

    2015-01-01

    Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins. PMID:26270677

  18. Bacterial-based Systems for Expression and Purification of Recombinant Lassa Virus Proteins of Immunological Relevance

    DTIC Science & Technology

    2008-06-06

    Biowarfare potential further jus- tifies the development of countermeasures against this highly virulent class of viruses . Methods Virus , cells, plasmids...BioMed CentralVirology Journal ssOpen AcceResearch Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of...recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for

  19. Detection of Vaccinia Virus DNA, but not Infectious Virus, in the Blood of Smallpox Vaccine Recipients

    PubMed Central

    Cohen, Jeffrey I.; Hohman, Patricia; Preuss, Jeanne C.; Li, Li; Fischer, Steven H.; Fedorko, Daniel P.

    2007-01-01

    The authors of a recent study suggested that the duration of deferral for blood donations by smallpox vaccinees should be extended, based on detection of vaccinia virus DNA in 5 blood samples by PCR and the potential for viremia. We found that 4 of 202 blood specimens (from 3 of 27 smallpox vaccinees) were positive for vaccinia virus DNA by PCR; none were positive for virus by culture. Throat swabs were negative by PCR and culture. Thus, while some blood specimens contained vaccinia virus DNA, infectious virus was not detected. Current guidelines for deferral of blood donation in vaccinees seem appropriate. PMID:17493714

  20. Solid-to-fluid – like DNA transition in viruses facilitates infection

    SciTech Connect

    Liu, Ting; Sae-Ueng, Udom; Li, Dong; Lander, Gabriel C.; Zuo, Xiaobing; Jonsson, Bengt; Rau, Donald; Shefer, Ivetta; Evilevitch, Alex

    2014-10-14

    Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single- molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ -DNA undergoes a solid-to-fluid – like disordering transition as a function of temperature, resultin g locally in less densely packed DNA, reducing DNADNA repulsions. This p rocess leads to a sig- nificant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkab le physical adaptation of bac- terial viruses to the environment of Escherichia coli cells in a human host.

  1. [Paradoxes of replication of RNA of a bacterial virus].

    PubMed

    Chetverin, A B

    2011-01-01

    The extraordinary ability of the bacteriophage Qbeta replicase to amplify RNA outside the cell attracted attention of molecular biologists in the late 60's-early 70's. However, at that time, a number of puzzling properties of the enzyme did not received a rational explanation. Only recently, Qbeta-replicase began to uncover its secrets, promising to give a key not only to understanding the mechanism of replication of the genome of the bacterial virus, but also to the solution of more general fundamental and applied problems.

  2. Mechanics of DNA packaging in viruses

    PubMed Central

    Purohit, Prashant K.; Kondev, Jané; Phillips, Rob

    2003-01-01

    A new generation of single-molecule experiments has opened up the possibility of reexamining many of the fundamental processes of biochemistry and molecular biology from a unique and quantitative perspective. One technique producing a host of intriguing results is the use of optical tweezers to measure the mechanical forces exerted by molecular motors during key processes such as the transcription of DNA or the packing of a viral genome into its capsid. The objective of the current article is to respond to such measurements on viruses and to use the theory of elasticity and a simple model of charge and hydration forces to derive the force required to pack DNA into a viral capsid as a function of the fraction of the viral genome that has been packed. The results are found to be in excellent accord with recent measurements and complement previous theoretical work. Because the packing of DNA in viral capsids occurs under circumstances of high internal pressure, we also compute how much pressure a capsid can sustain without rupture. PMID:12629206

  3. Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

    PubMed Central

    Kang, Chil-Yong; Temin, Howard M.

    1974-01-01

    Reticuloendotheliosis virus 60S RNA labeled with 125I, or reticuloendotheliosis virus complementary DNA labeled with 3H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and 125I-labeled viral RNAs and by determination of melting temperatures. In contrast, 125I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck. PMID:4372393

  4. Viral Carcinogenesis: Factors Inducing DNA Damage and Virus Integration

    PubMed Central

    Chen, Yan; Williams, Vonetta; Filippova, Maria; Filippov, Valery; Duerksen-Hughes, Penelope

    2014-01-01

    Viruses are the causative agents of 10%–15% of human cancers worldwide. The most common outcome for virus-induced reprogramming is genomic instability, including accumulation of mutations, aberrations and DNA damage. Although each virus has its own specific mechanism for promoting carcinogenesis, the majority of DNA oncogenic viruses encode oncogenes that transform infected cells, frequently by targeting p53 and pRB. In addition, integration of viral DNA into the human genome can also play an important role in promoting tumor development for several viruses, including HBV and HPV. Because viral integration requires the breakage of both the viral and the host DNA, the integration rate is believed to be linked to the levels of DNA damage. DNA damage can be caused by both endogenous and exogenous factors, including inflammation induced by either the virus itself or by co-infections with other agents, environmental agents and other factors. Typically, cancer develops years to decades following the initial infection. A better understanding of virus-mediated carcinogenesis, the networking of pathways involved in transformation and the relevant risk factors, particularly in those cases where tumorigenesis proceeds by way of virus integration, will help to suggest prophylactic and therapeutic strategies to reduce the risk of virus-mediated cancer. PMID:25340830

  5. Comparative interactomics for virus-human protein-protein interactions: DNA viruses versus RNA viruses.

    PubMed

    Durmuş, Saliha; Ülgen, Kutlu Ö

    2017-01-01

    Viruses are obligatory intracellular pathogens and completely depend on their hosts for survival and reproduction. The strategies adopted by viruses to exploit host cell processes and to evade host immune systems during infections may differ largely with the type of the viral genetic material. An improved understanding of these viral infection mechanisms is only possible through a better understanding of the pathogen-host interactions (PHIs) that enable viruses to enter into the host cells and manipulate the cellular mechanisms to their own advantage. Experimentally-verified protein-protein interaction (PPI) data of pathogen-host systems only became available at large scale within the last decade. In this study, we comparatively analyzed the current PHI networks belonging to DNA and RNA viruses and their human host, to get insights into the infection strategies used by these viral groups. We investigated the functional properties of human proteins in the PHI networks, to observe and compare the attack strategies of DNA and RNA viruses. We observed that DNA viruses are able to attack both human cellular and metabolic processes simultaneously during infections. On the other hand, RNA viruses preferentially interact with human proteins functioning in specific cellular processes as well as in intracellular transport and localization within the cell. Observing virus-targeted human proteins, we propose heterogeneous nuclear ribonucleoproteins and transporter proteins as potential antiviral therapeutic targets. The observed common and specific infection mechanisms in terms of viral strategies to attack human proteins may provide crucial information for further design of broad and specific next-generation antiviral therapeutics.

  6. Distinct DNA Exit and Packaging Portals in the Virus Acanthamoeba polyphaga mimivirus

    PubMed Central

    Zauberman, Nathan; Mutsafi, Yael; Halevy, Daniel Ben; Shimoni, Eyal; Klein, Eugenia; Xiao, Chuan; Sun, Siyang; Minsky, Abraham

    2008-01-01

    Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses. PMID:18479185

  7. Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus.

    PubMed

    Zauberman, Nathan; Mutsafi, Yael; Halevy, Daniel Ben; Shimoni, Eyal; Klein, Eugenia; Xiao, Chuan; Sun, Siyang; Minsky, Abraham

    2008-05-13

    Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the "stargate", allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane-containing viruses.

  8. Can variola-like viruses be derived from monkeypox virus? An investigation based on DNA mapping*

    PubMed Central

    Esposito, Joseph J.; Nakano, James H.; Obijeski, John F.

    1985-01-01

    The results are presented of a special study to determine whether variola-like “whitepox” viruses could arise as white pock variants of monkeypox virus after one or a few mutations. DNA mapping by cross-hybridization of restriction endonuclease DNA fragments was carried out on 18 orthopoxviruses relevant to this study, including variola and monkeypox viruses and white (non-haemorrhagic) pock producers recovered from chorioallantoic membranes infected with red (haemorrhagic) pock-producing monkeypox viruses. The distinctiveness of the DNA maps of true variola and monkeypox viruses indicated that spontaneous production of “whitepox” from monkeypox virus was genetically impossible. These and other observations led to the conclusion that the “whitepox” viruses recovered from monkeypox virus stocks had an exogenous origin. PMID:3002651

  9. Recovery and identification of bacterial DNA from illicit drugs.

    PubMed

    Cho, Kaymann T; Richardson, Michelle M; Kirkbride, K Paul; McNevin, Dennis; Nelson, Michelle; Pianca, Dennis; Roffey, Paul; Gahan, Michelle E

    2014-02-01

    Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002-2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated. Methods based on the ChargeSwitch(®)gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS). An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch(®) method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed. This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug

  10. The bacterial DnaC helicase loader is a DnaB ring breaker

    PubMed Central

    Arias-Palomo, Ernesto; O’Shea, Valerie L.; Hood, Iris V.; Berger, James M.

    2013-01-01

    Summary Dedicated AAA+ ATPases help deposit hexameric ring-shaped helicases onto DNA to promote replication in cellular organisms. To understand how loading occurs, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the ATP-bound structure of the intact E. coli DnaB•DnaC helicase/loader complex. The 480 kDa dodecamer forms a three-tiered assembly, in which DnaC adopts a spiral configuration that remodels N-terminal scaffolding and C-terminal motor regions of DnaB to produce a clear break in the helicase ring. Surprisingly, DnaC’s AAA+ fold is dispensable for ring remodeling, as the isolated helicase-binding domain of DnaC can both load DnaB onto DNA and increase the efficiency by which the helicase acts on substrates in vitro. Our data demonstrate that DnaC opens DnaB by a mechanism akin to that of polymerase clamp loaders, and indicate that bacterial replicative helicases, like their eukaryotic counterparts, possess auto-regulatory elements that influence how the hexameric motor domains are loaded onto and unwind DNA. PMID:23562643

  11. Top-down controls on bacterial community structure: microbial network analysis of bacteria, T4-like viruses and protists.

    PubMed

    Chow, Cheryl-Emiliane T; Kim, Diane Y; Sachdeva, Rohan; Caron, David A; Fuhrman, Jed A

    2014-04-01

    Characterizing ecological relationships between viruses, bacteria and protists in the ocean are critical to understanding ecosystem function, yet these relationships are infrequently investigated together. We evaluated these relationships through microbial association network analysis of samples collected approximately monthly from March 2008 to January 2011 in the surface ocean (0-5 m) at the San Pedro Ocean Time series station. Bacterial, T4-like myoviral and protistan communities were described by Automated Ribosomal Intergenic Spacer Analysis and terminal restriction fragment length polymorphism of the gene encoding the major capsid protein (g23) and 18S ribosomal DNA, respectively. Concurrent shifts in community structure suggested similar timing of responses to environmental and biological parameters. We linked T4-like myoviral, bacterial and protistan operational taxonomic units by local similarity correlations, which were then visualized as association networks. Network links (correlations) potentially represent synergistic and antagonistic relationships such as viral lysis, grazing, competition or other interactions. We found that virus-bacteria relationships were more cross-linked than protist-bacteria relationships, suggestive of increased taxonomic specificity in virus-bacteria relationships. We also found that 80% of bacterial-protist and 74% of bacterial-viral correlations were positive, with the latter suggesting that at monthly and seasonal timescales, viruses may be following their hosts more often than controlling host abundance.

  12. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    SciTech Connect

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  13. On the question of the integration of exogenous bacterial DNA into plant DNA.

    PubMed Central

    Kleinhofs, A; Eden, F C; Chilton, M D; Bendich, A J

    1975-01-01

    Extensive studies with pea, tomato, and barley failed to confirm the evidence presented by previous investigators for integration or replication of exogenously applied bacterial DNA in these plants. Labeled DNA of buoyant density in CsCl intermediate between that of high density donor bacterial DNA and of plant DNA was never observed with axenic plants. Intermediate peaks, similar to those used as evidence for recombination by earlier investigators, were observed only when the plants were contaminated with bacteria. Plant DNA prepared by a published procedure [Ledoux, L. & Huart, R. (1969) J. Mol. Biol. 43, 243-262] was found to be contaminated with unidentified impurities. Such DNA was partially protected from the action of DNase and produced aberrant banding patterns in CsCl after shearing. Much of the published evidence for integration of foreign DNA in plants is based upon experiments with plant DNA prepared by this procedure. We conclude that contamination is the likely explanation for what has been interpreted as evidence for integration. PMID:809769

  14. Tracking plant, fungal, and bacterial DNA in honey specimens.

    PubMed

    Olivieri, Cristina; Marota, Isolina; Rollo, Franco; Luciani, Stefania

    2012-01-01

    Consuming honey can result in adverse effects owing to poisoning by bacterial (botulism) or plant toxins. We have devised a method to extract polymerase chain reaction (PCR) amplifiable DNA of up to c. 400 bp in length based on dialysis of a 15-mL honey sample for 18 h against deionized water followed by sequential extraction using phenol, phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol, and ether. Sequence analysis of PCR products obtained using "universal" plant, fungal, and bacterial primers targeted to the ribosomal RNA genes has allowed us to identify six different orders of plants (Apiales, Fabales, Asterales, Solanales, Brassicales, and Sapindales), two orders of fungi (Entylomatales and Saccharomycetales), and six orders of bacteria (Sphingomonadales, Burkholderiales, Pseudomonadales, Enterobacteriales, Actinomycetales, and Bifidobacteriales) in a single honey specimen.

  15. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

    PubMed

    Pentland, Ieisha; Parish, Joanna L

    2015-07-06

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  16. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses

    PubMed Central

    Pentland, Ieisha; Parish, Joanna L.

    2015-01-01

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions. PMID:26154016

  17. Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction

    PubMed Central

    Wu, Xiao-Ting; Xiao, Lu-Jia; Li, Xing-Quan; Li, Jie-Shou

    1998-01-01

    AIM: To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS: DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP-PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification. RESULTS: The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11 (11/14) patients, and more than 90% in 10 (10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones. E. coli-related DNA fragments appeared in the stones of 8 (26.67%) patients; propionibacteria type DNA in 7 (23.33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes. A more heterogenous sequence collection was found in 7 (23.33%) patients, which could belong to multiple bacterial infections. Two (6.67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria. CONCLUSION: Most cholesterol gallstones harbor bacterial DNA. It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation. PMID:11819284

  18. Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD)

    PubMed Central

    2012-01-01

    Background The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA - random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method. Results A novel Getah virus was identified by VIDISCR from suckling mice exposed to mosquitoes (Aedes albopictus) collected in Yunnan Province, China. The non-structural protein gene, nsP3, the structural protein gene, the capsid protein gene, and the 3'-untranslated region (UTR) of the novel Getah virus isolate were cloned and sequenced. Nucleotide sequence identities of each gene were determined to be 97.1–99.3%, 94.9–99.4%, and 93.6–99.9%, respectively, when compared with the genomes of 10 other representative strains of Getah virus. Conclusions The VIDISCR method was able to identify known virus isolates and a novel isolate of Getah virus from infected mice. Phylogenetic analysis indicated that the YN08 isolate was more closely related to the Hebei HB0234 strain than the YN0540 strain, and more genetically distinct from the MM2021 Malaysia primitive strain. PMID:23268691

  19. Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD).

    PubMed

    Hu, Tingsong; Zheng, Ying; Zhang, Yan; Li, Gangshan; Qiu, Wei; Yu, Jing; Cui, Qinghua; Wang, Yiyin; Zhang, Chaoxiong; Zhou, Xiaofang; Feng, Ziliang; Zhou, Weiguo; Fan, Quanshui; Zhang, Fuqiang

    2012-12-27

    The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA--random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method. A novel Getah virus was identified by VIDISCR from suckling mice exposed to mosquitoes (Aedes albopictus) collected in Yunnan Province, China. The non-structural protein gene, nsP3, the structural protein gene, the capsid protein gene, and the 3'-untranslated region (UTR) of the novel Getah virus isolate were cloned and sequenced. Nucleotide sequence identities of each gene were determined to be 97.1-99.3%, 94.9-99.4%, and 93.6-99.9%, respectively, when compared with the genomes of 10 other representative strains of Getah virus. The VIDISCR method was able to identify known virus isolates and a novel isolate of Getah virus from infected mice. Phylogenetic analysis indicated that the YN08 isolate was more closely related to the Hebei HB0234 strain than the YN0540 strain, and more genetically distinct from the MM2021 Malaysia primitive strain.

  20. Phylogenetic evidence for extensive lateral acquisition of cellular genes by Nucleocytoplasmic large DNA viruses

    PubMed Central

    2008-01-01

    Background Nucleo-Cytoplasmic Large DNA viruses (NCLDV), a diverse group that infects a wide range of eukaryotic hosts, exhibit a large heterogeneity in genome size (between 100 kb and 1.2 Mb) but have been suggested to form a monophyletic group on the basis of a small subset of approximately 30 conserved genes. NCLDV were proposed to have evolved by simplification from cellular organism although some of the giant NCLDV have clearly grown by gene accretion from a bacterial origin. Results We demonstrate here that many NCLDV lineages appear to have undergone frequent gene exchange in two different ways. Viruses which infect protists directly (Mimivirus) or algae which exist as intracellular protists symbionts (Phycodnaviruses) acquire genes from a bacterial source. Metazoan viruses such as the Poxviruses show a predominant acquisition of host genes. In both cases, the laterally acquired genes show a strong tendency to be positioned at the tip of the genome. Surprisingly, several core genes believed to be ancestral in the family appear to have undergone lateral gene transfers, suggesting that the NCLDV ancestor might have had a smaller genome than previously believed. Moreover, our data show that the larger the genome, the higher is the number of laterally acquired genes. This pattern is incompatible with a genome reduction from a cellular ancestor. Conclusion We propose that the NCLDV viruses have evolved by significant growth of a simple DNA virus by gene acquisition from cellular sources. PMID:19036122

  1. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-06

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses.

  2. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  3. Virus and Bacterial Cell Chemical Analysis by NanoSIMS

    SciTech Connect

    Weber, P; Holt, J

    2008-07-28

    In past work for the Department of Homeland Security, the LLNL NanoSIMS team has succeeded in extracting quantitative elemental composition at sub-micron resolution from bacterial spores using nanometer-scale secondary ion mass spectrometry (NanoSIMS). The purpose of this task is to test our NanoSIMS capabilities on viruses and bacterial cells. This initial work has proven successful. We imaged Tobacco Mosaic Virus (TMV) and Bacillus anthracis Sterne cells using scanning electron microscopy (SEM) and then analyzed those samples by NanoSIMS. We were able resolve individual viral particles ({approx}18 nm by 300 nm) in the SEM and extract correlated elemental composition in the NanoSIMS. The phosphorous/carbon ratio observed in TMV is comparable to that seen in bacterial spores (0.033), as was the chlorine/carbon ratio (0.11). TMV elemental composition is consistent from spot to spot, and TMV is readily distinguished from debris by NanoSIMS analysis. Bacterial cells were readily identified in the SEM and relocated in the NanoSIMS for elemental analysis. The Ba Sterne cells were observed to have a measurably lower phosphorous/carbon ratio (0.005), as compared to the spores produced in the same run (0.02). The chlorine/carbon ratio was approximately 2.5X larger in the cells (0.2) versus the spores (0.08), while the fluorine/carbon ratio was approximately 10X lower in the cells (0.008) than the spores (0.08). Silicon/carbon ratios for both cells and spores encompassed a comparable range. The initial data in this study suggest that high resolution analysis is useful because it allows the target agent to be analyzed separate from particulates and other debris. High resolution analysis would also be useful for trace sample analysis. The next step in this work is to determine the potential utility of elemental signatures in these kinds of samples. We recommend bulk analyses of media and agent samples to determine the range of media compositions in use, and to determine how

  4. Assessing the Epidemic Potential of RNA and DNA Viruses

    PubMed Central

    Brierley, Liam; McCaffery, Chris; Lycett, Sam

    2016-01-01

    Many new and emerging RNA and DNA viruses are zoonotic or have zoonotic origins in an animal reservoir that is usually mammalian and sometimes avian. Not all zoonotic viruses are transmissible (directly or by an arthropod vector) between human hosts. Virus genome sequence data provide the best evidence of transmission. Of human transmissible virus, 37 species have so far been restricted to self-limiting outbreaks. These viruses are priorities for surveillance because relatively minor changes in their epidemiologies can potentially lead to major changes in the threat they pose to public health. On the basis of comparisons across all recognized human viruses, we consider the characteristics of these priority viruses and assess the likelihood that they will further emerge in human populations. We also assess the likelihood that a virus that can infect humans but is not capable of transmission (directly or by a vector) between human hosts can acquire that capability. PMID:27869592

  5. Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature.

    PubMed

    Verreault, Daniel; Marcoux-Voiselle, Mélissa; Turgeon, Nathalie; Moineau, Sylvain; Duchaine, Caroline

    2015-10-01

    The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies.

  6. Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature

    PubMed Central

    Verreault, Daniel; Marcoux-Voiselle, Mélissa; Turgeon, Nathalie; Moineau, Sylvain

    2015-01-01

    The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies. PMID:26253683

  7. VUV absorption spectroscopy of bacterial spores and DNA components

    NASA Astrophysics Data System (ADS)

    Fiebrandt, Marcel; Lackmann, Jan-Wilm; Raguse, Marina; Moeller, Ralf; Awakowicz, Peter; Stapelmann, Katharina

    2017-01-01

    Low-pressure plasmas can be used to inactivate bacterial spores and sterilize goods for medical and pharmaceutical applications. A crucial factor are damages induced by UV and VUV radiation emitted by the plasma. To analyze inactivation processes and protection strategies of spores, absorption spectra of two B. subtilis strains are measured. The results indicate, that the inner and outer coat of the spore significantly contribute to the absorption of UV-C and also of the VUV, protecting the spore against radiation based damages. As the sample preparation can significantly influence the absorption spectra due to salt residues, the cleaning procedure and sample deposition is tested for its reproducibility by measuring DNA oligomers and pUC18 plasmid DNA. The measurements are compared and discussed with results from the literature, showing a strong decrease of the salt content enabling the detection of absorption structures in the samples.

  8. The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer

    PubMed Central

    Fernandez, Agustin F.; Rosales, Cecilia; Lopez-Nieva, Pilar; Graña, Osvaldo; Ballestar, Esteban; Ropero, Santiago; Espada, Jesus; Melo, Sonia A.; Lujambio, Amaia; Fraga, Mario F.; Pino, Irene; Javierre, Biola; Carmona, Francisco J.; Acquadro, Francesco; Steenbergen, Renske D.M.; Snijders, Peter J.F.; Meijer, Chris J.; Pineau, Pascal; Dejean, Anne; Lloveras, Belen; Capella, Gabriel; Quer, Josep; Buti, Maria; Esteban, Juan-Ignacio; Allende, Helena; Rodriguez-Frias, Francisco; Castellsague, Xavier; Minarovits, Janos; Ponce, Jordi; Capello, Daniela; Gaidano, Gianluca; Cigudosa, Juan Cruz; Gomez-Lopez, Gonzalo; Pisano, David G.; Valencia, Alfonso; Piris, Miguel Angel; Bosch, Francesc X.; Cahir-McFarland, Ellen; Kieff, Elliott; Esteller, Manel

    2009-01-01

    The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses. PMID:19208682

  9. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    SciTech Connect

    Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

    2016-03-15

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  10. Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses

    PubMed Central

    Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

    2015-01-01

    Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order “Megavirales,” adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order “Megavirales,” and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order “Megavirales,” refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life. PMID:25727355

  11. Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses.

    PubMed

    Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

    2015-04-01

    Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order "Megavirales," adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order "Megavirales," and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order "Megavirales," refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life.

  12. DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

    PubMed Central

    Aron, G M; Purifoy, D J; Schaffer, P A

    1975-01-01

    Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies. PMID:169388

  13. The role of respiratory viruses in the etiology of bacterial pneumonia

    PubMed Central

    Lee, Kyu Han; Gordon, Aubree; Foxman, Betsy

    2016-01-01

    Pneumonia is the leading cause of death among children less than 5 years old worldwide. A wide range of viral, bacterial and fungal agents can cause pneumonia: although viruses are the most common etiologic agent, the severity of clinical symptoms associated with bacterial pneumonia and increasing antibiotic resistance makes bacterial pneumonia a major public health concern. Bacterial pneumonia can follow upper respiratory viral infection and complicate lower respiratory viral infection. Secondary bacterial pneumonia is a major cause of influenza-related deaths. In this review, we evaluate the following hypotheses: (i) respiratory viruses influence the etiology of pneumonia by altering bacterial community structure in the upper respiratory tract (URT) and (ii) respiratory viruses promote or inhibit colonization of the lower respiratory tract (LRT) by certain bacterial species residing in the URT. We conducted a systematic review of the literature to examine temporal associations between respiratory viruses and bacteria and a targeted review to identify potential mechanisms of interactions. We conclude that viruses both alter the bacterial community in the URT and promote bacterial colonization of the LRT. However, it is uncertain whether changes in the URT bacterial community play a substantial role in pneumonia etiology. The exception is Streptococcus pneumoniae where a strong link between viral co-infection, increased carriage and pneumococcal pneumonia has been established. PMID:26884414

  14. Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting.

    PubMed

    Crawford, A M; Buchanan, F C; Fraser, K M; Robinson, A J; Hill, D F

    1991-01-01

    In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.

  15. Hepatitis viruses exploitation of host DNA methyltransferases functions.

    PubMed

    Pazienza, Valerio; Panebianco, Concetta; Andriulli, Angelo

    2016-08-01

    Hepatitis B virus (HBV), hepatitis C virus (HCV) and Delta (HDV) infections are a global health burden. With different routes of infection and biology, HBV, HCV and HDV are capable to induce liver cirrhosis and cancer by impinging on epigenetic mechanisms altering host cell's pathways. In the present manuscript, we reviewed the published studies taking into account the relationship between the hepatitis viruses and the DNA methyltransferases proteins.

  16. Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

    PubMed

    Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

    2011-08-01

    Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both

  17. Viral hemorrhagic fevers of animals caused by DNA viruses

    USDA-ARS?s Scientific Manuscript database

    Here we outline serious diseases of food and fiber animals that cause damaging economic effects on producers all over the world. The only vector-borne DNA virus is included here (i.e., African swine fever virus), and the herpesviruses discussed have a complex epidemiology characterized by outbreaks ...

  18. Human Cytomegalovirus Induces JC Virus DNA Replication in Human Fibroblasts

    NASA Astrophysics Data System (ADS)

    Heilbronn, Regine; Albrecht, Ingrid; Stephan, Sonja; Burkle, Alexander; Zur Hausen, Harald

    1993-12-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML.

  19. Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

    PubMed

    Reddy, Sanjay M; Sun, Aijun; Khan, Owais A; Lee, Lucy F; Lupiani, Blanca

    2013-06-01

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

  20. Supercoiled circular DNA of an insect granulosis virus

    PubMed Central

    Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

    1977-01-01

    The DNA of the granulosis virus of the Indian meal moth, Plodia interpunctella, was characterized by physical chemical and electron microscopic techniques. Twenty-five percent of the DNA extracted from purified virus was isolated as supercoiled circular molecules. The remaining 75% consisted of relaxed circular molecules. These molecular forms were indicated by the production of two radioactive bands during sedimentation of 3H-labeled granulosis virus DNA in alkaline sucrose gradients or in equilibrium density gradients of neutral cesium chloride/propidium iodide. Electron microscopic visualization of the DNA that banded at the higher density in the latter gradients revealed supercoiled structures whereas that of DNA that banded at the lower density demonstrated relaxed circular molecules. The superhelical molecules were converted to relaxed circles by treatment with pancreatic DNase. The molecular weight of the viral DNA was calculated to be 81 × 106 by sedimentation in neutral sucrose and 78 × 106 by sedimentation in alkaline sucrose. The molecular weight estimated from length measurements in electron micrographs was 76 × 106. The buoyant density of the granulosis virus DNA was 1.703 g/cm3 and that of its insect host DNA was 1.697 g/cm3. Equilibrium sedimentation in cesium chloride and thermal denaturation indicated G + C contents of 44% and 39% for the viral and host DNA, respectively. Images PMID:198791

  1. Paleozoic origin of insect large dsDNA viruses.

    PubMed

    Thézé, Julien; Bézier, Annie; Periquet, Georges; Drezen, Jean-Michel; Herniou, Elisabeth A

    2011-09-20

    To understand how extant viruses interact with their hosts, we need a historical framework of their evolutionary association. Akin to retrovirus or hepadnavirus viral fossils present in eukaryotic genomes, bracoviruses are integrated in braconid wasp genomes and are transmitted by Mendelian inheritance. However, unlike viral genomic fossils, they have retained functional machineries homologous to those of large dsDNA viruses pathogenic to arthropods. Using a phylogenomic approach, we resolved the relationships between bracoviruses and their closest free relatives: baculoviruses and nudiviruses. The phylogeny showed that bracoviruses are nested within the nudivirus clade. Bracoviruses establish a bridge between the virus and animal worlds. Their inclusion in a virus phylogeny allowed us to relate free viruses to fossils. The ages of the wasps were used to calibrate the virus phylogeny. Bayesian analyses revealed that insect dsDNA viruses first evolved at ∼310 Mya in the Paleozoic Era during the Carboniferous Period with the first insects. Furthermore the virus diversification time frame during the Mesozoic Era appears linked to the diversification of insect orders; baculoviruses that infect larvae evolved at the same period as holometabolous insects. These results imply ancient coevolution by resource tracking between several insect dsDNA virus families and their hosts, dating back to 310 Mya.

  2. Analysis of bacterial populations in the environment using two-dimensional gel electrophoresis of genomic DNA and complementary DNA.

    PubMed

    Liu, Guo-Hua; Nakamura, Tatsuo; Amemiya, Takashi; Rajendran, Narasimmalu; Itoh, Kiminori

    2011-01-01

    Two-dimensional gel electrophoresis (2-DGE) mapping of genomic DNA and complementary DNA (cDNA) amplicons was attempted to analyze total and active bacterial populations within soil and activated sludge samples. Distinct differences in the number and species of bacterial populations and those that were metabolically active at the time of sampling were visually observed especially for the soil community. Statistical analyses and sequencing based on the 2-DGE data further revealed the relationships between total and active bacterial populations within each community. This high-resolution technique would be useful for obtaining a better understanding of bacterial population structures in the environment.

  3. Detection of vaccinia virus DNA, but not infectious virus, in the blood of smallpox vaccine recipients.

    PubMed

    Cohen, Jeffrey I; Hohman, Patricia; Preuss, Jeanne C; Li, Li; Fischer, Steven H; Fedorko, Daniel P

    2007-06-06

    The authors of a recent study [Savona MR, Dela Cruz WP, Jones MS, Thornton JA, Xia D, Hadfield TL, et al. Detection of vaccinia DNA in the blood following smallpox vaccination. JAMA 2006; 295:1898-1900] suggested that the duration of deferral for blood donations by smallpox vaccinees should be extended, based on detection of vaccinia virus DNA in five blood samples by polymerase chain reaction (PCR) and the potential for viremia. We found that 4 of 202 blood specimens (from 3 of 27 smallpox vaccinees) were positive for vaccinia virus DNA by PCR; none were positive for virus by culture. Throat swabs were negative by PCR and culture. Thus, while some blood specimens contained vaccinia virus DNA, infectious virus was not detected. Current guidelines for deferral of blood donation in vaccinees seem appropriate.

  4. Viruses and the DNA Damage Response: Activation and Antagonism.

    PubMed

    Luftig, Micah A

    2014-11-01

    Viruses must interact with their hosts in order to replicate; these interactions often provoke the evolutionarily conserved response to DNA damage, known as the DNA damage response (DDR). The DDR can be activated by incoming viral DNA, during the integration of retroviruses, or in response to the aberrant DNA structures generated upon replication of DNA viruses. Furthermore, DNA and RNA viral proteins can induce the DDR by promoting inappropriate S phase entry, by modifying cellular DDR factors directly, or by unintentionally targeting host DNA. The DDR may be antiviral, although viruses often require proximal DDR activation of repair and recombination factors to facilitate replication as well as downstream DDR signaling suppression to ensure cell survival. An unintended consequence of DDR attenuation during infection is the long-term survival and proliferation of precancerous cells. Therefore, the molecular basis for DDR activation and attenuation by viruses remains an important area of study that will likely provide key insights into how viruses have evolved with their hosts.

  5. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  6. The Role of Bacterial Chaperones in the Circulative Transmission of Plant Viruses by Insect Vectors

    PubMed Central

    Kliot, Adi; Ghanim, Murad

    2013-01-01

    Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV) by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV) by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed. PMID:23783810

  7. Detection of hepatitis A virus and bacterial contamination in raw oysters in Thailand.

    PubMed

    Kittigul, Leera; Pombubpa, Kannika; Sukonthalux, Suntharee; Rattanatham, Tippawan; Utrarachkij, Fuangfa; Diraphat, Pornphan

    2010-01-01

    This study was conducted to determine the presence of hepatitis A virus (HAV) in raw oysters (Crassostrea belcheri) using a virus concentration method and reverse transcription-nested polymerase chain reaction (RT-nested PCR). A total of 220 oyster samples were collected from oyster farms and local markets in Thailand. HAV was found in three oyster samples. Nested PCR products of HAV detected in oysters were characterized further by DNA sequencing of the VP1/2A region and subjected to phylogenetic analysis. All HAV sequences (168 basepairs) were associated with human HAV subgenotype IB (GIB). Fecal coliforms and Escherichia coli were determined using the multiple tube fermentation method, to assess the microbiological quality of collected oysters. Among oyster samples tested, 65% had fecal coliforms higher than the standard level for raw shellfish [< 20 Most Probable Numbers (MPN)/g]; MPN values in the range of 21.0-4.6 x 10(4)/g. Most oyster samples (85%) were contaminated with E. coli in the range of 3.0-4.6 x 10(4) MPN/g. One oyster sample with an acceptable level of fecal coliforms contained HAV GIB. E. coli was found in all HAV-positive oyster samples. The results suggest a significant presence of HAV and bacterial indicators of fecal contamination in raw oysters, which are a health risk for consumers and a source of gastrointestinal illness. Enteric viruses should also be tested to assess the microbiological quality of oysters.

  8. Regulation of bacterial metabolic activity by dissolved organic carbon and viruses

    NASA Astrophysics Data System (ADS)

    Xu, Jie; Jing, Hongmei; Sun, Mingming; Harrison, Paul J.; Liu, Hongbin

    2013-12-01

    regulation of bacterial metabolic activity by viruses and dissolved organic carbon (DOC) was examined using natural microbial communities in three treatments (active viruses, inactive viruses, and virus free) at two contrasting coastal sites (pristine vs. eutrophic) with substantial differences in environmental conditions during the wet and dry seasons. Our results showed that net growth rates and production of bacterioplankton were reduced primarily by viruses via repressing metabolically active bacteria with high nucleic acid (HNA) content which had a high capacity for incorporating carbon, while bacterial respiration was primarily regulated by DOC lability. The quality of organic matter played a more important role in regulating bacterial growth efficiency (BGE) than the supply of organic matter in eutrophic coastal waters. The lack of HMW-DOC and high carbon demand in the virus-free treatment resulted in a significant increase in cell-specific bacterial respiration, which was responsible for the lowest bacterial growth efficiency among the three treatments. The presence of viruses did not necessarily lower bacterial growth efficiency since virus-induced mortality alleviated bacterial carbon demand and enhanced carbon cycling. Virus-induced mortality was greater in relatively pristine waters than eutrophic waters, likely since the high supply of substrates alleviated the pressure of viral infection, through extracellular proteases produced by bacteria, which might result in the hydrolytic destruction or modification of viral capsids. An important implication of our results was that the input of riverine DOC and nutrients improved bacterial metabolic activity by alleviating virus-induced mortality of bacteria in estuarine and coastal waters.

  9. Cloning of full length genome of varicella-zoster virus vaccine strain into a bacterial artificial chromosome and reconstitution of infectious virus.

    PubMed

    Yoshii, Hironori; Somboonthum, Pranee; Takahashi, Michiaki; Yamanishi, Koichi; Mori, Yasuko

    2007-06-28

    The complete genome of the varicella-zoster virus (VZV) Oka vaccine strain (vOka) has been cloned into a bacterial artificial chromosome (BAC), and several BAC clones with the vOka genome have been obtained. Infectious viruses were reconstituted in MRC-5 cells transfected with the vOka-BAC DNA clones. The clones were distributed into two groups based on differences in amino acids found in ORF 62/71 region among the vOka-BAC clones. The recombinant vOka (rvOka) grew slower than recombinant Oka parental virus (rpOka), pOka and vOka. This is the first report that the vOka genome was cloned into BAC vector. The rvOka-BAC system would be useful as a vector for construction of recombinant live vaccines.

  10. Infectious Maize rayado fino virus from cloned cDNA

    USDA-ARS?s Scientific Manuscript database

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  11. DNA confinement drives uncoating of the HIV Virus

    NASA Astrophysics Data System (ADS)

    Rouzina, I.; Bruinsma, R.

    2014-09-01

    The enzyme reverse transcriptase converts single-stranded RNA molecules into double-stranded DNA molecules inside mature HIV viral capsids. We present a model for the uncoating of the HIV virus where the capsid uncoating process is driven by the confinement force exerted on the capsid wall porduced to the double-stranded DNA generated by reverse transcriptase.

  12. Identification of a novel circular DNA virus in pig feces

    USDA-ARS?s Scientific Manuscript database

    Metagenomic analysis of fecal samples collected from a swine with diarrhea detected sequences encoding a replicase (Rep) protein typically found in small circular Rep-encoding ssDNA (CRESS-DNA) viruses. The complete 3,062 nucleotide genome was generated and found to encode two bi-directionally trans...

  13. Diverse small circular DNA viruses circulating amongst estuarine molluscs.

    PubMed

    Dayaram, Anisha; Goldstien, Sharyn; Argüello-Astorga, Gerardo R; Zawar-Reza, Peyman; Gomez, Christopher; Harding, Jon S; Varsani, Arvind

    2015-04-01

    Our understanding of the diversity and abundance of circular replication associated protein (Rep) - encoding single stranded (CRESS) DNA viruses has increased considerably over the last few years due to a combination of modern sequencing technologies and new molecular tools. Studies have used these to identify and recover CRESS DNA viruses from a range of different marine organisms, including copepods, shrimp and molluscs. In our study we identified 79 novel CRESS DNA viruses from three mollusc species (Austrovenus stutchburyi, Paphies subtriangulata and Amphibola crenata) and benthic sediments from the Avon-Heathcote estuary in Christchurch, New Zealand. The genomes recovered have varying genome architectures, with all encoding at least two major ORFs that have either unidirectional or bidirectional organisation. Analysis of the Reps of the viral genomes showed they are all highly diverse, with only one Rep sequence sharing 65% amino acid identity with the Rep of gastropod-associated circular DNA virus (GaCSV). Our study adds significantly to the wealth of CRESS DNA viruses recovered from freshwater and marine environments and extends our knowledge of the distribution of these viruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Bacterial DNA Detected in Japanese Rice Wines and the Fermentation Starters.

    PubMed

    Terasaki, Momoka; Fukuyama, Akari; Takahashi, Yurika; Yamada, Masato; Nishida, Hiromi

    2017-08-22

    As Japanese rice wine (sake) brewing is not done aseptically, bacterial contamination is conceivable during the process of sake production. There are two types of the fermentation starter, sokujo-moto and yamahai-moto (kimoto). We identified bacterial DNA found in various sakes, the sokujo-moto and the yamahai-moto making just after sake yeast addition. Each sake has a unique variety of bacterial DNA not observed in other sakes. Although most bacterial DNA sequences detected in the sokujo-moto were found in sakes of different sake breweries, most bacterial DNA sequences detected in the yamahai-moto at the early stage of the starter fermentation were not detected in any sakes. Our findings demonstrate that various bacteria grow and then die during the process of sake brewing, as indicated by the presence of trace levels of bacterial DNA.

  15. Frequency of varicella zoster virus DNA in human adrenal glands.

    PubMed

    Badani, Hussain; White, Teresa; Schulick, Nicole; Raeburn, Christopher D; Topkaya, Ibrahim; Gilden, Don; Nagel, Maria A

    2016-06-01

    Varicella zoster virus (VZV) becomes latent in ganglionic neurons derived from neural crest cells. Because the adrenal gland also contains medullary chromaffin cells of neural crest origin, we examined human adrenal glands and medullary chromaffin cell tumors (pheochromocytomas) for VZV and herpes simplex virus type 1 (HSV-1). We found VZV, but not HSV-1, DNA in 4/63 (6 %) normal adrenal glands. No VZV transcripts or antigens were detected in the 4 VZV DNA-positive samples. No VZV or HSV-1 DNA was found in 21 pheochromocytomas.

  16. Origin of giant viruses from smaller DNA viruses not from a fourth domain of cellular life.

    PubMed

    Yutin, Natalya; Wolf, Yuri I; Koonin, Eugene V

    2014-10-01

    The numerous and diverse eukaryotic viruses with large double-stranded DNA genomes that at least partially reproduce in the cytoplasm of infected cells apparently evolved from a single virus ancestor. This major group of viruses is known as Nucleocytoplasmic Large DNA Viruses (NCLDV) or the proposed order Megavirales. Among the "Megavirales", there are three groups of giant viruses with genomes exceeding 500kb, namely Mimiviruses, Pithoviruses, and Pandoraviruses that hold the current record of viral genome size, about 2.5Mb. Phylogenetic analysis of conserved, ancestral NLCDV genes clearly shows that these three groups of giant viruses have three distinct origins within the "Megavirales". The Mimiviruses constitute a distinct family that is distantly related to Phycodnaviridae, Pandoraviruses originate from a common ancestor with Coccolithoviruses within the Phycodnaviridae family, and Pithoviruses are related to Iridoviridae and Marseilleviridae. Maximum likelihood reconstruction of gene gain and loss events during the evolution of the "Megavirales" indicates that each group of giant viruses evolved from viruses with substantially smaller and simpler gene repertoires. Initial phylogenetic analysis of universal genes, such as translation system components, encoded by some giant viruses, in particular Mimiviruses, has led to the hypothesis that giant viruses descend from a fourth, probably extinct domain of cellular life. The results of our comprehensive phylogenomic analysis of giant viruses refute the fourth domain hypothesis and instead indicate that the universal genes have been independently acquired by different giant viruses from their eukaryotic hosts.

  17. Origin of giant viruses from smaller DNA viruses not from a fourth domain of cellular life

    PubMed Central

    Yutin, Natalya; Wolf, Yuri I.; Koonin, Eugene V.

    2015-01-01

    The numerous and diverse eukaryotic viruses with large double-stranded DNA genomes that at least partially reproduce in the cytoplasm of infected cells apparently evolved from a single virus ancestor. This major group of viruses is known as Nucleocytoplasmic Large DNA Viruses (NCLDV) or the proposed order Megavirales. Among the “Megavirales”, there are three groups of giant viruses with genomes exceeding 500 kb, namely Mimiviruses, Pithoviruses, and Pandoraviruses that hold the current record of viral genome size, about 2.5 Mb. Phylogenetic analysis of conserved, ancestral NLCDV genes clearly shows that these three groups of giant viruses have three distinct origins within the “Megavirales”. The Mimiviruses constitute a distinct family that is distantly related to Phycodnaviridae, Pandoraviruses originate from a common ancestor with Coccolithoviruses within the Phycodnaviridae family, and Pithoviruses are related to Iridoviridae and Marseilleviridae. Maximum likelihood reconstruction of gene gain and loss events during the evolution of the “Megavirales” indicates that each group of giant viruses evolved from viruses with substantially smaller and simpler gene repertoires. Initial phylogenetic analysis of universal genes, such as translation system components, encoded by some giant viruses, in particular Mimiviruses, has led to the hypothesis that giant viruses descend from a fourth, probably extinct domain of cellular life. The results of our comprehensive phylogenomic analysis of giant viruses refute the fourth domain hypothesis and instead indicate that the universal genes have been independently acquired by different giant viruses from their eukaryotic hosts. PMID:25042053

  18. Identification of torque teno virus in culture-negative endophthalmitis by representational deep DNA sequencing.

    PubMed

    Lee, Aaron Y; Akileswaran, Lakshmi; Tibbetts, Michael D; Garg, Sunir J; Van Gelder, Russell N

    2015-03-01

    To test the hypothesis that uncultured organisms may be present in cases of culture-negative endophthalmitis by use of deep DNA sequencing of vitreous biopsies. Single-center, consecutive, prospective, observational study. Aqueous or vitreous biopsies from 21 consecutive patients presenting with presumed infectious endophthalmitis and 7 vitreous samples from patients undergoing surgery for noninfectious retinal disorders. Traditional bacterial and fungal culture, 16S quantitative polymerase chain reaction (qPCR), and a representational deep-sequencing method (biome representational in silico karyotyping [BRiSK]) were applied in parallel to samples to identify DNA sequences corresponding to potential pathogens. Presence of potential pathogen DNA in ocular samples. Zero of 7 control eyes undergoing routine vitreous surgery yielded positive results for bacteria or virus by culture or 16S polymerase chain reaction (PCR). A total of 14 of the 21 samples (66.7%) from eyes harboring suspected infectious endophthalmitis were culture-positive, the most common being Staphylococcal and Streptococcal species. There was good agreement among culture, 16S bacterial PCR, and BRiSK methodologies for culture-positive cases (Fleiss' kappa of 0.621). 16S PCR did not yield a recognizable pathogen sequence in any culture-negative sample, whereas BRiSK suggested the presence of Streptococcus in 1 culture-negative sample. With the use of BRiSK, 57.1% of culture-positive and 100% of culture-negative samples demonstrated the presence of torque teno virus (TTV) sequences, compared with none in the controls (P=0.0005, Fisher exact test). The presence of TTV viral DNA was confirmed in 7 cases by qPCR. No other known viruses or potential pathogens were identified in these samples. Culture, 16S qPCR, and BRiSK provide complementary information in presumed infectious endophthalmitis. The majority of culture-negative endophthalmitis samples did not contain significant levels of bacterial DNA

  19. Identification of torque teno virus in culture-negative endophthalmitis by representational deep-DNA sequencing

    PubMed Central

    Lee, Aaron Y.; Akileswaran, Lakshmi; Tibbetts, Michael D.; Garg, Sunir J.; Van Gelder, Russell N.

    2014-01-01

    Purpose To test the hypothesis that uncultured organisms may be present in cases of culture-negative endophthalmitis, by use of deep DNA sequencing of vitreous biopsies. Design Single center consecutive prospective observational study. Participants and Controls Aqueous or vitreous biopsies from 21 consecutive patients presenting with presumed infectious endophthalmitis, and seven vitreous samples from patients undergoing surgery for non-infectious retinal disorders. Methods Traditional bacterial and fungal culture, 16S quantitative polymerase chain reaction (qPCR) and a representational deep-sequencing method (Biome Representational in Silico Karyotyping [BRiSK]) were applied in parallel to samples to identify DNA sequences corresponding to potential pathogens. Main Outcome Measures Presence of potential pathogen DNA in ocular samples. Results None of 7 control eyes undergoing routine vitreous surgery yielded positive results for bacteria or virus by culture or 16S PCR. Fourteen of the 21 samples (66.7%) from eyes harboring suspected infectious endophthalmitis were culture-positive, the most common being Staphylococcal and Streptococcal species. There was good agreement among culture, 16S bacterial PCR, and BRiSK methodologies for culture-positive cases (Fleiss’ kappa of 0.621). 16S PCR did not yield a recognizable pathogen sequence in any culture-negative sample, while BRiSK suggested presence of Steptococcus in one culture-negative sample. Surprisingly, using BRiSK, 57.1% of culture-positive and 100% of culture-negative samples demonstrated presence of Torque Teno Virus (TTV) sequences, compared to none in the controls (Fisher exact, p = 0.0005). Presence of TTV viral DNA was confirmed in seven cases by qPCR. No other known viruses or potential pathogens were identified in these samples. Conclusion Culture, 16S qPCR, and BRiSK provide complementary information in presumed infectious endophthalmitis. The majority of culture-negative endophthalmitis samples did

  20. Use of whole genome DNA spectrograms in bacterial classification.

    PubMed

    Kubicova, Vladimira; Provaznik, Ivo

    2016-02-01

    A spectrogram reflects the arrangement of nucleotides through the whole chromosome or genome. Our previous study suggested that the spectrogram of whole genome DNA sequences is a suitable tool for the determination of relationships among bacteria. Related bacteria have similar spectrograms, and similarity in spectrograms was measured using a color layout descriptor. Several parameters, such as the mapping of four bases into a spectrogram, the number of considered elements in the color layout descriptor, the color model of the image and the building tree method, can be changed. This study addresses the use of parameter selection to ensure the best classification results. The quality of the classification was measured by Matthew's correlation coefficient (MCC). The proposed method with optimal parameters (called SpectCMP-Spectrogram CoMParison method) achieved an average MCC of 0.73 at the phylum level. The SpectCMP method was also tested at the order level; the average MCC in the classification of class Gammaproteobacteria was 0.76. The success of a classification with respect to the correct phyla was compared to three methods that are used in bacterial phylogeny: the CVTree method, OGTree method and moment vector method. The results show that the SpectCMP method can be used in bacterial classification at various taxonomic levels.

  1. Design stars: how small DNA viruses remodel the host nucleus.

    PubMed

    Jiang, Mengxi; Imperiale, Michael J

    2012-05-01

    Numerous host components are encountered by viruses during the infection process. While some of these host structures are left unchanged, others may go through dramatic remodeling processes. In this review, we summarize these host changes that occur during small DNA virus infections, with a focus on host nuclear components and pathways. Although these viruses differ significantly in their genome structures and infectious pathways, there are common nuclear targets that are altered by various viral factors. Accumulating evidence suggests that these nuclear remodeling processes are often essential for productive viral infections and/or viral-induced transformation. Understanding the complex interactions between viruses and these host structures and pathways will help to build a more integrated network of how the virus completes its life cycle and point toward the design of novel therapeutic regimens that either prevent harmful viral infections or employ viruses as nontraditional treatment options or molecular tools.

  2. Plasma Bacterial and Mitochondrial DNA Distinguish Bacterial Sepsis from Sterile SIRS and Quantify Inflammatory Tissue Injury in Nonhuman Primates

    PubMed Central

    Sursal, Tolga; Stearns-Kurosawa, Deborah J; Itagaki, Kiyoshi; Oh, Sun-Young; Sun, Shiqin; Kurosawa, Shinichiro; Hauser, Carl J

    2012-01-01

    Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. SIRS can cause organ dysfunction and death but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS and post-infective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern (DAMP) reflecting cellular injury. Circulating bacterial 16S-DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed qPCR assays to quantify these markers and predicted their plasma levels might help distinguish sterile injury from infection. To study these events in primates we assayed banked serum from papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis deltaSterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to “severe” sepsis and death we studied animals where anthrax was pretreated with drotrecogin alfa (aPC), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against non-lethal E.coli bacteremia and against sterile tissue injury from Shiga-like toxin-1 (Stx1). bDNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Stx1 increased mtDNA but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sub-lethal E.coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal anthrax challenge (n=4) bDNA increased transiently but mtDNA levels remained elevated until death

  3. Bacterial DNA microarrays for clinical microbiology: the early logarithmic phase.

    PubMed

    Cassone, Marco; Giordano, Antonio; Pozzi, Gianni

    2007-01-01

    In this era of coexistence of high-throughput sequencing technologies and serious difficulties in the management of both common and novel infectious syndromes, new techniques which improve the study of micro-organisms is timely. In bacteriology, the most important subjects are bacterial pathogenicity, discovery of the genomic complexity of bacteria, and the epidemiology of antimicrobial resistance traits. From the clinical point of view, genetic testing is flanking phenotypic testing for the assessment of new, difficult to test antibiotic resistance traits, and for correlations with the microbial behaviour in vivo. The demand for faster, comprehensive and highly parallel microbial diagnostics is also cogent even at the basic laboratory level, where the ultimate objective is saving lives. In this setting, DNA microarrays offer a pivotal contribution by allowing performance of hybridization experiments in highly parallel formats, with an increasing reliability. Not only they are useful in deciphering host and microbial pathophysiology, they can also make the difference in the management of prognostic and therapeutic aspects of many diseases. Here, we provide an overview of the current use and the potential of DNA microarrays in clinical bacteriology, and several applications and technical solutions are discussed.

  4. Bithionol blocks pathogenicity of bacterial toxins, ricin, and Zika virus

    PubMed Central

    Leonardi, William; Zilbermintz, Leeor; Cheng, Luisa W.; Zozaya, Josue; Tran, Sharon H.; Elliott, Jeffrey H.; Polukhina, Kseniya; Manasherob, Robert; Li, Amy; Chi, Xiaoli; Gharaibeh, Dima; Kenny, Tara; Zamani, Rouzbeh; Soloveva, Veronica; Haddow, Andrew D.; Nasar, Farooq; Bavari, Sina; Bassik, Michael C.; Cohen, Stanley N.; Levitin, Anastasia; Martchenko, Mikhail

    2016-01-01

    Diverse pathogenic agents often utilize overlapping host networks, and hub proteins within these networks represent attractive targets for broad-spectrum drugs. Using bacterial toxins, we describe a new approach for discovering broad-spectrum therapies capable of inhibiting host proteins that mediate multiple pathogenic pathways. This approach can be widely used, as it combines genetic-based target identification with cell survival-based and protein function-based multiplex drug screens, and concurrently discovers therapeutic compounds and their protein targets. Using B-lymphoblastoid cells derived from the HapMap Project cohort of persons of African, European, and Asian ancestry we identified host caspases as hub proteins that mediate the lethality of multiple pathogenic agents. We discovered that an approved drug, Bithionol, inhibits host caspases and also reduces the detrimental effects of anthrax lethal toxin, diphtheria toxin, cholera toxin, Pseudomonas aeruginosa exotoxin A, Botulinum neurotoxin, ricin, and Zika virus. Our study reveals the practicality of identifying host proteins that mediate multiple disease pathways and discovering broad-spectrum therapies that target these hub proteins. PMID:27686742

  5. Bithionol blocks pathogenicity of bacterial toxins, ricin, and Zika virus.

    PubMed

    Leonardi, William; Zilbermintz, Leeor; Cheng, Luisa W; Zozaya, Josue; Tran, Sharon H; Elliott, Jeffrey H; Polukhina, Kseniya; Manasherob, Robert; Li, Amy; Chi, Xiaoli; Gharaibeh, Dima; Kenny, Tara; Zamani, Rouzbeh; Soloveva, Veronica; Haddow, Andrew D; Nasar, Farooq; Bavari, Sina; Bassik, Michael C; Cohen, Stanley N; Levitin, Anastasia; Martchenko, Mikhail

    2016-09-30

    Diverse pathogenic agents often utilize overlapping host networks, and hub proteins within these networks represent attractive targets for broad-spectrum drugs. Using bacterial toxins, we describe a new approach for discovering broad-spectrum therapies capable of inhibiting host proteins that mediate multiple pathogenic pathways. This approach can be widely used, as it combines genetic-based target identification with cell survival-based and protein function-based multiplex drug screens, and concurrently discovers therapeutic compounds and their protein targets. Using B-lymphoblastoid cells derived from the HapMap Project cohort of persons of African, European, and Asian ancestry we identified host caspases as hub proteins that mediate the lethality of multiple pathogenic agents. We discovered that an approved drug, Bithionol, inhibits host caspases and also reduces the detrimental effects of anthrax lethal toxin, diphtheria toxin, cholera toxin, Pseudomonas aeruginosa exotoxin A, Botulinum neurotoxin, ricin, and Zika virus. Our study reveals the practicality of identifying host proteins that mediate multiple disease pathways and discovering broad-spectrum therapies that target these hub proteins.

  6. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata.

    PubMed

    Breitbart, Mya; Benner, Bayleigh E; Jernigan, Parker E; Rosario, Karyna; Birsa, Laura M; Harbeitner, Rachel C; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B; Berger, Stella A; Nejstgaard, Jens C

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton.

  7. Subversion and piracy: DNA viruses and immune evasion.

    PubMed

    Haig, D M

    2001-06-01

    During the co-evolution of viruses with their vertebrate hosts, the DNA viruses have acquired an impressive array of immunomodulatory genes to combat host immune responses and their hosts have developed a sophisticated immune system to contain virus infections. In order to replicate, the viruses have evolved mechanisms to inhibit key host anti-virus responses that include apoptosis, interferon production, chemokine production, inflammatory cytokine production, and the activity of cytotoxic T-cells, natural killer cells and antibody. In addition, some of the viruses encode cytokine or chemokine homologues that recruit or expand cell numbers for infection or that subvert the host cellular response from a protective response to a benign one. The specificity of the viral immunomodulatory molecules reflects the life cycle and the pathogenesis of the viruses. Herpesviruses achieve latency in host cells by inducing cell survival and protecting infected cells from immune recognition. This involves interference with cell signal transduction pathways. Many of the viral immunomodulatory proteins are homologues of host proteins that appear to have been pirated from the host and reassorted in the virus genomes. Some of these have unique functions and indicate novel or important aspects of both viral pathogenesis and host immunity to viruses. The specific example of orf virus infection of sheep is described.

  8. Metagenomic analysis of bacterial infections by means of high-throughput DNA sequencing.

    PubMed

    Nakamura, Shota; Nakaya, Takaaki; Iida, Tetsuya

    2011-08-01

    DNA sequencing technology has undergone a remarkable and continuous improvement in recent years. The so-called 'high-throughput DNA sequencers' can determine hundreds of megabases of DNA sequences per run. We have been applying these new sequencers to the analysis of infectious diseases, especially bacterial infections. We review the efficacy of these sequencers, mainly based on our own experiences. The approach described here can be viewed as a metagenomic analysis of infectious diseases. The approach is in principle a method that does not depend on the type of target pathogens, so that it is possible to analyze various microorganisms, including bacteria, viruses, fungi and parasites, with a single common protocol. Applying this novel approach to cases in which infectious diseases are suspected because of environmental evidence, but the causative agent has not been identified, may lead to the discovery of unknown, novel pathogens. Also, the approach enables us to conduct an unbiased analysis of dynamics of the pathogen and associated microbiota in human specimens during the course of infectious diseases.

  9. A virus of hyperthermophilic archaea with a unique architecture among DNA viruses

    PubMed Central

    Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

    2016-01-01

    Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle. PMID:26884161

  10. A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

    PubMed

    Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

    2016-03-01

    Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

  11. DNA Polymerase in Virions of a Reptilian Type C Virus

    PubMed Central

    Twardzik, Daniel R.; Papas, Takis S.; Portugal, Frank H.

    1974-01-01

    A study was made of the DNA polymerase of reptilian type C virus isolated from Russell's viper spleen cells. Simultaneous detection experiments demonstrated the presence of 70S RNA and RNA-dependent DNA polymerase activity in reptilian type C virions. The endogenous activity was dependent on the addition of all four deoxynucleotide triphosphates and demonstrated an absolute requirement for a divalent cation. The reptilian viral DNA polymerase elutes from phosphocellulose at 0.22 M salt. In this respect, it is similar to the avian (avian myeloblastosis virus; AMV) viral enzyme but is different from the mammalian (Rauscher leukemia virus; RLV) viral enzyme which elutes at 0.4 M salt. The molecular weight of the viper DNA polymerase as estimated from glycerol gradient centrifugation is 109,000. It is a smaller enzyme than the AMV DNA polymerase (180,000 daltons) and somewhat larger than the RLV enzyme (70,000 daltons). A comparison of other properties of the type C reptilian DNA polymerase with the enzyme found in other type C oncogenic viruses is made. PMID:4129837

  12. DNA viruses: the really big ones (giruses).

    PubMed

    Van Etten, James L; Lane, Leslie C; Dunigan, David D

    2010-01-01

    Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences than on extensive details about individual viruses.

  13. DNA Viruses: The Really Big Ones (Giruses)

    PubMed Central

    Van Etten, James L.; Lane, Leslie C.; Dunigan, David D.

    2010-01-01

    Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences rather than provides extensive details about individual viruses. PMID:20690825

  14. Bacterial neuraminidase rescues influenza virus replication from inhibition by a neuraminidase inhibitor.

    PubMed

    Nishikawa, Tomoko; Shimizu, Kazufumi; Tanaka, Torahiko; Kuroda, Kazumichi; Takayama, Tadatoshi; Yamamoto, Tatsuo; Hanada, Nobuhiro; Hamada, Yoshiki

    2012-01-01

    Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may

  15. Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

    PubMed Central

    Nishikawa, Tomoko; Shimizu, Kazufumi; Tanaka, Torahiko; Kuroda, Kazumichi; Takayama, Tadatoshi; Yamamoto, Tatsuo; Hanada, Nobuhiro; Hamada, Yoshiki

    2012-01-01

    Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may

  16. Evolution of Eukaryotic DNA Polymerases via Interaction Between Cells and Large DNA Viruses.

    PubMed

    Takemura, Masaharu; Yokobori, Shin-ichi; Ogata, Hiroyuki

    2015-08-01

    B-family DNA-directed DNA polymerases are DNA replication enzymes found in Eukaryota, Archaea, large DNA viruses, and in some, but not all, bacteria. Several polymerase domains are conserved among the B-family DNA polymerases from these organisms, suggesting that the B-family DNA polymerases evolved from a common ancestor. Eukaryotes retain at least three replicative B-family DNA polymerases, DNA polymerase α, δ, and ε, and one translesion B-family DNA polymerase, DNA polymerase ζ. Here, we present molecular evolutionary evidence that suggests DNA polymerase genes evolved through horizontal gene transfer between the viral and archaeal-eukaryotic lineages. Molecular phylogenetic analyses of the B-family DNA polymerases from nucleo-cytoplasmic large DNA viruses (NCLDVs), eukaryotes, and archaea suggest that different NCLDV lineages such as Poxviridae and Mimiviridae were involved in the evolution of different DNA polymerases (pol-α-, δ-, ε-, and ζ-like genes) in archaeal-eukaryotic cell lineages, putatively through horizontal gene transfer. These results support existing theories that link the evolution of NCLDVs and the origin of the eukaryotic nucleus.

  17. A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

    PubMed

    Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

    2015-01-01

    A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria.

  18. Absence of bacterial and viral DNA in bladder biopsies from patients with interstitial cystitis/chronic pelvic pain syndrome.

    PubMed

    Al-Hadithi, Hiba N; Williams, Helen; Hart, C Anthony; Frazer, Malcolm; Adams, Elisabeth J; Richmond, David H; Tincello, Douglas G

    2005-07-01

    We examined bladder biopsies from women with interstitial cystitis/chronic pelvic pain syndrome (IC/CPPS) for the presence of bacterial and viral DNA sequences using polymerase chain reaction. Bladder biopsies were taken during cystoscopy from patients under investigation for IC/CPPS, or controls undergoing colposuspension for stress incontinence. Biopsies were snap frozen to -70C. After DNA extraction, polymerase chain reaction (PCR) using specific primers for the hypoxanthine-guanine phosphoribosyl transferase gene confirmed the presence of human DNA. PCR for bacterial and viral gene sequences was performed using specific primers. Positive reactions were repeated to confirm the signal. A total of 92 patients with IC/CPPS (12 who met the National Institute of Diabetes and Digestive and Kidney Diseases criteria and 80 who did not) and 91 controls were recruited. PCR for hypoxanthine-guanine phosphoribosyl transferase gene was positive in all samples. PCR for the 16S ribosomal RNA gene, as well as for adenovirus, cytomegalovirus, herpes simplex virus types I and II, human papillomavirus (all subtypes) and Chlamydia trachomatis were negative in all samples. IC/CPPS is not associated with persistence of viral and bacterial DNA in the bladder. A chronic infective etiology for the condition is excluded by these findings.

  19. Two distinct SSB protein families in nucleo-cytoplasmic large DNA viruses

    PubMed Central

    Venclovas, Česlovas

    2012-01-01

    Motivation: Eukaryote-infecting nucleo-cytoplasmic large DNA viruses (NCLDVs) feature some of the largest genomes in the viral world. These viruses typically do not strongly depend on the host DNA replication systems. In line with this observation, a number of essential DNA replication proteins, such as DNA polymerases, primases, helicases and ligases, have been identified in the NCLDVs. One other ubiquitous component of DNA replisomes is the single-stranded DNA-binding (SSB) protein. Intriguingly, no NCLDV homologs of canonical OB-fold-containing SSB proteins had previously been detected. Only in poxviruses, one of seven NCLDV families, I3 was identified as the SSB protein. However, whether I3 is related to any known protein structure has not yet been established. Results: Here, we addressed the case of ‘missing’ canonical SSB proteins in the NCLDVs and also probed evolutionary origins of the I3 family. Using advanced computational methods, in four NCLDV families, we detected homologs of the bacteriophage T7 SSB protein (gp2.5). We found the properties of these homologs to be consistent with the SSB function. Moreover, we implicated specific residues in single-stranded DNA binding. At the same time, we found no evolutionary link between the T7 gp2.5-like NCLDV SSB homologs and the poxviral SSB protein (I3). Instead, we identified a distant relationship between I3 and small protein B (SmpB), a bacterial RNA-binding protein. Thus, apparently, the NCLDVs have the two major distinct sets of SSB proteins having bacteriophage and bacterial origins, respectively. Contact: venclovas@ibt.lt Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23097418

  20. Hairpin loop structure of African swine fever virus DNA.

    PubMed Central

    González, A; Talavera, A; Almendral, J M; Viñuela, E

    1986-01-01

    The ends of African swine fever virus genome are formed by a 37 nucleotide-long hairpin loop composed, almost entirely, of incompletely paired A and T residues. The loops at each DNA end were present in two equimolar forms that, when compared in opposite polarities, were inverted and complementary (flip-flop), as in the case of poxvirus DNA. The hairpin loops of African swine fever and vaccinia virus DNAs had no homology, but both DNAs had a 16 nucleotide-long sequence, close to the hairpin loops, with an homology of about 80%. An analysis of African swine fever virus replicating DNA showed head-to-head and tail-to-tail linked molecules that may be replicative intermediates. Images PMID:3763393

  1. Lactulose reduces bacterial DNA translocation, which worsens neurocognitive shape in cirrhotic patients with minimal hepatic encephalopathy.

    PubMed

    Moratalla, Alba; Ampuero, Javier; Bellot, Pablo; Gallego-Durán, Rocío; Zapater, Pedro; Roger, Manuela; Figueruela, Blanca; Martínez-Moreno, Belén; González-Navajas, José M; Such, José; Romero-Gómez, Manuel; Francés, Rubén

    2017-02-01

    Minimal hepatic encephalopathy is associated with poor prognosis and mortality in patients with cirrhosis. We aimed at investigating whether bacterial-DNA translocation affects hyperammonaemia and neurocognitive scores in patients with mHE according to the use of lactulose. Observational study including 72 mHE cirrhotic patients, as defined by a psychometric hepatic encephalopathy score (PHES)<-4 and/or a critical flicker frequency (CFF)<39 Hz. Bacterial-DNA, serum ammonia, pro-inflammatory cytokines and nitric oxide levels were evaluated. A second cohort of 40 lactulose-untreated patients were evaluated before and 6-month after lactulose administration (30-60 mL/d). In the first cohort, bacterial-DNA rate was significantly higher in patients without lactulose (39% vs 23%, P=.03). Serum ammonia and inflammatory markers were significantly increased in patients with bacterial-DNA, regardless the use of lactulose, and correlated with the amount of amplified bacterial-DNA. Neurocognitive scores were significantly worse in bacterial-DNA positive vs negative patients (PHES -7.6±1.1 vs -5.5±1.0; CFF 32.5±2.6 vs 36.2±2.8, P=.01). Lactulose was associated with improved neurocognitive scores in patients without bacterial-DNA. Serum ammonia levels inversely correlated with neurocognitive scores in patients with bacterial-DNA (PHES r=-.84; CFF r=-.72, P=.001). In the second cohort, lactulose reduced bacterial-DNA translocation (36%-16%, P=.02). Neurocognitive scores were significantly improved in bacterial-DNA positive patients who cleared bacterial-DNA during the period on lactulose. Serum ammonia levels correlated with both neurocognitive scores in patients with bacterial-DNA, either before or after lactulose. Bacterial-DNA translocation worsens neurocognitive scores in mHE patients and it is reduced by lactulose, enhancing the relevance of controlling bacterial antigen translocation in these patients. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Reconstruction of a Bacterial Genome from DNA Cassettes

    SciTech Connect

    Christopher Dupont; John Glass; Laura Sheahan; Shibu Yooseph; Lisa Zeigler Allen; Mathangi Thiagarajan; Andrew Allen; Robert Friedman; J. Craig Venter

    2011-12-31

    This basic research program comprised two major areas: (1) acquisition and analysis of marine microbial metagenomic data and development of genomic analysis tools for broad, external community use; (2) development of a minimal bacterial genome. Our Marine Metagenomic Diversity effort generated and analyzed shotgun sequencing data from microbial communities sampled from over 250 sites around the world. About 40% of the 26 Gbp of sequence data has been made publicly available to date with a complete release anticipated in six months. Our results and those mining the deposited data have revealed a vast diversity of genes coding for critical metabolic processes whose phylogenetic and geographic distributions will enable a deeper understanding of carbon and nutrient cycling, microbial ecology, and rapid rate evolutionary processes such as horizontal gene transfer by viruses and plasmids. A global assembly of the generated dataset resulted in a massive set (5Gbp) of genome fragments that provide context to the majority of the generated data that originated from uncultivated organisms. Our Synthetic Biology team has made significant progress towards the goal of synthesizing a minimal mycoplasma genome that will have all of the machinery for independent life. This project, once completed, will provide fundamentally new knowledge about requirements for microbial life and help to lay a basic research foundation for developing microbiological approaches to bioenergy.

  3. Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses

    PubMed Central

    2014-01-01

    Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages. PMID:25105011

  4. Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome.

    PubMed

    Gray, Wayne L; Zhou, Fuchun; Noffke, Juliane; Tischer, B Karsten

    2011-05-01

    Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.

  5. The hepatitis B virus and its DNA polymerase: the prototype three-D virus.

    PubMed

    Hirschman, S Z

    1979-07-15

    The hepatitis B virus (HBV), the causal agent of serum hepatitis, has a diameter of 42 nm and is comprised of an outer surface coat and a 27 nm core. A unique DNA-dependent DNA polymerase is associated with the core of the virus. The core also houses a circular DNA that contains both double-stranded and single-stranded regions. In the endogenous reaction, the DNA polymerase repairs the single-stranded gaps of the viral DNA. The surface protein of the virus, called hepatitis B surface antigen, contains both lipid and carbohydrate, and is often present in particulate form in the blood of infected patients. In Asia and Africa HBV infection is associated with subsequent development of primary hepatocellular carcinoma. Although most patients recover completely from acute illness, the hepatitis B virus may cause chronic infection. Recently, a virus similar to human HBV was discovered in woodchucks. HBV has not yet been propagated in a cell culture system and the mode of replication of this unusual virus in hepatocytes is still moot. Although reliable therapy has not yet been provided, the problem of this world-wide infection has led to many interesting approaches to both vaccine production and anti-viral chemotherapy.

  6. Molecular genetics of DNA viruses: recombinant virus technology.

    PubMed

    Neuhierl, Bernhard; Delecluse, Henri-Jacques

    2005-01-01

    Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecular genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All available techniques are based on homologous recombination between a targeting vector carrying the mutated version of the gene of interest and the recombinant virus. After modification, the mutant viral genome is stably introduced into eukaryotic cells permissive for viral lytic replication. In these cells, mutant viral genomes can be packaged into infectious particles to evaluate the effect of these mutations in the context of the complete genome.

  7. Influence of Calcium in Extracellular DNA Mediated Bacterial Aggregation and Biofilm Formation

    PubMed Central

    Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

    2014-01-01

    Calcium (Ca2+) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation. PMID:24651318

  8. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

    PubMed

    Das, Theerthankar; Sehar, Shama; Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

    2014-01-01

    Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.

  9. Electromagnetic signals are produced by aqueous nanostructures derived from bacterial DNA sequences.

    PubMed

    Montagnier, Luc; Aïssa, Jamal; Ferris, Stéphane; Montagnier, Jean-Luc; Lavallée, Claude

    2009-06-01

    A novel property of DNA is described: the capacity of some bacterial DNA sequences to induce electromagnetic waves at high aqueous dilutions. It appears to be a resonance phenomenon triggered by the ambient electromagnetic background of very low frequency waves. The genomic DNA of most pathogenic bacteria contains sequences which are able to generate such signals. This opens the way to the development of highly sensitive detection system for chronic bacterial infections in human and animal diseases.

  10. DNA-mediated anisotropic mechanical reinforcement of a virus

    PubMed Central

    Carrasco, C.; Carreira, A.; Schaap, I. A. T.; Serena, P. A.; Gómez-Herrero, J.; Mateu, M. G.; de Pablo, P. J.

    2006-01-01

    In this work, we provide evidence of a mechanism to reinforce the strength of an icosahedral virus by using its genomic DNA as a structural element. The mechanical properties of individual empty capsids and DNA-containing virions of the minute virus of mice are investigated by using atomic force microscopy. The stiffness of the empty capsid is found to be isotropic. Remarkably, the presence of the DNA inside the virion leads to an anisotropic reinforcement of the virus stiffness by ≈3%, 40%, and 140% along the fivefold, threefold, and twofold symmetry axes, respectively. A finite element model of the virus indicates that this anisotropic mechanical reinforcement is due to DNA stretches bound to 60 concavities of the capsid. These results, together with evidence of biologically relevant conformational rearrangements of the capsid around pores located at the fivefold symmetry axes, suggest that the bound DNA may reinforce the overall stiffness of the viral particle without canceling the conformational changes needed for its infectivity. PMID:16945903

  11. Next Generation Sequencing of DNA-Launched Chikungunya Vaccine Virus

    PubMed Central

    Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

    2016-01-01

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3’ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. PMID:26855330

  12. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment.

    PubMed

    Kennedy, Edward M; Cullen, Bryan R

    2015-05-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  13. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    PubMed Central

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  14. Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta).

    PubMed

    Rosario, Karyna; Dayaram, Anisha; Marinov, Milen; Ware, Jessica; Kraberger, Simona; Stainton, Daisy; Breitbart, Mya; Varsani, Arvind

    2012-12-01

    Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.

  15. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array-based multiplex assay.

    PubMed

    Lievens, B; Frans, I; Heusdens, C; Justé, A; Jonstrup, S P; Lieffrig, F; Willems, K A

    2011-11-01

    Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study. © 2011 Blackwell Publishing Ltd.

  16. Computational analysis of DNA replicases in double-stranded DNA viruses: relationship with the genome size

    PubMed Central

    Kazlauskas, Darius; Venclovas, Česlovas

    2011-01-01

    Genome duplication in free-living cellular organisms is performed by DNA replicases that always include a DNA polymerase, a DNA sliding clamp and a clamp loader. What are the evolutionary solutions for DNA replicases associated with smaller genomes? Are there some general principles? To address these questions we analyzed DNA replicases of double-stranded (ds) DNA viruses. In the process we discovered highly divergent B-family DNA polymerases in phiKZ-like phages and remote sliding clamp homologs in Ascoviridae family and Ma-LMM01 phage. The analysis revealed a clear dependency between DNA replicase components and the viral genome size. As the genome size increases, viruses universally encode their own DNA polymerases and frequently have homologs of DNA sliding clamps, which sometimes are accompanied by clamp loader subunits. This pattern is highly non-random. The absence of sliding clamps in large viral genomes usually coincides with the presence of atypical polymerases. Meanwhile, sliding clamp homologs, not accompanied by clamp loaders, have an elevated positive electrostatic potential, characteristic of non-ring viral processivity factors that bind the DNA directly. Unexpectedly, we found that similar electrostatic properties are shared by the eukaryotic 9-1-1 clamp subunits, Hus1 and, to a lesser extent, Rad9, also suggesting the possibility of direct DNA binding. PMID:21742758

  17. Herpes simplex virus induces the replication of foreign DNA

    SciTech Connect

    Danovich, R.M.; Frenkel, N.

    1988-08-01

    Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions. HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.

  18. Myocardial injury and bacterial pneumonia contribute to the pathogenesis of fatal influenza B virus infection.

    PubMed

    Paddock, Christopher D; Liu, Lindy; Denison, Amy M; Bartlett, Jeanine H; Holman, Robert C; Deleon-Carnes, Marlene; Emery, Shannon L; Drew, Clifton P; Shieh, Wun-Ju; Uyeki, Timothy M; Zaki, Sherif R

    2012-03-15

    Influenza B virus infection causes rates of hospitalization and influenza-associated pneumonia similar to seasonal influenza A virus infection and accounts for a substantial percentage of all influenza-related hospitalizations and deaths among those aged <18 years; however, the pathogenesis of fatal influenza B virus infection is poorly described. Tissue samples obtained at autopsy from 45 case patients with fatal influenza B virus infection were evaluated by light microscopy and immunohistochemical assays for influenza B virus, various bacterial pathogens, and complement components C4d and C9, to identify the cellular tropism of influenza B virus, characterize concomitant bacterial pneumonia, and describe the spectrum of cardiopulmonary injury. Viral antigens were localized to ciliated respiratory epithelium and cells of submucosal glands and ducts. Concomitant bacterial pneumonia, caused predominantly by Staphylococcus aureus, was identified in 38% of case patients and occurred with significantly greater frequency in those aged >18 years. Pathologic evidence of myocardial injury was identified in 69% of case patients for whom cardiac tissue samples were available for examination, predominantly in case patients aged <18 years. Our findings suggest that bacterial pneumonia and cardiac injury contribute to fatal outcomes after infection with influenza B virus and that the frequency of these manifestations may be age related.

  19. Biodiversity and distribution of polar freshwater DNA viruses

    PubMed Central

    Aguirre de Cárcer, Daniel; López-Bueno, Alberto; Pearce, David A.; Alcamí, Antonio

    2015-01-01

    Viruses constitute the most abundant biological entities and a large reservoir of genetic diversity on Earth. Despite the recent surge in their study, our knowledge on their actual biodiversity and distribution remains sparse. We report the first metagenomic analysis of Arctic freshwater viral DNA communities and a comparative analysis with other freshwater environments. Arctic viromes are dominated by unknown and single-stranded DNA viruses with no close relatives in the database. These unique viral DNA communities mostly relate to each other and present some minor genetic overlap with other environments studied, including an Arctic Ocean virome. Despite common environmental conditions in polar ecosystems, the Arctic and Antarctic DNA viromes differ at the fine-grain genetic level while sharing a similar taxonomic composition. The study uncovers some viral lineages with a bipolar distribution, suggesting a global dispersal capacity for viruses, and seemingly indicates that viruses do not follow the latitudinal diversity gradient known for macroorganisms. Our study sheds light into the global biogeography and connectivity of viral communities. PMID:26601189

  20. Structure of faustovirus, a large dsDNA virus

    DOE PAGES

    Klose, Thomas; Reteno, Dorine G.; Benamar, Samia; ...

    2016-05-16

    Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. In this paper, we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unitmore » of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. Finally, this complexity might help the virus to rapidly adapt to new environments or hosts.« less

  1. Structure of faustovirus, a large dsDNA virus

    SciTech Connect

    Klose, Thomas; Reteno, Dorine G.; Benamar, Samia; Hollerbach, Adam; Colson, Philippe; La Scola, Bernard; Rossmann, Michael G.

    2016-05-16

    Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. In this paper, we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unit of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. Finally, this complexity might help the virus to rapidly adapt to new environments or hosts.

  2. Structure of faustovirus, a large dsDNA virus

    PubMed Central

    Klose, Thomas; Reteno, Dorine G.; Benamar, Samia; Hollerbach, Adam; Colson, Philippe; La Scola, Bernard; Rossmann, Michael G.

    2016-01-01

    Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. Here we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unit of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. This complexity might help the virus to rapidly adapt to new environments or hosts. PMID:27185929

  3. Structure of faustovirus, a large dsDNA virus

    SciTech Connect

    Klose, Thomas; Reteno, Dorine G.; Benamar, Samia; Hollerbach, Adam; Colson, Philippe; La Scola, Bernard; Rossmann, Michael G.

    2016-05-16

    Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. In this paper, we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unit of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. Finally, this complexity might help the virus to rapidly adapt to new environments or hosts.

  4. Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli.

    PubMed

    Korycka-Machala, Malgorzata; Nowosielski, Marcin; Kuron, Aneta; Rykowski, Sebastian; Olejniczak, Agnieszka; Hoffmann, Marcin; Dziadek, Jaroslaw

    2017-01-17

    The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use β-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5'-triphosphate. This fact leads to the conclusion that NAD⁺-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD⁺ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide) inhibited the activity of M. tuberculosis NAD⁺-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage) DNA LigT₄ was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM.

  5. Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

    PubMed Central

    Roux, Simon; Solonenko, Natalie E.; Dang, Vinh T.; Poulos, Bonnie T.; Schwenck, Sarah M.; Goldsmith, Dawn B.; Coleman, Maureen L.; Breitbart, Mya

    2016-01-01

    Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems. PMID:28003936

  6. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGES

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  7. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

  8. DNA viruses associated with diseases of marine and anadromous fish

    NASA Astrophysics Data System (ADS)

    Hetrick, F. M.

    1984-03-01

    The association of DNA-containing viruses with diseases of marine and anadromous fish is reviewed. One section of the review describes those diseases with a proven viral etiology. Available information on the physical, chemical, and biological properties of the viruses is included. Another section deals with those diseases where a viral etiology is suspected but not established. The primary evidence associating viruses with many of these diseases is the observation of virus particles in electron micrographs of thin sections of tissue samples from diseased fish. Finally, the possible role of pollutants, and other stress factors, in predisposing fish to viral infection is discussed as are the problems associated with studying diseases of wild fish populations.

  9. Bovine Leukemia Virus DNA in Human Breast Tissue

    PubMed Central

    Shen, Hua Min; Jensen, Hanne M.; Choi, K. Yeon; Sun, Dejun; Nuovo, Gerard

    2014-01-01

    Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease. PMID:24750974

  10. Bovine leukemia virus DNA in human breast tissue.

    PubMed

    Buehring, Gertrude Case; Shen, Hua Min; Jensen, Hanne M; Choi, K Yeon; Sun, Dejun; Nuovo, Gerard

    2014-05-01

    Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease.

  11. DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

    PubMed Central

    Yamaguchi, M; DePamphilis, M L

    1986-01-01

    Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites. Images PMID:3006062

  12. Small RNAs tackle large viruses: RNA interference-based antiviral defense against DNA viruses in insects.

    PubMed

    Bronkhorst, Alfred W; Miesen, Pascal; van Rij, Ronald P

    2013-01-01

    The antiviral RNA interference (RNAi) pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNA) that guide the recognition and cleavage of complementary viral target RNAs. In RNA virus infections, viral replication intermediates, dsRNA genomes or viral structured RNAs have been implicated as Dicer-2 substrates. In a recent publication, we demonstrated that a double-stranded DNA virus, Invertebrate iridescent virus 6, is a target of the Drosophila RNAi machinery, and we proposed that overlapping converging transcripts base pair to form the dsRNA substrates for vsiRNA biogenesis. Here, we discuss the role of RNAi in antiviral defense to DNA viruses in Drosophila and other invertebrate model systems.

  13. The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses

    PubMed Central

    Hull, R.; Sadler, J.; Longstaff, M.

    1986-01-01

    Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin. PMID:16453731

  14. Recombination promoted by DNA viruses: phage λ to herpes simplex virus.

    PubMed

    Weller, Sandra K; Sawitzke, James A

    2014-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies.

  15. Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

    PubMed Central

    Weller, Sandra K.; Sawitzke, James A.

    2015-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

  16. The Role of Virus Infection in Deregulating the Cytokine Response to Secondary Bacterial Infection.

    PubMed

    Mehta, Divya; Petes, Carlene; Gee, Katrina; Basta, Sameh

    2015-12-01

    Proinflammatory cytokines are produced by macrophages and dendritic cells (DCs) after infection to stimulate T helper (Th) cells, linking innate and adaptive immunity. Virus infections can deregulate the proinflammatory cytokine response like tumor necrosis factor-α and interleukin (IL)-2, making the host more susceptible to secondary bacterial infections. Studies using various viruses such as lymphocytic choriomeningitis virus, influenza A virus, and human immunodeficiency virus have revealed several intriguing mechanisms that account for the increased susceptibility to several prevalent bacterial infections. In particular, type I interferons induced during a virus infection have been observed to play a role in suppressing the production of some key antibacterial proinflammatory cytokines such as IL-23 and IL-17. Other suppressive mechanisms as a result of cytokine deregulation by viral infections include reduced function of immune cells such as DC, macrophage, natural killer, CD4(+), and CD8(+) T cells leading to impaired clearance of secondary bacterial infections. In this study, we highlight some of the immune mechanisms that become deregulated by viral infections, and can thus become defective during secondary bacterial infections.

  17. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

    PubMed Central

    Smale, S T; Tjian, R

    1986-01-01

    We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication. Images PMID:3025630

  18. Statistical properties of DNA sequences revisited: the role of inverse bilateral symmetry in bacterial chromosomes

    NASA Astrophysics Data System (ADS)

    José, Marco V.; Govezensky, Tzipe; Bobadilla, Juan R.

    2005-06-01

    Herein it is shown that in order to study the statistical properties of DNA sequences in bacterial chromosomes it suffices to consider only one half of the chromosome because they are similar to its corresponding complementary sequence in the other half. This is due to the inverse bilateral symmetry of bacterial chromosomes. Contrary to the classical result that DNA coding regions of bacterial genomes are purely uncorrelated random sequences, here it is shown, via a renormalization group approach, that DNA random fluctuations of single bases are modulated by log-periodic variations. Distance series of triplets display long-range correlations in each half of the intact chromosome and in protein-coding sequences, or both long-range correlations and log-periodic modulations along the whole chromosome. Hence scaling analyses of distance series of DNA sequences have to consider the functional units of bacterial chromosomes.

  19. Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

    PubMed Central

    Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

    2014-01-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

  20. A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

    PubMed Central

    Gammon, Don B; Duraffour, Sophie; Rozelle, Daniel K; Hehnly, Heidi; Sharma, Rita; Sparks, Michael E; West, Cara C; Chen, Ying; Moresco, James J; Andrei, Graciela; Connor, John H; Conte, Darryl; Gundersen-Rindal, Dawn E; Marshall, William L; Yates, John R; Silverman, Neal; Mello, Craig C

    2014-01-01

    Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems. DOI: http://dx.doi.org/10.7554/eLife.02910.001 PMID:24966209

  1. Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

    PubMed Central

    Sèle, Céleste; Gabel, Frank; Gutsche, Irina; Ivanov, Ivan; Burmeister, Wim P.

    2013-01-01

    Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages. PMID:23175373

  2. Structures of herpes simplex virus type 1 genes required for replication of virus DNA.

    PubMed Central

    McGeoch, D J; Dalrymple, M A; Dolan, A; McNab, D; Perry, L J; Taylor, P; Challberg, M D

    1988-01-01

    Recently, a method has been developed to identify regions in the genome of herpes simplex virus type 1 (HSV-1) which contain genes required for DNA synthesis from an HSV-1 origin of DNA replication, and seven genomic loci have been identified as representing the necessary and sufficient gene set for such replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). Two of the loci represent the well-known genes for DNA polymerase and major DNA-binding protein, but the remainder had little or no previous characterization. In this report we present the DNA sequences of the five newly identified genes and their deduced transcript organizations and encoded amino acid sequences. These genes were designated UL5, UL8, UL9, UL42, and UL52 and were predicted to encode proteins with molecular weights of, respectively, 99,000, 80,000, 94,000, 51,000, and 114,000. All of these genes had clear counterparts in the genome of the related alphaherpesvirus varicella-zoster virus, but only UL5 and UL52 were detectably conserved in the distantly related gammaherpesvirus Epstein-Barr virus, as judged by amino acid sequence similarity. The sequence of the UL5 protein, and of its counterparts in the other viruses, contained a region closely resembling known ATP-binding sites; this could be indicative, for instance, of a helicase or primase activity. PMID:2826807

  3. Effects of viruses on bacterial functions under contrasting nutritional conditions for four species of bacteria isolated from Hong Kong waters.

    PubMed

    Liu, Hao; Yuan, Xiangcheng; Xu, Jie; Harrison, Paul J; He, Lei; Yin, Kedong

    2015-09-25

    Free living viruses are ubiquitous in marine waters and concentrations are usually several times higher than the bacterial abundance. These viruses are capable of lysing host bacteria and therefore, play an important role in the microbial loop in oligotrophic waters. However, few studies have been conducted to compare the role of viruses in regulating bacterial abundance and heterotrophic activities between natural oligotrophic waters and anthropogenic influenced eutrophic waters. In this study, we examined viral effects on bacterial functions of four single bacterial species incubated with natural viral assemblages in seawater samples from eutrophic and oligotrophic waters. The viral-lysis of bacteria was significantly higher in eutrophic than oligotrophic waters. This suggests that viruses were capable of controlling bacterial abundance, respiration and production in the eutrophic waters. Cellular bacterial respiration and production was higher with viruses than without viruses, which was more evident in the oligotrophic waters. These results indicate that viruses can slow down bacterial consumption of oxygen and reduce bacteria-induced eutrophication effects in anthropogenic eutrophic waters, but switch to the role of sustaining the bacterial population when nutrients are limiting. There were bacterial species differences in resisting viral attack, which can influence the dominance and biodiversity of bacterial species in coastal waters.

  4. Effects of viruses on bacterial functions under contrasting nutritional conditions for four species of bacteria isolated from Hong Kong waters

    NASA Astrophysics Data System (ADS)

    Liu, Hao; Yuan, Xiangcheng; Xu, Jie; Harrison, Paul J.; He, Lei; Yin, Kedong

    2015-09-01

    Free living viruses are ubiquitous in marine waters and concentrations are usually several times higher than the bacterial abundance. These viruses are capable of lysing host bacteria and therefore, play an important role in the microbial loop in oligotrophic waters. However, few studies have been conducted to compare the role of viruses in regulating bacterial abundance and heterotrophic activities between natural oligotrophic waters and anthropogenic influenced eutrophic waters. In this study, we examined viral effects on bacterial functions of four single bacterial species incubated with natural viral assemblages in seawater samples from eutrophic and oligotrophic waters. The viral-lysis of bacteria was significantly higher in eutrophic than oligotrophic waters. This suggests that viruses were capable of controlling bacterial abundance, respiration and production in the eutrophic waters. Cellular bacterial respiration and production was higher with viruses than without viruses, which was more evident in the oligotrophic waters. These results indicate that viruses can slow down bacterial consumption of oxygen and reduce bacteria-induced eutrophication effects in anthropogenic eutrophic waters, but switch to the role of sustaining the bacterial population when nutrients are limiting. There were bacterial species differences in resisting viral attack, which can influence the dominance and biodiversity of bacterial species in coastal waters.

  5. Effects of viruses on bacterial functions under contrasting nutritional conditions for four species of bacteria isolated from Hong Kong waters

    PubMed Central

    Liu, Hao; Yuan, Xiangcheng; Xu, Jie; Harrison, Paul J.; He, Lei; Yin, Kedong

    2015-01-01

    Free living viruses are ubiquitous in marine waters and concentrations are usually several times higher than the bacterial abundance. These viruses are capable of lysing host bacteria and therefore, play an important role in the microbial loop in oligotrophic waters. However, few studies have been conducted to compare the role of viruses in regulating bacterial abundance and heterotrophic activities between natural oligotrophic waters and anthropogenic influenced eutrophic waters. In this study, we examined viral effects on bacterial functions of four single bacterial species incubated with natural viral assemblages in seawater samples from eutrophic and oligotrophic waters. The viral-lysis of bacteria was significantly higher in eutrophic than oligotrophic waters. This suggests that viruses were capable of controlling bacterial abundance, respiration and production in the eutrophic waters. Cellular bacterial respiration and production was higher with viruses than without viruses, which was more evident in the oligotrophic waters. These results indicate that viruses can slow down bacterial consumption of oxygen and reduce bacteria-induced eutrophication effects in anthropogenic eutrophic waters, but switch to the role of sustaining the bacterial population when nutrients are limiting. There were bacterial species differences in resisting viral attack, which can influence the dominance and biodiversity of bacterial species in coastal waters. PMID:26404394

  6. Mechanisms of Severe Mortality-Associated Bacterial Co-infections Following Influenza Virus Infection.

    PubMed

    Jia, Leili; Xie, Jing; Zhao, Jiangyun; Cao, Dekang; Liang, Yuan; Hou, Xuexin; Wang, Ligui; Li, Zhenjun

    2017-01-01

    Influenza virus infection remains one of the largest disease burdens on humans. Influenza-associated bacterial co-infections contribute to severe disease and mortality during pandemic and seasonal influenza episodes. The mechanisms of severe morbidity following influenza-bacteria co-infections mainly include failure of an antibacterial immune response and pathogen synergy. Moreover, failure to resume function and tolerance might be one of the main reasons for excessive mortality. In this review, recent advances in the study of mechanisms of severe disease, caused by bacterial co-infections following influenza virus pathogenesis, are summarized. Therefore, understanding the synergy between viruses and bacteria will facilitate the design of novel therapeutic approaches to prevent mortality associated with bacterial co-infections.

  7. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed Central

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-01-01

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region. PMID:3671088

  8. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-10-26

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region.

  9. Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute.

    PubMed

    Swarts, Daan C; Szczepaniak, Malwina; Sheng, Gang; Chandradoss, Stanley D; Zhu, Yifan; Timmers, Elizabeth M; Zhang, Yong; Zhao, Hongtu; Lou, Jizhong; Wang, Yanli; Joo, Chirlmin; van der Oost, John

    2017-03-16

    Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

  10. DNA intercalator stimulates influenza transcription and virus replication.

    PubMed

    Li, Olive T W; Poon, Leo L M

    2011-03-15

    Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII). In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD), was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPII(a) in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPII(a)) to hyperphosphorylated RNAPII (RNAPII(o)).

  11. Application of gelatin-coated magnetic particles for isolation of genomic DNA from bacterial cells.

    PubMed

    Intorasoot, Sorasak; Srirung, Rujira; Intorasoot, Amornrat; Ngamratanapaiboon, Surachai

    2009-03-15

    Gelatin-coated magnetic particles were implemented for bacterial genomic DNA isolation in this study. Based on structural differences in the cell wall, the standard strains Staphylococcus aureus and Escherichia coli were selected. The quantity, quality, and timing process for DNA extraction using gelatin-coated magnetite were compared to reference phenol-chloroform extraction and a commercially available kit. Approximately twice as much DNA was recovered with the use of coated magnetite, providing greater yields than other DNA extraction methods. In addition, the DNA quality was determined using 16S ribosomal DNA (rDNA) gene amplification by polymerase chain reaction (PCR). The described technique is rapid, simple, and a well-suited method to use with PCR for diagnosis of bacterial infections.

  12. Evolution and Phylogeny of Large DNA Viruses, Mimiviridae and Phycodnaviridae Including Newly Characterized Heterosigma akashiwo Virus

    PubMed Central

    Maruyama, Fumito; Ueki, Shoko

    2016-01-01

    Nucleocytoplasmic DNA viruses are a large group of viruses that harbor double-stranded DNA genomes with sizes of several 100 kbp, challenging the traditional concept of viruses as small, simple ‘organisms at the edge of life.’ The most intriguing questions about them may be their origin and evolution, which have yielded the variety we see today. Specifically, the phyletic relationship between two giant dsDNA virus families that are presumed to be close, Mimiviridae, which infect Acanthamoeba, and Phycodnaviridae, which infect algae, is still obscure and needs to be clarified by in-depth analysis. Here, we studied Mimiviridae–Phycodnaviridae phylogeny including the newly identified Heterosigma akashiwo virus strain HaV53. Gene-to-gene comparison of HaV53 with other giant dsDNA viruses showed that only a small proportion of HaV53 genes show similarities with the others, revealing its uniqueness among Phycodnaviridae. Phylogenetic/genomic analysis of Phycodnaviridae including HaV53 revealed that the family can be classified into four distinctive subfamilies, namely, Megaviridae (Mimivirus-like), Chlorovirus-type, and Coccolitho/Phaeovirus-type groups, and HaV53 independent of the other three groups. Several orthologs found in specific subfamilies while absent from the others were identified, providing potential family marker genes. Finally, reconstruction of the evolutionary history of Phycodnaviridae and Mimiviridae revealed that these viruses are descended from a common ancestor with a small set of genes and reached their current diversity by differentially acquiring gene sets during the course of evolution. Our study illustrates the phylogeny and evolution of Mimiviridae–Phycodnaviridae and proposes classifications that better represent phyletic relationships among the family members. PMID:27965659

  13. Evolution and Phylogeny of Large DNA Viruses, Mimiviridae and Phycodnaviridae Including Newly Characterized Heterosigma akashiwo Virus.

    PubMed

    Maruyama, Fumito; Ueki, Shoko

    2016-01-01

    Nucleocytoplasmic DNA viruses are a large group of viruses that harbor double-stranded DNA genomes with sizes of several 100 kbp, challenging the traditional concept of viruses as small, simple 'organisms at the edge of life.' The most intriguing questions about them may be their origin and evolution, which have yielded the variety we see today. Specifically, the phyletic relationship between two giant dsDNA virus families that are presumed to be close, Mimiviridae, which infect Acanthamoeba, and Phycodnaviridae, which infect algae, is still obscure and needs to be clarified by in-depth analysis. Here, we studied Mimiviridae-Phycodnaviridae phylogeny including the newly identified Heterosigma akashiwo virus strain HaV53. Gene-to-gene comparison of HaV53 with other giant dsDNA viruses showed that only a small proportion of HaV53 genes show similarities with the others, revealing its uniqueness among Phycodnaviridae. Phylogenetic/genomic analysis of Phycodnaviridae including HaV53 revealed that the family can be classified into four distinctive subfamilies, namely, Megaviridae (Mimivirus-like), Chlorovirus-type, and Coccolitho/Phaeovirus-type groups, and HaV53 independent of the other three groups. Several orthologs found in specific subfamilies while absent from the others were identified, providing potential family marker genes. Finally, reconstruction of the evolutionary history of Phycodnaviridae and Mimiviridae revealed that these viruses are descended from a common ancestor with a small set of genes and reached their current diversity by differentially acquiring gene sets during the course of evolution. Our study illustrates the phylogeny and evolution of Mimiviridae-Phycodnaviridae and proposes classifications that better represent phyletic relationships among the family members.

  14. Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus.

    PubMed

    Robinson, Christopher M; Jesudhasan, Palmy R; Pfeiffer, Julie K

    2014-01-15

    Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication, and pathogenesis in mice are equivalent, VP1-T99K poliovirus was unstable in feces following peroral inoculation of mice. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host.

  15. Low-energy plasma immersion ion implantation to induce DNA transfer into bacterial E. coli

    NASA Astrophysics Data System (ADS)

    Sangwijit, K.; Yu, L. D.; Sarapirom, S.; Pitakrattananukool, S.; Anuntalabhochai, S.

    2015-12-01

    Plasma immersion ion implantation (PIII) at low energy was for the first time applied as a novel biotechnology to induce DNA transfer into bacterial cells. Argon or nitrogen PIII at low bias voltages of 2.5, 5 and 10 kV and fluences ranging from 1 × 1012 to 1 × 1017 ions/cm2 treated cells of Escherichia coli (E. coli). Subsequently, DNA transfer was operated by mixing the PIII-treated cells with DNA. Successes in PIII-induced DNA transfer were demonstrated by marker gene expressions. The induction of DNA transfer was ion-energy, fluence and DNA-size dependent. The DNA transferred in the cells was confirmed functioning. Mechanisms of the PIII-induced DNA transfer were investigated and discussed in terms of the E. coli cell envelope anatomy. Compared with conventional ion-beam-induced DNA transfer, PIII-induced DNA transfer was simpler with lower cost but higher efficiency.

  16. Evolution of DNA ligases of Nucleo-Cytoplasmic Large DNA viruses of eukaryotes: a case of hidden complexity

    PubMed Central

    2009-01-01

    Background Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for these enzymes were already present in the last common ancestor of the NCLDV. However, the details of the evolution of these genes that bear on the complexity of the putative ancestral NCLDV and on the evolutionary relationships between viruses and their hosts are not well understood. Results Phylogenetic analysis of the ATP-dependent and NAD-dependent DNA ligases encoded by the NCLDV reveals an unexpectedly complex evolutionary history. The NAD-dependent ligases are encoded only by a minority of NCLDV (including mimiviruses, some iridoviruses and entomopoxviruses) but phylogenetic analysis clearly indicated that all viral NAD-dependent ligases are monophyletic. Combined with the topology of the NCLDV tree derived by consensus of trees for universally conserved genes suggests that this enzyme was represented in the ancestral NCLDV. Phylogenetic analysis of ATP-dependent ligases that are encoded by chordopoxviruses, most of the phycodnaviruses and Marseillevirus failed to demonstrate monophyly and instead revealed an unexpectedly complex evolutionary trajectory. The ligases of the majority of phycodnaviruses and Marseillevirus seem to have evolved from bacteriophage or bacterial homologs; the ligase of one phycodnavirus, Emiliana huxlei virus, belongs to the eukaryotic DNA ligase I branch; and ligases of chordopoxviruses unequivocally cluster with eukaryotic DNA ligase III. Conclusions Examination of phyletic patterns and phylogenetic analysis of DNA ligases of the NCLDV suggest that the common ancestor of the extant NCLDV encoded an NAD-dependent ligase that most likely was acquired from a bacteriophage at the early stages of evolution of eukaryotes. By contrast, ATP-dependent ligases from different prokaryotic and eukaryotic sources displaced the ancestral NAD-dependent ligase at different

  17. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences

    NASA Technical Reports Server (NTRS)

    Nordheim, A.; Rich, A.

    1983-01-01

    Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50-80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

  18. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences

    NASA Technical Reports Server (NTRS)

    Nordheim, A.; Rich, A.

    1983-01-01

    Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50-80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

  19. Seasonal Dynamics of Haptophytes and dsDNA Algal Viruses Suggest Complex Virus-Host Relationship.

    PubMed

    Johannessen, Torill Vik; Larsen, Aud; Bratbak, Gunnar; Pagarete, António; Edvardsen, Bente; Egge, Elianne D; Sandaa, Ruth-Anne

    2017-04-20

    Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host-virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae, showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species.

  20. Seasonal Dynamics of Haptophytes and dsDNA Algal Viruses Suggest Complex Virus-Host Relationship

    PubMed Central

    Johannessen, Torill Vik; Larsen, Aud; Bratbak, Gunnar; Pagarete, António; Edvardsen, Bente; Egge, Elianne D.; Sandaa, Ruth-Anne

    2017-01-01

    Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host–virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae, showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species. PMID:28425942

  1. Halobacteriovorax, an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality

    PubMed Central

    Williams, Henry N; Lymperopoulou, Despoina S; Athar, Rana; Chauhan, Ashvini; Dickerson, Tamar L; Chen, Huan; Laws, Edward; Berhane, Timkhite-Kulu; Flowers, Adrienne R; Bradley, Nadine; Young, Shanterial; Blackwood, Denene; Murray, Jacqueline; Mustapha, Oladipupo; Blackwell, Cory; Tung, Yahsuan; Noble, Rachel T

    2016-01-01

    Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients. PMID:26251870

  2. Halobacteriovorax, an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality.

    PubMed

    Williams, Henry N; Lymperopoulou, Despoina S; Athar, Rana; Chauhan, Ashvini; Dickerson, Tamar L; Chen, Huan; Laws, Edward; Berhane, Timkhite-Kulu; Flowers, Adrienne R; Bradley, Nadine; Young, Shanterial; Blackwood, Denene; Murray, Jacqueline; Mustapha, Oladipupo; Blackwell, Cory; Tung, Yahsuan; Noble, Rachel T

    2016-02-01

    Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.

  3. Abundance of Virus-Sized Non-DNase-Digestible DNA (Coated DNA) in Eutrophic Seawater

    PubMed Central

    Maruyama, A.; Oda, M.; Higashihara, T.

    1993-01-01

    Total DNA concentration in 0.2-μm-pore-size Nuclepore filter filtrates (<0.2-μm fraction) of Tokyo Bay water was estimated to be 9 to 19 ng/ml by an immunochemical quantification method. Almost 90% of the DNA in the <0.2-μm fraction was found in the size fractions larger than 3.0 × 105 Da and 0.03 μm, and most was not susceptible to DNase digestion, that is, consisted of non-DNase-digestible DNA (coated DNA). A significant amount of DNA was obtained from the <0.2-μm fraction of the seawater by three different methods: polyethylene glycol precipitation, direct ethanol precipitation, and ultrafilter concentration. Gel electrophoresis analysis of the isolated DNAs showed that they consisted mainly of coated DNAs with a similar molecular sizes (20 to 30 kb [1.3 × 107 to 2.0 × 107 Da). The abundance of the ultramicron virus-sized coated DNA in natural seawater suggests that these DNA-rich particles can be attributed to marine DNA virus assemblages and that they may be a significant phosphorus reservoir in the environment. Images PMID:16348887

  4. Hepatitis E virus DNA vaccine elicits immunologic memory in mice.

    PubMed

    He, J; Hayes, C G; Binn, L N; Seriwatana, J; Vaughn, D W; Kuschner, R A; Innis, B L

    2001-01-01

    Injection of an expression vector pJHEV containing hepatitis E virus (HEV) structural protein open reading frame 2 gene generates a strong antibody response in BALB/c mice that can bind to and agglutinate HEV. In this study, we tested for immunologic memory in immunized mice whose current levels of IgG to HEV were low or undetectable despite 3 doses of HEV DNA vaccine 18 months earlier. Mice previously vaccinated with vector alone were controls. All mice were administered a dose of HEV DNA vaccine to simulate an infectious challenge with HEV. The endpoint was IgG to HEV determined by ELISA. Ten days after the vaccine dose, 5 of 9 mice previously immunized with HEV DNA vaccine had a slight increase in IgG to HEV. By 40 days after the vaccine dose, the level of IgG to HEV had increased dramatically in all 9 mice (108-fold increase in geometric mean titer). In contrast, no control mice became seropositive. These results indicate that mice vaccinated with 3 doses of HEV DNA vaccine retain immunologic memory. In response to a small antigenic challenge delivered as DNA, possibly less than delivered by a human infective dose of virus, mice with memory were able to generate high levels of antibody in less time than the usual incubation period of hepatitis E. We speculate that this type of response could protect a human from overt disease.

  5. Rapid and label-free electrochemical DNA biosensor for detecting hepatitis A virus.

    PubMed

    Manzano, Marisa; Viezzi, Sara; Mazerat, Sandra; Marks, Robert S; Vidic, Jasmina

    2017-08-19

    Diagnostic systems that can deliver highly specific and sensitive detection of hepatitis A virus (HAV) in food and water are of particular interest in many fields including food safety, biosecurity and control of outbreaks. Our aim was the development of an electrochemical method based on DNA hybridization to detect HAV. A ssDNA probe specific for HAV (capture probe) was designed and tested on DNAs from various viral and bacterial samples using Nested-Reverse Transcription Polymerase Chain Reaction (nRT-PCR). To develop the electrochemical device, a disposable gold electrode was functionalized with the specific capture probe and tested on complementary ssDNA and on HAV cDNA. The DNA hybridization on the electrode was measured through the monitoring of the oxidative peak potential of the indicator tripropylamine by cyclic voltammetry. To prevent non-specific binding the gold surface was treated with 3% BSA before detection. High resolution atomic force microscopy (AFM) confirmed the efficiency of electrode functionalization and on-electrode hybridization. The proposed device showed a limit of detection of 0.65pM for the complementary ssDNA and 6.94fg/µL for viral cDNA. For a comparison, nRT-PCR quantified the target HAV cDNA with a limit of detection of 6.4fg/µL. The DNA-sensor developed can be adapted to a portable format to be adopted as an easy-to- use and low cost method for screening HAV in contaminated food and water. In addition, it can be useful for rapid control of HAV infections as it takes only a few minutes to provide the results. Copyright © 2017. Published by Elsevier B.V.

  6. Relationships Between Environmental Factors, Bacterial Indicators, and the Occurrence of Enteric Viruses in Estuarine Sediments

    PubMed Central

    LaBelle, Raymond L.; Gerba, Charles P.; Goyal, Sagar M.; Melnick, Joseph L.; Cech, Irina; Bogdan, Gregory F.

    1980-01-01

    Current standards for evaluation of the public health safety of recreational and shellfish-harvesting waters are based upon bacteriological analysis, but do not include an evaluation of the number of viruses. The objective of this study was to determine the occurrence of enteric viruses in estuarine sediments and to find a relationship, if any, between the presence of viruses in seawater or sediment or both and various biological and physicochemical characteristics of the environment. Viruses were found in greater numbers in sediment than in overlying seawater on a volume basis. Several types of enteroviruses were isolated: coxsackievirus types A16, B1, and B5, echovirus type 1, and poliovirus type 2. On several occasions, viruses were isolated from sediments when overlying seawaters met bacteriological water quality standards for recreational use. Statistical analysis of the relationship between viruses in seawater or in sediment and other variables measured yielded only one significant association: the number of viruses in sediment was found to be positively correlated with the number of fecal coliforms in sediment. No other physical, chemical, or biological characteristic of seawater or sediment that was measured showed statistically significant association with viral numbers. No correlation was found between bacterial indicators and virus in the overlying waters. The data indicated that evaluation of the presence of bacteria and viruses in sediment may provide additional insight into long-term water quality conditions and that indicator bacteria in water are not reflective of the concentration of enteric viruses in marine waters. PMID:6247974

  7. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues

    USDA-ARS?s Scientific Manuscript database

    Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...

  8. A robust universal method for extraction of genomic DNA from bacterial species.

    PubMed

    Atashpaz, Sina; Khani, Sajjad; Barzegari, Abolfazl; Barar, Jaleh; Vahed, Sepideh Zununi; Azarbaijani, Reza; Omidi, Yadollah

    2010-01-01

    The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCI. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g., RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method f r large-scale DNA isolation fromvarious bacterial species.

  9. Infectious Maize rayado fino virus from Cloned cDNA.

    PubMed

    Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

    2015-06-01

    A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

  10. Origin and Evolution of Eukaryotic Large Nucleo-Cytoplasmic DNA Viruses

    PubMed Central

    Koonin, Eugene V.; Yutin, Natalya

    2010-01-01

    Background/Aims The nucleo-cytoplasmic large DNA viruses (NCLDV) constitute an apparently monophyletic group that consists of 6 families of viruses infecting a broad variety of eukaryotes. A comprehensive genome comparison and maximum-likelihood reconstruction of NCLDV evolution reveal a set of approximately 50 conserved genes that can be tentatively mapped to the genome of the common ancestor of this class of eukaryotic viruses. We address the origins and evolution of NCLDV. Results Phylogenetic analysis indicates that some of the major clades of NCLDV infect diverse animals and protists, suggestive of early radiation of the NCLDV, possibly concomitant with eukaryogenesis. The core NCLDV genes seem to have originated from different sources including homologous genes of bacteriophages, bacteria and eukaryotes. These observations are compatible with a scenario of the origin of the NCLDV at an early stage of the evolution of eukaryotes through extensive mixing of genes from widely different genomes. Conclusions The common ancestor of the NCLDV probably evolved from a bacteriophage as a result of recruitment of numerous eukaryotic and some bacterial genes, and concomitant loss of the majority of phage genes except for a small core of genes coding for proteins essential for virus genome replication and virion formation. PMID:20551680

  11. Carriage of λ Latent Virus Is Costly for Its Bacterial Host due to Frequent Reactivation in Monoxenic Mouse Intestine

    PubMed Central

    De Paepe, Marianne; Tournier, Laurent; Moncaut, Elisabeth; Son, Olivier; Langella, Philippe; Petit, Marie-Agnès

    2016-01-01

    Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts’ fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity. PMID:26871586

  12. Recombinant expression and purification of "virus-like" bacterial encapsulin protein cages.

    PubMed

    Rurup, W Frederik; Cornelissen, Jeroen J L M; Koay, Melissa S T

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules and organelles, the rate of migration can be used as a tool for purification. Here we describe a detailed protocol for the purification of recently discovered virus-like assemblies called bacterial encapsulins from Thermotoga maritima and Brevibacterium linens.

  13. Rapid cleanup of bacterial DNA from samples containing aerosol contaminants

    NASA Astrophysics Data System (ADS)

    Menking, Darrell E.; Kracke, Suzanne K.; Emanuel, Peter A.; Valdes, James J.

    1999-01-01

    Polymerase Chain Reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of DNA using gene probes allows for absolute identification not only of specific organisms, but also of genetic material in recombinant organisms. PCR is an exquisite biological method for detecting bacteria in aerosol samples. A major challenge facing detection of DNA from field samples is that they are almost sure to contain impurities, especially impurities that inhibit amplification through PCR. DNA is being extracted from air, sewage/stool samples, food, sputum, a water and sediment; however, multi- step, time consuming methods are required to isolate the DNA from the surrounding contamination. This research focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhibitors from 'dirty samples' to produce purified, PCR-ready DNA. GeneReleaser produces PCR-ready DNA in a rapid five-minute protocol. GeneReleaser resin was able to clean up sample contain micrograms of typical aerosol and water contaminants. The advantages of using GR are that it is rapid, inexpensive, requires one-step, uses no hazardous material and produces PCR-ready DNA.

  14. Effect of DNA extraction and sample preservation method on rumen bacterial population.

    PubMed

    Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kopečný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

    2014-10-01

    The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. ChIP-seq for genome-scale analysis of bacterial DNA-binding proteins.

    PubMed

    Bonocora, Richard P; Wade, Joseph T

    2015-01-01

    Protein-DNA interactions are central to many basic biological processes, including transcription regulation, DNA replication, and DNA repair. Chromatin Immunoprecipitation (ChIP) is used to determine the position and strength of protein-DNA interactions in vivo. Coupling ChIP with microarrays (ChIP-chip), and more recently with deep sequencing (ChIP-seq), has allowed genome-wide profiling of DNA binding events in vivo. In this chapter we outline the steps to generate ChIP-seq libraries from bacterial samples and briefly discuss basic analysis of the data.

  16. Lateral Gene Transfer of Family A DNA Polymerases between Thermophilic Viruses, Aquificae, and Apicomplexa

    PubMed Central

    Schoenfeld, Thomas W.; Murugapiran, Senthil K.; Dodsworth, Jeremy A.; Floyd, Sally; Lodes, Michael; Mead, David A.; Hedlund, Brian P.

    2013-01-01

    Bioinformatics and functional screens identified a group of Family A-type DNA Polymerase (polA) genes encoded by viruses inhabiting circumneutral and alkaline hot springs in Yellowstone National Park and the US Great Basin. The proteins encoded by these viral polA genes (PolAs) shared no significant sequence similarity with any known viral proteins but were remarkably similar to PolAs encoded by two of three families of the bacterial phylum Aquificae and by several apicoplast-targeted PolA-like proteins found in the eukaryotic phylum Apicomplexa, which includes the obligate parasites Plasmodium, Babesia, and Toxoplasma. The viral gene products share signature elements previously associated only with Aquificae and Apicomplexa PolA-like proteins and were similar to proteins encoded by prophage elements of a variety of otherwise unrelated Bacteria, each of which additionally encoded a prototypical bacterial PolA. Unique among known viral DNA polymerases, the viral PolA proteins of this study share with the Apicomplexa proteins large amino-terminal domains with putative helicase/primase elements but low primary sequence similarity. The genomic context and distribution, phylogeny, and biochemistry of these PolA proteins suggest that thermophilic viruses transferred polA genes to the Apicomplexa, likely through secondary endosymbiosis of a virus-infected proto-apicoplast, and to the common ancestor of two of three Aquificae families, where they displaced the orthologous cellular polA gene. On the basis of biochemical activity, gene structure, and sequence similarity, we speculate that the xenologous viral-type polA genes may have functions associated with diversity-generating recombination in both Bacteria and Apicomplexa. PMID:23608703

  17. New tools to convert bacterial artificial chromosomes to a self-excising design and their application to a herpes simplex virus type 1 infectious clone.

    PubMed

    Richards, Alexsia L; Sollars, Patricia J; Smith, Gregory A

    2016-08-31

    Infectious clones are fundamental tools for the study of many viruses, allowing for efficient mutagenesis and reproducible production of genetically-defined strains. For the large dsDNA genomes of the herpesviridae, bacterial artificial chromosomes have become the cloning vector of choice due to their capacity to house full-length herpesvirus genomes as single contiguous inserts. Furthermore, while maintained as plasmids in Escherichia coli, the clones can be mutated using robust prokaryotic recombination systems. An important consideration in the design of these clones is the means by which the vector backbone is removed from the virus genome upon delivery into mammalian cells. A common approach to vector excision is to encode loxP sites flanking the vector sequences and rely on Cre recombinase expression from a transformed cell line. Here we examine the efficiency of vector removal using this method, and describe a "self-excising" infectious clone of HSV-1 strain F that offers enhancements in virus production and utility. Insertion of a fluorescent protein expression cassette into the vector backbone of the HSV-1 strain F clone, pYEbac102, demonstrated that 2 serial passages on cells expressing Cre recombinase was required to achieve > 95 % vector removal from the virus population, with 3 serial passages resulting in undetectable vector retention. This requirement was eliminated by replacing the reporter coding sequence with the CREin gene, which consists of a Cre coding sequence disrupted by a synthetic intron. This self-excising variant of the infectious clone produced virus that propagated with wild-type kinetics in culture and lacked vector attenuation in a mouse neurovirulence model. Conversion of a herpesvirus infectious clone into a self-excising variant enables rapid production of viruses lacking bacterial vector sequences, and removes the requirement to initially propagate viruses in cells that express Cre recombinase. The self-excising bacterial

  18. Viruses

    USDA-ARS?s Scientific Manuscript database

    Lytic bacteriophages, viruses which infect and lyse bacterial cells, can provide a natural method to reduce bacterial pathogens on produce commodities. The use of multi-phage cocktails is most likely to be effective against bacterial pathogens on produce commodities, and minimize the development of...

  19. Impact of bacterial colonization on the severity, and accompanying airway inflammation, of virus-induced wheezing in children.

    PubMed

    Yu, D; Wei, L; Zhengxiu, L; Jian, L; Lijia, W; Wei, L; Xiqiang, Y; Xiaodong, Z; Zhou, F; Enmei, L

    2010-09-01

    It is reported that bacterial colonization of the airway in neonates affects the likelihood and severity of subsequent wheezing in childhood. This study aimed to explore the impact of bacterial colonization on the severity of virus-induced wheezing, and accompanying airway inflammation. Nasopharyngeal aspirates (NPAs) from 68 hospitalized children with bronchiolitis and 85 children with recurrent wheezing were obtained. Eleven common respiratory viruses were sought by PCR and/or direct fluorescence assay. Bacteria were isolated from NPAs by routine culture methods. Cell numbers and concentrations of cytokines/chemokines in the NPAs were measured, and nucleated cells were characterized. The frequency of bacterial colonization in children with recurrent wheezing was significantly higher than in children with an initial attack of bronchiolitis. Bacterial colonization accompanying virus infection had no effect on clinical manifestations, duration of hospitalization, concentrations of cytokines/chemokines (except interleukin-10 (IL-10)) or cellularity in the children with bronchiolitis; however, among the children with recurrent wheezing, those who had coexistent non-invasive bacterial colonization and virus infection presented more frequent cyanosis, longer duration of hospitalization, a higher concentration of IL-10 and a higher percentage of neutrophils in NPAs than those with virus infection but without bacterial colonization. Bacterial colonization was common in children with virus-induced wheezing, particularly in the situation of recurrent wheezing. To some extent, bacterial colonization accompanying virus infection may contribute to the severity of the wheezing because of its impact on airway inflammation.

  20. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata

    PubMed Central

    Breitbart, Mya; Benner, Bayleigh E.; Jernigan, Parker E.; Rosario, Karyna; Birsa, Laura M.; Harbeitner, Rachel C.; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B.; Berger, Stella A.; Nejstgaard, Jens C.

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton. PMID:26733971

  1. Using Time-Structured Data to Estimate Evolutionary Rates of Double-Stranded DNA Viruses

    PubMed Central

    Firth, Cadhla; Kitchen, Andrew; Shapiro, Beth; Suchard, Marc A.; Holmes, Edward C.; Rambaut, Andrew

    2010-01-01

    Double-stranded (ds) DNA viruses are often described as evolving through long-term codivergent associations with their hosts, a pattern that is expected to be associated with low rates of nucleotide substitution. However, the hypothesis of codivergence between dsDNA viruses and their hosts has rarely been rigorously tested, even though the vast majority of nucleotide substitution rate estimates for dsDNA viruses are based upon this assumption. It is therefore important to estimate the evolutionary rates of dsDNA viruses independent of the assumption of host-virus codivergence. Here, we explore the use of temporally structured sequence data within a Bayesian framework to estimate the evolutionary rates for seven human dsDNA viruses, including variola virus (VARV) (the causative agent of smallpox) and herpes simplex virus-1. Our analyses reveal that although the VARV genome is likely to evolve at a rate of approximately 1 × 10−5 substitutions/site/year and hence approaching that of many RNA viruses, the evolutionary rates of many other dsDNA viruses remain problematic to estimate. Synthetic data sets were constructed to inform our interpretation of the substitution rates estimated for these dsDNA viruses and the analysis of these demonstrated that given a sequence data set of appropriate length and sampling depth, it is possible to use time-structured analyses to estimate the substitution rates of many dsDNA viruses independently from the assumption of host-virus codivergence. Finally, the discovery that some dsDNA viruses may evolve at rates approaching those of RNA viruses has important implications for our understanding of the long-term evolutionary history and emergence potential of this major group of viruses. PMID:20363828

  2. Bithionol blocks pathogenicity of bacterial toxins, ricin, and Zika virus

    USDA-ARS?s Scientific Manuscript database

    Disease pathways form overlapping networks, and hub proteins represent attractive targets for broad-spectrum drugs. Using bacterial toxins as a proof of concept, we describe a new approach of discovering broad-spectrum therapies capable of inhibiting host proteins that mediate multiple pathogenic pa...

  3. The assembly of papaya mosaic virus coat protein with DNA.

    PubMed

    Erickson, J W; Bancroft, J B

    1980-01-01

    Products of specific (pH 8.0-8.5) and nonspecific (pH 6.0) assembly reactions of papaya mosaic virus (PMV) coat protein with DNA are described. The strandedness, topology, and sugar moiety of the nucleic acid are important parameters for assembly in nonspecific conditions. The linear, single-stranded form of lambda DNA, but not the double-stranded form, reacted with PMV protein to form multiply initiated particles whose helical segments apparently annealed to produce continuous tubular particles. With the circular, single-stranded DNA of phi X174, partially tubular, partially extended particles were made. Poly(dA), unlike poly(A) [Erickson JW, AbouHaidar M, Bancroft JB: Virology 90:60, 1978], was not encapsidated by PMV protein under specific assembly conditions. With all DNAs tested, extended particles were the only products formed in specific conditions at pH 8.5.

  4. DNase I sensitivity of integrated simian virus 40 DNA.

    PubMed Central

    Blanck, G; Chen, S; Pollack, R

    1984-01-01

    We undertook an analysis of integrated simian virus 40 (SV40) DNA to learn whether the DNase I-sensitive region is retained in the integrated array of mouse transformants. Our results indicate that full-length integrated SV40 chromatin retains a DNase I-hypersensitive region at the same point as in nonintegrated SV40 chromatin. Thus, the lack of a DNase I-hypersensitive region is not likely to be the reason for nonpermissivity of SV40 in mouse cells. In addition, results reported here indicate that a deletion of about 200 base pairs of DNA in the region of the DNase I-hypersensitive site severely reduces the sensitivity of integrated SV40 chromatin. This result is similar to a previously reported result obtained with deletion mutants of SV40 analyzed in the lytic cycle. It is the first report of a DNA lesion affecting DNase I hypersensitivity of a mammalian chromosome. Images PMID:6325884

  5. Necessity of Purification during Bacterial DNA Extraction with Environmental Soils.

    PubMed

    Lim, Hyun Jeong; Choi, Jung-Hyun; Son, Ahjeong

    2017-08-08

    Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for PCR assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium) showed that sand samples containing less than 10 ug/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of magnesium ion was different from other inhibitors due to the complexation interaction of magnesium ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 ug/g of humic acids, less than 70% clay content and less than 0.01% magnesium ion content.

  6. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response

    PubMed Central

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  7. Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

    PubMed

    Christen, Matthias; Deutsch, Samuel; Christen, Beat

    2015-08-21

    Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

  8. Comparison of seven methods for extraction of bacterial DNA from fecal and cecal samples of mice.

    PubMed

    Ferrand, Janina; Patron, Kevin; Legrand-Frossi, Christine; Frippiat, Jean-Pol; Merlin, Christophe; Alauzet, Corentine; Lozniewski, Alain

    2014-10-01

    Analysis of bacterial DNA from fecal samples of mice is commonly performed in experimental studies. Although DNA extraction is a critical step in various molecular approaches, the efficiency of methods that may be used for DNA extraction from mice fecal samples has never been evaluated. We compared the efficiencies of six widely used commercial kits (MasterPure™ Gram Positive DNA Purification Kit, QIAamp® DNA Stool Mini Kit; NucliSENS® easyMAG®, ZR Fecal DNA MiniPrep™, FastDNA® SPIN Kit for Feces and FastDNA® SPIN Kit for Soil) and a non-commercial method for DNA isolation from mice feces and cecal contents. DNA quantity and quality were assessed by fluorometry, spectrophotometry, gel electrophoresis and qPCR. Cell lysis efficiencies were evaluated by qPCR targeting three relevant bacteria in spiked specimens. For both feces and intestinal contents, the most efficient extraction method was the FastDNA® SPIN Kit for Soil.

  9. The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection

    PubMed Central

    Schliehe, Christopher; Swaminanthan, Savitha; Bosnjak, Berislav; Bauer, Lisa; Kandasamy, Richard K.; Griesshammer, Isabel M.; Kosack, Lindsay; Schmitz, Frank; Litvak, Vladimir; Sissons, James; Lercher, Alexander; Bhattacharya, Anannya; Khamina, Kseniya; Trivett, Anna L.; Tessarollo, Lino; Mesteri, Ildiko; Hladik, Anastasiya; Merkler, Doron; Kubicek, Stefan; Knapp, Sylvia; Epstein, Michelle M.; Bergthaler, Andreas

    2014-01-01

    Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that SET domain bifurcated 2 (Setdb2) was the only protein lysine methyltransferase induced during influenza virus infection. Setdb2 expression depended on type-I interferon signaling and it repressed the expression of the neutrophil attractant Cxcl1 and other NF-κB target genes. This coincided with Setdb2 occupancy at the Cxcl1 promoter, which in the absence of Setdb2 displayed reduced H3K9 tri-methylation. Setdb2 hypomorphic gene-trap mice exhibited increased neutrophil infiltration in sterile lung inflammation and were less sensitive to bacterial superinfection upon influenza virus infection. This suggests that a Setdb2-mediated regulatory crosstalk between the type-I interferon and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection. PMID:25419628

  10. Bacterial DNA translocation holds increased insulin resistance and systemic inflammatory levels in morbid obese patients.

    PubMed

    Ortiz, Sergio; Zapater, Pedro; Estrada, José Luis; Enriquez, Pablo; Rey, Monica; Abad, Angel; Such, José; Lluis, Félix; Francés, Rubén

    2014-07-01

    Morbidly obese patients show several common comorbidities associated with immunological alterations such as a sustained low-level proinflammatory profile. Bacterial product translocation is frequent in inflammation-related diseases and may aggravate patients' clinical outcome. Consecutively admitted morbidly obese patients who presented indications for bariatric surgery were studied. Before surgery, patients were subjected to a modified fasting diet. Patients underwent surgery by sleeve gastrectomy or laparoscopic Roux-en-Y gastric bypass. Clinical and analytical parameters were recorded. Blood samples were collected at baseline, at the end of a 3-month modified fasting period, and 3, 6, and 12 months after surgery. Serum cytokine and endotoxin levels were evaluated by flow cytometry and ELISA, respectively. Bacterial DNA was identified in blood by broad-range PCR of prokaryote 16SrRNA gene and partial sequencing analysis. Fifty-eight patients were included in the study. All patients showed a significantly reduced weight and body mass index at each time-point. Postoperative mortality was null. Bacterial DNA translocation rate was 32.8% (19 of 58) at baseline; 13.8% (8 of 58) after the modified fasting period; and 13.8% (8 of 58), 1.8% (1 of 58), and 5.2% (3 of 58) at 3, 6, and 12 months after surgery. Proinflammatory cytokines, serum endotoxin levels, and insulin resistance remained increased in patients with bacterial DNA despite weight loss and were individually affected by the appearance/clearance of bacterial DNA in blood. Multivariate analyses revealed bacterial DNA as an independent significant factor, explaining the systemic cytokine response and the insulin resistance levels in the studied population. Bacterial DNA translocation holds increased insulin resistance and systemic inflammatory levels in morbidly obese patients despite significant weight loss.

  11. A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

    PubMed Central

    Maciel, Milton; Cruz, Fábia da Silva Pereira; Cordeiro, Marli Tenório; da Motta, Márcia Archer; Cassemiro, Klécia Marília Soares de Melo; Maia, Rita de Cássia Carvalho; de Figueiredo, Regina Célia Bressan Queiroz; Galler, Ricardo; Freire, Marcos da Silva; August, Joseph Thomas; Marques, Ernesto T. A.; Dhalia, Rafael

    2015-01-01

    Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies. PMID:25875109

  12. A DNA vaccine against yellow fever virus: development and evaluation.

    PubMed

    Maciel, Milton; Cruz, Fábia da Silva Pereira; Cordeiro, Marli Tenório; da Motta, Márcia Archer; Cassemiro, Klécia Marília Soares de Melo; Maia, Rita de Cássia Carvalho; de Figueiredo, Regina Célia Bressan Queiroz; Galler, Ricardo; Freire, Marcos da Silva; August, Joseph Thomas; Marques, Ernesto T A; Dhalia, Rafael

    2015-04-01

    Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.

  13. A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses.

    PubMed

    Diemer, Geoffrey S; Stedman, Kenneth M

    2012-06-11

    Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis. Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar to those found only in single-stranded RNA viruses. The presence and circular configuration of the complete virus genome was confirmed by inverse PCR amplification from native DNA extracted from lake sediment. The virus genome appears to be the result of a RNA-DNA recombination event between two ostensibly unrelated virus groups. Environmental sequence databases were examined for homologous genes arranged in similar configurations and three similar putative virus genomes from marine environments were identified. This result indicates the existence of a widespread but previously undetected group of viruses. This unique viral genome carries implications for theories of virus emergence and evolution, as no mechanism for interviral RNA-DNA recombination has yet been identified, and only scant evidence exists that genetic exchange occurs between such distinct virus lineages. This article was reviewed by EK, MK (nominated by PF) and AM. For the full reviews, please go to the Reviewers' comments section.

  14. REVIEW ARTICLE: DNA protein interactions and bacterial chromosome architecture

    NASA Astrophysics Data System (ADS)

    Stavans, Joel; Oppenheim, Amos

    2006-12-01

    Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

  15. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    NASA Astrophysics Data System (ADS)

    Yu, L. D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

    2014-05-01

    As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10-20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  16. The inflammasome as a target of modulation by DNA viruses

    PubMed Central

    DeFilippis, Victor

    2014-01-01

    The cellular innate immune response represents the initial reaction of a host against infecting pathogens. Host cells detect incoming microbes by way of a large and expanding array of receptors that react with evolutionarily conserved molecular patterns exhibited by microbial intruders. These receptors are responsible for initiating signaling that leads to both transcriptional activation of immunologically important genes as well as protease-dependent processing of cellular proteins. The inflammasome refers to a protein complex that functions as an activation platform for the cysteine protease caspase-1, which then processes inflammatory molecules such as IL-1β and IL-18 into functional forms. Assembly of this complex is triggered following receptor-mediated detection of pathogen-associated molecules. Receptors have been identified that are essential to inflammasome activation in response to numerous molecular patterns including virus-associated molecules such as DNA. In fact, the importance of cytoplasmic DNA as an immune stimulus is exemplified by the existence of at least nine distinct cellular receptors capable of initiating innate reactivity in response to this molecule. Viruses that employ DNA as genomic material include herpesviruses, poxviruses and adenoviruses. Each has been described as capable of inducing inflammasome-mediated activity. Interestingly, however, the cellular molecules responsible for these responses appear to vary according to host species, cell type and even viral strain. Secretion of IL-1β and IL-18 are important components of antimicrobial immunity and, as a result, pathogens have evolved factors to evade or counteract this response. This includes DNA-based viruses, many of which encode multiple redundant counteractive molecules. However, it is clear that such phenotypes are only beginning to be uncovered. The purpose of this review is to describe what is known regarding the activation of inflammasome-mediated processes in response to

  17. Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication.

    PubMed

    Muylaert, Isabella; Elias, Per

    2007-04-13

    Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.

  18. New BK virus episomal vector for complementary DNA expression in human cells.

    PubMed

    Grossi, M P; Caputo, A; Rimessi, P; Chiccoli, L; Balboni, P G; Barbanti-Brodano, G

    1988-01-01

    The properties of pRP-c, a new vector for complementary DNA (cDNA) expression, are described. The vector contains the early region and replication origin of BK virus (BKV), a human papovavirus. Due to the presence of these BKV sequences, pRP-c replicates in human cells allowing amplification of inserted cDNAs. The promoter, intron and polyadenylation region for cDNA expression are separated by unique restriction sites and can therefore be individually excised and substituted with different transcription signals. Coding sequences of the bacterial genes for chloramphenicol-acetyl transferase (CAT) or neomycin phosphotransferase (neo) were inserted into the cDNA cloning site of pRP-c and expressed in human cells in transient assays or stable clones. In both cases expression of the inserted sequences was significantly more efficient than by using the integration vectors pSV2CAT and pSV2neo, demonstrating the advantages of episomal expression vectors in human cells. Possible uses of pRP-c to express viral and cellular cDNAs in human cells are discussed.

  19. Bacterial and fungal DNA extraction from blood samples: manual protocols.

    PubMed

    Lorenz, Michael G; Mühl, Helge; Disqué, Claudia

    2015-01-01

    A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

  20. Bones hold the key to DNA virus history and epidemiology.

    PubMed

    Toppinen, M; Perdomo, M F; Palo, J U; Simmonds, P; Lycett, S J; Söderlund-Venermo, M; Sajantila, A; Hedman, K

    2015-11-27

    DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970´s, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20(th) century and provides the first evidence of the suitability of bone for exploration of DNA viruses.

  1. Host DNA damage response facilitates African swine fever virus infection.

    PubMed

    Simões, Margarida; Martins, Carlos; Ferreira, Fernando

    2013-07-26

    Studies with different viral infection models on virus interactions with the host cell nucleus have opened new perspectives on our understanding of the molecular basis of these interactions in African swine fever virus (ASFV) infection. The present study aims to characterize the host DNA damage response (DDR) occurring upon in vitro infection with the ASFV-Ba71V isolate. We evaluated protein levels during ASFV time-course infection, of several signalling cascade factors belonging to DDR pathways involved in double strand break repair - Ataxia Telangiectasia Mutated (ATM), ATM-Rad 3 related (ATR) and DNA dependent protein kinase catalytic subunit (DNA-PKcs). DDR inhibitory trials using caffeine and wortmannin and ATR inducible-expression cell lines were used to confirm specific pathway activation during viral infection. Our results show that ASFV specifically elicits ATR-mediated pathway activation from the early phase of infection with increased levels of H2AX, RPA32, p53, ATR and Chk1 phosphorylated forms. Viral p72 synthesis was abrogated by ATR kinase inhibitors and also in ATR-kd cells. Furthermore, a reduction of viral progeny was identified in these cells when compared to the outcome of infection in ATR-wt. Overall, our results strongly suggest that the ATR pathway plays an essential role for successful ASFV infection of host cells.

  2. The influenza virus nucleoprotein synthesized from cloned DNA in a simian virus 40 vector is detected in the nucleus.

    PubMed Central

    Lin, B C; Lai, C J

    1983-01-01

    We obtained DNA sequences coding for the nucleoprotein (NP) of an influenza A virus by reverse transcription of virion RNA with synthetic oligonucleotide primers. Terminal sequence analysis showed that the cloned gene contained a full-length copy of the virion RNA segment. The NP-specific DNA was inserted into the late region of a simian virus 40 vector, and the DNA recombinant was propagated in the presence of an early simian virus 40 temperature-sensitive mutant helper. Infection of African green monkey kidney cells with the recombinant produced a polypeptide immunoprecipitable with NP-specific antisera. The polypeptide product had a molecular weight of 56,000, identical to that of the nucleoprotein of influenza virus as estimated on polyacrylamide gels. The putative NP was detected in the nucleus of infected primate cells by an immunofluorescence assay. This nuclear localization of NP from recombinant DNA was similar to that seen during influenza virus infection. Images PMID:6296449

  3. Altered Virome and Bacterial Microbiome in Human Immunodeficiency Virus-Associated Acquired Immunodeficiency Syndrome.

    PubMed

    Monaco, Cynthia L; Gootenberg, David B; Zhao, Guoyan; Handley, Scott A; Ghebremichael, Musie S; Lim, Efrem S; Lankowski, Alex; Baldridge, Megan T; Wilen, Craig B; Flagg, Meaghan; Norman, Jason M; Keller, Brian C; Luévano, Jesús Mario; Wang, David; Boum, Yap; Martin, Jeffrey N; Hunt, Peter W; Bangsberg, David R; Siedner, Mark J; Kwon, Douglas S; Virgin, Herbert W

    2016-03-09

    Human immunodeficiency virus (HIV) infection is associated with increased intestinal translocation of microbial products and enteropathy as well as alterations in gut bacterial communities. However, whether the enteric virome contributes to this infection and resulting immunodeficiency remains unknown. We characterized the enteric virome and bacterial microbiome in a cohort of Ugandan patients, including HIV-uninfected or HIV-infected subjects and those either treated with anti-retroviral therapy (ART) or untreated. Low peripheral CD4 T cell counts were associated with an expansion of enteric adenovirus sequences and this increase was independent of ART treatment. Additionally, the enteric bacterial microbiome of patients with lower CD4 T counts exhibited reduced phylogenetic diversity and richness with specific bacteria showing differential abundance, including increases in Enterobacteriaceae, which have been associated with inflammation. Thus, immunodeficiency in progressive HIV infection is associated with alterations in the enteric virome and bacterial microbiome, which may contribute to AIDS-associated enteropathy and disease progression.

  4. The DNA damage response induced by infection with human cytomegalovirus and other viruses.

    PubMed

    Xiaofei, E; Kowalik, Timothy F

    2014-05-23

    Viruses use different strategies to overcome the host defense system. Recent studies have shown that viruses can induce DNA damage response (DDR). Many of these viruses use DDR signaling to benefit their replication, while other viruses block or inactivate DDR signaling. This review focuses on the effects of DDR and DNA repair on human cytomegalovirus (HCMV) replication. Here, we review the DDR induced by HCMV infection and its similarities and differences to DDR induced by other viruses. As DDR signaling pathways are critical for the replication of many viruses, blocking these pathways may represent novel therapeutic opportunities for the treatment of certain infectious diseases. Lastly, future perspectives in the field are discussed.

  5. Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency

    PubMed Central

    Zhang, Yong-Yuan

    2015-01-01

    Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells. PMID:26713436

  6. Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

    SciTech Connect

    Jiang, X.; Estes, M.K.; Metcalf, T.G.; Melnick, J.L

    1986-10-01

    The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10/sup 4/ physical particlels of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hydbridization stringency, /sup 32/P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.

  7. Bacterial DNA persists for extended periods after cell death.

    PubMed

    Young, Geoffrey; Turner, Sally; Davies, John K; Sundqvist, Göran; Figdor, David

    2007-12-01

    The fate of DNA from bacteria that infect the root canal but cannot survive is currently unknown, yet such information is essential in establishing the validity of polymerase chain reaction (PCR)-based identification methods for root canal samples. This in vitro study tested the hypothesis that PCR-detectable DNA from dead bacteria might persist after cell death and investigated the efficiency of sodium hypochlorite (NaOCl) as a field decontamination agent. Using heat-killed Enterococcus faecalis, the persistence of DNA encoding the 16S rRNA gene was monitored by PCR. While most probable number analysis showed an approximate 1000-fold decay in amplifiable template, E. faecalis DNA was still PCR-detectable 1 year after cell death. NaOCl (1%) eliminated amplifiable DNA within 60 seconds of exposure. Our findings also disclosed a previously overlooked problem of concentration-dependent inhibition of the PCR reaction by thiosulfate-inactivated NaOCl. These results highlight the challenges of reliably identifying the authentic living root canal flora with PCR techniques.

  8. Chromosome replication status and DNA content at any cell age in a bacterial cell cycle.

    PubMed

    Jiménez-Sánchez, Alfonso

    2015-09-07

    An algorithm is presented to determine the chromosome replication status, the rate of DNA synthesis per fork, and the amount of DNA in chromosome equivalents (G) per chromosome, per cell and per age throughout a bacterial cell cycle. This algorithm is the only attempt to study replication and the G value at any cell age since the general model of the bacterial cell cycle by Cooper and Helmstetter (1968, J. Mol. Biol. 31, 619-644). To help using it, two implementations are provided. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

    PubMed Central

    Reeves, Analise Z; Lesser, Cammie F

    2017-01-01

    Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives. PMID:28203614

  10. Whole genome molecular phylogeny of large dsDNA viruses using composition vector method

    PubMed Central

    Gao, Lei; Qi, Ji

    2007-01-01

    Background One important mechanism by which large DNA viruses increase their genome size is the addition of modules acquired from other viruses, host genomes or gene duplications. Phylogenetic analysis of large DNA viruses, especially using methods based on alignment, is often difficult due to the presence of horizontal gene transfer events. The recent composition vector approach, not sensitive to such events, is applied here to reconstruct the phylogeny of 124 large DNA viruses. Results The results are mostly consistent with the biologist's systematics with only a few outliers and can also provide some information for those unclassified viruses and cladistic relationships of several families. Conclusion With composition vector approach we obtained the phylogenetic tree of large DNA viruses, which not only give results comparable to biologist's systematics but also provide a new way for recovering the phylogeny of viruses. PMID:17359548

  11. Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes.

    PubMed

    Chen, Chao; Wang, Lianrong; Chen, Si; Wu, Xiaolin; Gu, Meijia; Chen, Xi; Jiang, Susu; Wang, Yunfu; Deng, Zixin; Dedon, Peter C; Chen, Shi

    2017-04-11

    Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, (6m)A methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(GPS(6m)A) dinucleotides in the GPS(6m)AAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(GPSA) motifs per genome, with (6m)A arising from a yet-to-be-identified methyltransferase. To further explore PT and (6m)A in another consensus sequence, GPS(6m)ATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in GPSATC) and Dam methyltransferase from E. coli DH10B ((6m)A in G(6m)ATC). Based on this model, in vitro studies revealed reduced Dam activity in GPSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed (6m)A in all 2,058 GPSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that (6m)A can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.

  12. Molecular interactions in viruses investigated by Raman spectroscopy: the chromosome and capsid of bacterial virus P22

    NASA Astrophysics Data System (ADS)

    Thomas, George J., Jr.

    1993-07-01

    Laser Raman scattering is the method of choice for probing macromolecular structures and interactions in complex biological assemblies. The Raman technique is a valuable complement to high-resolution structure methods applied to biological molecules in the crystal (x-ray crystallography) and to static and dynamic light scattering methods applicable to solutions. Raman spectroscopy is particularly well suited to investigating assembly mechanisms of viruses and conformations of their nucleic acid and protein constituents. Here we describe the use of digital difference Raman methods to probe structures of the double-stranded (ds) DNA genome of the icosahedral virus P22 in packaged and unpackaged states. The findings are compared with structural changes in protein subunits of the P22 viral capsid, attendant with capsid expansion and DNA packaging. We find no evidence in the Raman spectrum of specific intermolecular interactions involving capsid protein and major groove sites of the packaged DNA. The present results suggest a simple model for organization of condensed dsDNA chromosomes in icosahedral viruses.

  13. RNA silencing pathways of plants: silencing and its suppression by plant DNA viruses.

    PubMed

    Hohn, Thomas; Vazquez, Franck

    2011-01-01

    RNA silencing refers to processes that depend on small (s)RNAs to regulate the expression of eukaryotic genomes. In plants, these processes play critical roles in development, in responses to a wide array of stresses, in maintaining genome integrity and in defense against viral and bacterial pathogens. We provide here an updated view on the array of endogenous sRNA pathways, including microRNAs (miRNAs), discovered in the model plant Arabidopsis, which are also the basis for antiviral silencing. We emphasize the current knowledge as well as the recent advances made on understanding the defense and counter-defense strategies evolved in the arms race between plants and DNA viruses on both the nuclear and the cytoplasmic front. This article is part of a Special Issue entitled: MicroRNA's in viral gene regulation. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  15. Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    PubMed Central

    Vuyisich, Momchilo; Arefin, Ayesha; Davenport, Karen; Feng, Shihai; Gleasner, Cheryl; McMurry, Kim; Parson-Quintana, Beverly; Price, Jennifer; Scholz, Matthew; Chain, Patrick

    2014-01-01

    Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderia spp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic DNA is used. PMID:25478564

  16. Genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cDNA clone.

    PubMed

    Jengarn, Juggragarn; Wongthida, Phonphimon; Wanasen, Nanchaya; Frantz, Phanramphoei Namprachan; Wanitchang, Asawin; Jongkaewwattana, Anan

    2015-08-01

    Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDVAVCT12. Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDVAVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDVAVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate.

  17. Recovery of Pathogenic Measles Virus from Cloned cDNA

    PubMed Central

    Takeda, Makoto; Takeuchi, Kaoru; Miyajima, Naoko; Kobune, Fumio; Ami, Yasushi; Nagata, Noriyo; Suzaki, Yuriko; Nagai, Yoshiyuki; Tashiro, Masato

    2000-01-01

    Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700–705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773–5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis. PMID:10864679

  18. Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses.

    PubMed

    Xu, Xin-Gang; Chen, Guang-Da; Huang, Yong; Ding, Li; Li, Zhao-Cai; Chang, Ching-Dong; Wang, Chi-Young; Tong, De-Wen; Liu, Hung-Jen

    2012-07-01

    Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine.

  19. Rapid development of a DNA vaccine for Zika virus.

    PubMed

    Dowd, Kimberly A; Ko, Sung-Youl; Morabito, Kaitlyn M; Yang, Eun Sung; Pelc, Rebecca S; DeMaso, Christina R; Castilho, Leda R; Abbink, Peter; Boyd, Michael; Nityanandam, Ramya; Gordon, David N; Gallagher, John Robert; Chen, Xuejun; Todd, John-Paul; Tsybovsky, Yaroslav; Harris, Audray; Huang, Yan-Jang S; Higgs, Stephen; Vanlandingham, Dana L; Andersen, Hanne; Lewis, Mark G; De La Barrera, Rafael; Eckels, Kenneth H; Jarman, Richard G; Nason, Martha C; Barouch, Dan H; Roederer, Mario; Kong, Wing-Pui; Mascola, John R; Pierson, Theodore C; Graham, Barney S

    2016-10-14

    Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection. Copyright © 2016, American Association for the Advancement of Science.

  20. Stimulation of bacterial DNA synthesis by algal exudates in attached algal-bacterial consortia. [Amphora coffeaeformis; Vibrio proteolyticus

    SciTech Connect

    Murray, R.E.; Cooksey, K.E.; Priscu, J.C.

    1986-11-01

    Algal-bacterial consortia attached to polystyrene surfaces were prepared in the laboratory by using the marine diatom Amphora coffeaeformis and the marine bacterium Vibrio proteolytica (the approved name of this bacterium is Vibrio proteolyticus. The organisms were attached to the surfaces at cell densities of approximately 5 x 10/sup 4/ cells cm/sup -2/ (diatoms) and 5 x 10/sup 6/ cells cm/sup -2/ (bacteria). The algal-bacterial consortia consistently exhibited higher rates of (/sup 3/H)thymidine incorporation than did biofilms composed solely of bacteria. The rates of (/sup 3/H)thymidine incorporation by the algal-bacterial consortia were fourfold greater than the rates of incorporation by monobacterial biofilms 16 h after biofilm formation and were 16-fold greater 70 h after biofilm formation. Extracellular material released from the attached Amphora cells supported rates of bacterial activity (0.8 x 10/sup -21/ mol to 17.9 x 10/sup -21/ mol of (/sup 3/H)thymidine incorporated cell /sup -1/ h/sup -1/) and growth (doubling time, 29.5 to 1.4 days) comparable to values reported for a wide variety of marine and freshwater ecosystems. In the presence of sessile diatom populations, DNA synthesis by attached V. proteolytica cells was light dependent and increased with increasing algal abundance. The metabolic activity of diatoms thus appears to be the rate-limiting process in biofilm development on illuminated surfaces under conditions of low bulk-water dissolved organic carbon.

  1. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency

    PubMed Central

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A. Francis; Müller, Rolf; Zhang, Youming

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples. PMID:27095488

  2. Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA.

    PubMed

    Gourves, A S; Defais, M; Johnson, N P

    2001-03-30

    The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I. This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the presence of adenosine 5'-O-(thiotriphosphate) cofactor using fluorescence anisotropy. The RecA protein carried out DNA strand exchange with a 5'-fluorescein-labeled 32-mer oligonucleotide. The anisotropy signal was shown to measure oligonucleotide binding to RecA, and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described by a simple two-state equilibrium, K = 4.5 +/- 1.5 x 10(5) m(-1) (37 degrees C, 150 mm NaCl, pH 7.4). The reaction was enthalpy-driven and entropy-opposed. It depended on salt concentration and was sensitive to the type of monovalent anion, suggesting that anion-dependent protein conformations contribute to ssDNA binding at site II.

  3. Structure of the DNA-SspC Complex: Implications for DNA Packaging, Protection, and Repair in Bacterial Spores

    PubMed Central

    Frenkiel-Krispin, Daphna; Sack, Rinat; Englander, Joseph; Shimoni, Eyal; Eisenstein, Miriam; Bullitt, Esther; Horowitz-Scherer, Rachel; Hayes, Christopher S.; Setlow, Peter; Minsky, Abraham; Wolf, Sharon Grayer

    2004-01-01

    Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed α/β-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the α/β-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the α/β-type SASP. PMID:15150240

  4. Translation in Giant Viruses: A Unique Mixture of Bacterial and Eukaryotic Termination Schemes

    PubMed Central

    Jeudy, Sandra; Abergel, Chantal; Claverie, Jean-Michel; Legendre, Matthieu

    2012-01-01

    Mimivirus and Megavirus are the best characterized representatives of an expanding new family of giant viruses infecting Acanthamoeba. Their most distinctive features, megabase-sized genomes carried in particles of size comparable to that of small bacteria, fill the gap between the viral and cellular worlds. These giant viruses are also uniquely equipped with genes coding for central components of the translation apparatus. The presence of those genes, thought to be hallmarks of cellular organisms, revived fundamental interrogations on the evolutionary origin of these viruses and the link they might have with the emergence of eukaryotes. In this work, we focused on the Mimivirus-encoded translation termination factor gene, the detailed primary structure of which was elucidated using computational and experimental approaches. We demonstrated that the translation of this protein proceeds through two internal stop codons via two distinct recoding events: a frameshift and a readthrough, the combined occurrence of which is unique to these viruses. Unexpectedly, the viral gene carries an autoregulatory mechanism exclusively encountered in bacterial termination factors, though the viral sequence is related to the eukaryotic/archaeal class-I release factors. This finding is a hint that the virally-encoded translation functions may not be strictly redundant with the one provided by the host. Lastly, the perplexing occurrence of a bacterial-like regulatory mechanism in a eukaryotic/archaeal homologous gene is yet another oddity brought about by the study of giant viruses. PMID:23271980

  5. Translation in giant viruses: a unique mixture of bacterial and eukaryotic termination schemes.

    PubMed

    Jeudy, Sandra; Abergel, Chantal; Claverie, Jean-Michel; Legendre, Matthieu

    2012-01-01

    Mimivirus and Megavirus are the best characterized representatives of an expanding new family of giant viruses infecting Acanthamoeba. Their most distinctive features, megabase-sized genomes carried in particles of size comparable to that of small bacteria, fill the gap between the viral and cellular worlds. These giant viruses are also uniquely equipped with genes coding for central components of the translation apparatus. The presence of those genes, thought to be hallmarks of cellular organisms, revived fundamental interrogations on the evolutionary origin of these viruses and the link they might have with the emergence of eukaryotes. In this work, we focused on the Mimivirus-encoded translation termination factor gene, the detailed primary structure of which was elucidated using computational and experimental approaches. We demonstrated that the translation of this protein proceeds through two internal stop codons via two distinct recoding events: a frameshift and a readthrough, the combined occurrence of which is unique to these viruses. Unexpectedly, the viral gene carries an autoregulatory mechanism exclusively encountered in bacterial termination factors, though the viral sequence is related to the eukaryotic/archaeal class-I release factors. This finding is a hint that the virally-encoded translation functions may not be strictly redundant with the one provided by the host. Lastly, the perplexing occurrence of a bacterial-like regulatory mechanism in a eukaryotic/archaeal homologous gene is yet another oddity brought about by the study of giant viruses.

  6. Novel circular DNA viruses identified in Procordulia grayi and Xanthocnemis zealandica larvae using metagenomic approaches.

    PubMed

    Dayaram, Anisha; Galatowitsch, Mark; Harding, Jon S; Argüello-Astorga, Gerardo R; Varsani, Arvind

    2014-03-01

    Recent advances in sequencing and metagenomics have enabled the discovery of many novel single stranded DNA (ssDNA) viruses from various environments. We have previously demonstrated that adult dragonflies, as predatory insects, are useful indicators of ssDNA viruses in terrestrial ecosystems. Here we recover and characterise 13 viral genomes which represent 10 novel and diverse circular replication associated protein (Rep)-encoding single stranded (CRESS) DNA viruses (1628-2668nt) from Procordulia grayi and Xanthocnemis zealandica dragonfly larvae collected from four high-country lakes in the South Island of New Zealand. The dragonfly larvae associated CRESS DNA viruses have different genome architectures, however, they all encode two major open reading frames (ORFs) which either have bidirectional or unidirectional arrangement. The 13 viral genomes have a conserved NAGTATTAC nonanucleotide motif and in their predicted Rep proteins we identified the rolling circle replication (RCR) motif 1, 2 and 3, as well as superfamily 3 (SF3) helicase motifs. Maximum likelihood phylogenetic and pairwise identity analysis of the Rep amino acid sequences reveal that the dragonfly larvae novel CRESS DNA viruses share <63% pairwise amino acid identity to the Reps of other CRESS DNA viruses whose complete genomes have been determined and available in public databases and that these viruses are novel. CRESS DNA viruses are circulating in larval dragonfly populations; however, we are unable to ascertain whether these viruses are infecting the larvae directly or are transient within dragonflies via their diet. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. UV Radiation Damage and Bacterial DNA Repair Systems

    ERIC Educational Resources Information Center

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  8. UV Radiation Damage and Bacterial DNA Repair Systems

    ERIC Educational Resources Information Center

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  9. Bacterial DNA Sifted from the Trichoplax adhaerens (Animalia: Placozoa) Genome Project Reveals a Putative Rickettsial Endosymbiont

    PubMed Central

    Driscoll, Timothy; Gillespie, Joseph J.; Nordberg, Eric K.; Azad, Abdu F.; Sobral, Bruno W.

    2013-01-01

    Eukaryotic genome sequencing projects often yield bacterial DNA sequences, data typically considered as microbial contamination. However, these sequences may also indicate either symbiont genes or lateral gene transfer (LGT) to host genomes. These bacterial sequences can provide clues about eukaryote–microbe interactions. Here, we used the genome of the primitive animal Trichoplax adhaerens (Metazoa: Placozoa), which is known to harbor an uncharacterized Gram-negative endosymbiont, to search for the presence of bacterial DNA sequences. Bioinformatic and phylogenomic analyses of extracted data from the genome assembly (181 bacterial coding sequences [CDS]) and trace read archive (16S rDNA) revealed a dominant proteobacterial profile strongly skewed to Rickettsiales (Alphaproteobacteria) genomes. By way of phylogenetic analysis of 16S rDNA and 113 proteins conserved across proteobacterial genomes, as well as identification of 27 rickettsial signature genes, we propose a Rickettsiales endosymbiont of T. adhaerens (RETA). The majority (93%) of the identified bacterial CDS belongs to small scaffolds containing prokaryotic-like genes; however, 12 CDS were identified on large scaffolds comprised of eukaryotic-like genes, suggesting that T. adhaerens might have recently acquired bacterial genes. These putative LGTs may coincide with the placozoan’s aquatic niche and symbiosis with RETA. This work underscores the rich, and relatively untapped, resource of eukaryotic genome projects for harboring data pertinent to host–microbial interactions. The nature of unknown (or poorly characterized) bacterial species may only emerge via analysis of host genome sequencing projects, particularly if these species are resistant to cell culturing, as are many obligate intracellular microbes. Our work provides methodological insight for such an approach. PMID:23475938

  10. Compaction of bacterial genomic DNA: clarifying the concepts.

    PubMed

    Joyeux, Marc

    2015-09-30

    The unconstrained genomic DNA of bacteria forms a coil, whose volume exceeds 1000 times the volume of the cell. Since prokaryotes lack a membrane-bound nucleus, in sharp contrast with eukaryotes, the DNA may consequently be expected to occupy the whole available volume when constrained to fit in the cell. Still, it has been known for more than half a century that the DNA is localized in a well-defined region of the cell, called the nucleoid, which occupies only 15% to 25% of the total volume. Although this problem has focused the attention of many scientists in recent decades, there is still no certainty concerning the mechanism that enables such a dramatic compaction. The goal of this Topical Review is to take stock of our knowledge on this question by listing all possible compaction mechanisms with the proclaimed desire to clarify the physical principles they are based upon and discuss them in the light of experimental results and the results of simulations based on coarse-grained models. In particular, the fundamental differences between ψ-condensation and segregative phase separation and between the condensation by small and long polycations are highlighted. This review suggests that the importance of certain mechanisms, like supercoiling and the architectural properties of DNA-bridging and DNA-bending nucleoid proteins, may have been overestimated, whereas other mechanisms, like segregative phase separation and the self-association of nucleoid proteins, as well as the possible role of the synergy of two or more mechanisms, may conversely deserve more attention.

  11. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    PubMed

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

  12. Invertebrate Iridescent Virus 6, a DNA Virus, Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors

    PubMed Central

    Ahlers, Laura R. H.; Bastos, Reginaldo G.; Hiroyasu, Aoi

    2016-01-01

    Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-β secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection. PMID:27824940

  13. Gyramides prevent bacterial growth by inhibiting DNA gyrase and altering chromosome topology.

    PubMed

    Rajendram, Manohary; Hurley, Katherine A; Foss, Marie H; Thornton, Kelsey M; Moore, Jared T; Shaw, Jared T; Weibel, Douglas B

    2014-06-20

    Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.

  14. Gyramides Prevent Bacterial Growth by Inhibiting DNA Gyrase and Altering Chromosome Topology

    PubMed Central

    2015-01-01

    Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division. PMID:24712739

  15. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

    PubMed Central

    Versalovic, J; Koeuth, T; Lupski, J R

    1991-01-01

    Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes. Images PMID:1762913

  16. Engineering of bacterial strains and vectors for the production of plasmid DNA.

    PubMed

    Bower, Diana M; Prather, Kristala L J

    2009-04-01

    The demand for plasmid DNA (pDNA) is anticipated to increase significantly as DNA vaccines and non-viral gene therapies enter phase 3 clinical trials and are approved for use. This increased demand, along with renewed interest in pDNA as a therapeutic vector, has motivated research targeting the design of high-yield, cost-effective manufacturing processes. An important aspect of this research is engineering bacterial strains and plasmids that are specifically suited to the production of plasmid biopharmaceuticals. This review will survey recent innovations in strain and vector engineering that aim to improve plasmid stability, enhance product safety, increase yield, and facilitate downstream purification. While these innovations all seek to enhance pDNA production, they can vary in complexity from subtle alterations of the host genome or vector backbone to the investigation of non-traditional host strains for higher pDNA yields.

  17. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    PubMed

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.

  18. Herpes Simplex Virus Latency: The DNA Repair-Centered Pathway

    PubMed Central

    2017-01-01

    Like all herpesviruses, herpes simplex virus 1 (HSV1) is able to produce lytic or latent infections depending on the host cell type. Lytic infections occur in a broad range of cells while latency is highly specific for neurons. Although latency suggests itself as an attractive target for novel anti-HSV1 therapies, progress in their development has been slowed due in part to a lack of agreement about the basic biochemical mechanisms involved. Among the possibilities being considered is a pathway in which DNA repair mechanisms play a central role. Repair is suggested to be involved in both HSV1 entry into latency and reactivation from it. Here I describe the basic features of the DNA repair-centered pathway and discuss some of the experimental evidence supporting it. The pathway is particularly attractive because it is able to account for important features of the latent response, including the specificity for neurons, the specificity for neurons of the peripheral compared to the central nervous system, the high rate of genetic recombination in HSV1-infected cells, and the genetic identity of infecting and reactivated virus. PMID:28255301

  19. Construction and characterization of bacterial artificial chromosomes (BACs) containing herpes simplex virus full-length genomes.

    PubMed

    Nagel, Claus-Henning; Pohlmann, Anja; Sodeik, Beate

    2014-01-01

    Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models.

  20. Serum bacterial DNA detection in patients with cholangitis after Kasai procedure.

    PubMed

    Luo, Qianfu; Hao, Fabao; Zhang, Mingman; Guo, Chunbao

    2015-10-01

    The aim of this study was to investigate the detectability of bacterial DNA (bactDNA) and associated clinical factors in patients with cholangitis after the Kasai procedure through the identification of bactDNA in blood. A cross-sectional study of 110 patients who had undergone the Kasai procedure was carried out. A total of 77 patients had cholangitis and 33 did not. Blood samples were obtained on admission or on follow up at the outpatient visit. bactDNA was analyzed using a polymerase chain reaction-based method, DNA nucleotide sequencing, for identification of bacterial species. Rate of bactDNA positivity was higher using the 16SrDNA method than the culture method (58.4% vs 14.3%, P < 0.0001), and more patients with cholangitis were positive for bactDNA compared with the patients without cholangitis (58.4% vs 39.4%, P = 0.095). Bacteria identified on blood 16SrDNA primarily consisted of opportunistic pathogens derived from the gut, including Escherichia coli, Klebsiella pneumoniae, Shigella fexneri, and Enterobacteriaceae bacterium. Treatment with probiotics or antibiotics showed a trend toward reduced prevalence of bactDNA. Pediatric end-stage liver disease score, procalcitonin, C-reactive protein, and heart rate were all significantly associated with the presence of bactDNA. bactDNA is frequently detected in patients with cholangitis after the Kasai procedure compared with those without cholangitis (and controls), and the bacteria are similar to those in the gut, suggesting that it is useful as an early diagnostic tool for cholangitis and other infections (registration number in Chinese Clinical Trial Registry ChiCTR-ECC-13003759). © 2015 Japan Pediatric Society.

  1. Comprehensive analysis of DNA polymerase III α subunits and their homologs in bacterial genomes

    PubMed Central

    Timinskas, Kęstutis; Balvočiūtė, Monika; Timinskas, Albertas; Venclovas, Česlovas

    2014-01-01

    The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set. PMID:24106089

  2. Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation.

    PubMed

    Guo, Peixuan; Zhao, Zhengyi; Haak, Jeannie; Wang, Shaoying; Wu, Dong; Meng, Bing; Weitao, Tao

    2014-01-01

    Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic

  3. Non-classical phase diagram for virus bacterial coevolution mediated by clustered regularly interspaced short palindromic repeats.

    PubMed

    Han, Pu; Deem, Michael W

    2017-02-01

    CRISPR is a newly discovered prokaryotic immune system. Bacteria and archaea with this system incorporate genetic material from invading viruses into their genomes, providing protection against future infection by similar viruses. The condition for coexistence of prokaryots and viruses is an interesting problem in evolutionary biology. In this work, we show an intriguing phase diagram of the virus extinction probability, which is more complex than that of the classical predator-prey model. As the CRISPR incorporates genetic material, viruses are under pressure to evolve to escape recognition by CRISPR. When bacteria have a small rate of deleting spacers, a new parameter region in which bacteria and viruses can coexist arises, and it leads to a more complex coexistence patten for bacteria and viruses. For example, when the virus mutation rate is low, the virus extinction probability changes non-montonically with the bacterial exposure rate. The virus and bacteria coevolution not only alters the virus extinction probability, but also changes the bacterial population structure. Additionally, we show that recombination is a successful strategy for viruses to escape from CRISPR recognition when viruses have multiple proto-spacers, providing support for a recombination-mediated escape mechanism suggested experimentally. Finally, we suggest that the re-entrant phase diagram, in which phages can progress through three phases of extinction and two phases of abundance at low spacer deletion rates as a function of exposure rate to bacteria, is an experimentally testable phenomenon. © 2017 The Author(s).

  4. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  5. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  6. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  7. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  8. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  9. Discovery of bacterial NAD+-dependent DNA ligase inhibitors: optimization of antibacterial activity.

    PubMed

    Stokes, Suzanne S; Huynh, Hoan; Gowravaram, Madhusudhan; Albert, Robert; Cavero-Tomas, Marta; Chen, Brendan; Harang, Jenna; Loch, James T; Lu, Min; Mullen, George B; Zhao, Shannon; Liu, Ce-Feng; Mills, Scott D

    2011-08-01

    Optimization of adenosine analog inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. Antibacterial activity against Streptococcus pneumoniae and Staphylococcus aureus was improved by modification of the 2-position substituent on the adenine ring and 3'- and 5'-substituents on the ribose. Compounds with logD values 1.5-2.5 maximized potency and maintained drug-like physical properties.

  10. Electronic microarray analysis of 16S rDNA amplicons for bacterial detection.

    PubMed

    Barlaan, Edward A; Sugimori, Miho; Furukawa, Seiji; Takeuchi, Kazuhisa

    2005-01-12

    Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.

  11. Use of quantitative 16S ribosomal DNA detection for diagnosis of central vascular catheter-associated bacterial infection.

    PubMed

    Warwick, S; Wilks, M; Hennessy, E; Powell-Tuck, J; Small, M; Sharp, J; Millar, M R

    2004-04-01

    Many central vascular catheters (CVCs) are removed unnecessarily because current diagnostic methods for CVC-associated infection are unreliable. A quantitative PCR assay using primers and probe targeted to bacterial 16S ribosomal DNA was used to measure the levels of bacterial DNA in blood samples drawn through the CVC in a population of patients receiving intravenous nutrition. Bacterial DNA concentrations were raised in 16 of 16 blood samples taken during episodes of probable bacterial CVC-associated infection. Bacterial DNA concentrations were raised in 4 of 29 episodes in which bacterial CVC-associated infection was unlikely. The use of this technique has the potential to substantially reduce the unnecessary removal of CVCs.

  12. A magneto-DNA nanoparticle system for target specific bacterial identification

    PubMed Central

    Chung, Hyun Jung; Castro, Cesar M.; Im, Hyungsoon; Lee, Hakho; Weissleder, Ralph

    2013-01-01

    To date, while various diagnostic approaches for pathogen detection have been proposed, most are too expensive, lengthy or limited in specificity for clinical use. Nanoparticle systems with unique material properties, however, circumvent these problems and offer improved accuracy over current methods. Herein, we present novel magneto-DNA probes capable of rapid and specific profiling of pathogens directly in clinical samples. A nanoparticle hybridisation assay, involving ubiquitous and specific probes that target bacterial 16S rRNAs, was designed to detect amplified target DNAs using a miniaturised nuclear magnetic resonance device. Ultimately, the magneto-DNA platform allowed both universal and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. Furthermore, the assay was robust and rapid, simultaneously diagnosing a panel of 13 bacterial species in clinical specimens within 2 hours. The generic platform described could be used to rapidly identify and phenotype pathogens for a variety of applications. PMID:23644570

  13. Artifically inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes

    USDA-ARS?s Scientific Manuscript database

    The long terminal repeat (LTR) sequence of reticuloendotheliosis virus (REV) was inserted into the very virulent Marek’s disease virus (MDV) Md5 bacterial artificial chromosome clone. The insertion site was nearly identical to the REV LTR that was naturally inserted into the JM/102W strain of MDV fo...

  14. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  15. [Bacterial 16S rDNA sequence analysis of Siberian tiger faecal flora].

    PubMed

    Tu, Ya; Zhu, Wei-yun; Lu, Cheng-ping

    2005-10-01

    Bacterial 16S rDNA library of Siberian tiger was developed and 15 different clones were obtained using EcoR I and Hind III in restriction fragment length polymorphism analysis. DNA sequencing and similarity analysis showed that 10 clones matched corresponding Clostridium sequences, of which 6 sequences had over 99% similarity with Clostridium novyi type A, and 4 sequences had 97% similarity with Swine manure bacterium RT-18B, which identified as Peptostreptococcus spp. The other five 16S rDNA sequences had 94% - 95% similarity with Clostridium pascui, Clostridium tetani E88, Clostridium sp. 14505 Clostridium perfringens and Carnobacterium sp. R-7279 respectively.

  16. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus

    SciTech Connect

    Liu Yingjie; Wu Jinlu; Chen Hu; Hew, Choy Leong; Yan Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300 kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  17. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus.

    PubMed

    Liu, Yingjie; Wu, Jinlu; Chen, Hu; Hew, Choy Leong; Yan, Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  18. Immunogenicity of Combination DNA Vaccines for Rift Valley Fever Virus, Tick-Borne Encephalitis Virus, Hantaan Virus, and Crimean Congo Hemorrhagic Fever Virus

    DTIC Science & Technology

    2005-08-22

    genus of the family Bunyaviridae and is one of four hantaviruses known to cause hemorrhagic fever with renal syndrome (HFRS). HFRS caused by HTNV...infection is found exclusively in Asia, with most cases occurring in China (reviewed in [2]). Hantaviruses are transmitted to humans by exposure to...before in our studies of antavirus DNA vaccines. We showed that although DNA accines for two hantaviruses , HTNV and Seoul virus, are ighly immunogenic

  19. Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.

    PubMed

    Gill, Christina; van de Wijgert, Janneke H H M; Blow, Frances; Darby, Alistair C

    2016-01-01

    Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies

  20. Novel Circular Single-Stranded DNA Viruses among an Asteroid, Echinoid and Holothurian (Phylum: Echinodermata).

    PubMed

    Jackson, Elliot W; Bistolas, Kalia S I; Button, Jason B; Hewson, Ian

    2016-01-01

    Echinoderms are prone to large population fluctuations that can be mediated by pervasive disease events. For the majority of echinoderm disease events the causative pathogen is unknown. Viruses have only recently been explored as potential pathogens using culture-independent techniques though little information currently exists on echinoderm viruses. In this study, ten circular ssDNA viruses were discovered in tissues among an asteroid (Asterias forbesi), an echinoid (Strongylocentrotus droebachiensis) and a holothurian (Parastichopus californicus) using viral metagenomics. Genome architecture and sequence similarity place these viruses among the rapidly expanding circular rep-encoding single stranded (CRESS) DNA viral group. Multiple genomes from the same tissue were no more similar in sequence identity to each other than when compared to other known CRESS DNA viruses. The results from this study are the first to describe a virus from a holothurian and continue to show the ubiquity of these viruses among aquatic invertebrates.

  1. Novel Circular Single-Stranded DNA Viruses among an Asteroid, Echinoid and Holothurian (Phylum: Echinodermata)

    PubMed Central

    Jackson, Elliot W.; Bistolas, Kalia S. I.; Button, Jason B.; Hewson, Ian

    2016-01-01

    Echinoderms are prone to large population fluctuations that can be mediated by pervasive disease events. For the majority of echinoderm disease events the causative pathogen is unknown. Viruses have only recently been explored as potential pathogens using culture-independent techniques though little information currently exists on echinoderm viruses. In this study, ten circular ssDNA viruses were discovered in tissues among an asteroid (Asterias forbesi), an echinoid (Strongylocentrotus droebachiensis) and a holothurian (Parastichopus californicus) using viral metagenomics. Genome architecture and sequence similarity place these viruses among the rapidly expanding circular rep-encoding single stranded (CRESS) DNA viral group. Multiple genomes from the same tissue were no more similar in sequence identity to each other than when compared to other known CRESS DNA viruses. The results from this study are the first to describe a virus from a holothurian and continue to show the ubiquity of these viruses among aquatic invertebrates. PMID:27855181

  2. Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size

    PubMed Central

    2014-01-01

    Background Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. Results Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left

  3. Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size.

    PubMed

    De-Donatis, Gian Marco; Zhao, Zhengyi; Wang, Shaoying; Huang, Lisa P; Schwartz, Chad; Tsodikov, Oleg V; Zhang, Hui; Haque, Farzin; Guo, Peixuan

    2014-01-01

    Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is

  4. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    PubMed

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  5. Discovery of a novel circular DNA virus in the Forbes sea star, Asterias forbesi.

    PubMed

    Fahsbender, Elizabeth; Hewson, Ian; Rosario, Karyna; Tuttle, Allison D; Varsani, Arvind; Breitbart, Mya

    2015-09-01

    A single-stranded DNA (ssDNA) virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in a Forbes sea star displaying symptoms of sea star wasting disease (SSWD). The AfaCV genome organization is typical of circular Rep-encoding ssDNA (CRESS-DNA) viruses and is similar to that of members of the family Circoviridae. PCR-based surveys indicate that AfaCV is not clearly associated with SSWD, whereas the sea star-associated densovirus (SSaDV), recently implicated in SSWD in the Pacific, was prevalent in symptomatic specimens. AfaCV represents the first CRESS-DNA virus detected in echinoderms, adding to the growing diversity of these viruses recently recovered from invertebrates.

  6. DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes.

    PubMed

    Rasche, Frank; Lueders, Tillmann; Schloter, Michael; Schaefer, Sabine; Buegger, Franz; Gattinger, Andreas; Hood-Nowotny, Rebecca C; Sessitsch, Angela

    2009-03-01

    A (13)CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of (13)CO2 into (13)C-labeled metabolites by the plant, the most directly active, cultivar specific heterotrophic endophytic bacteria that consume these labeled metabolite scan be identified by DNA stable isotope probing (DNA-SIP).Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries.Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the 'light' fractions of (13)CO2-incubated potato cultivars and of potato cultivars not incubated with (13)CO2. In the 'heavy' fractions of the (13)CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée,respectively, indicating cultivar-dependent distinctions in (13)C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities.The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant-microbe interactions and plant-dependent microbial metabolisms within the endosphere.

  7. Bacterial lysates improve the protective antibody response against respiratory viruses through Toll-like receptor 4

    PubMed Central

    Coviello, Silvina; Wimmenauer, Vera; Polack, Fernando P; Irusta, Pablo M

    2014-01-01

    Respiratory viruses cause significant morbidity and mortality in infants and young children worldwide. Current strategies to modulate the immune system and prevent or treat respiratory viral infections in this age group have shown limited success. Here, we demonstrate that a lysate derived from Gram-positive and Gram-negative organisms positively modulates protective antibody responses against both respiratory syncytial virus (RSV) and influenza virus in murine models of infection. Interestingly, despite the complex mixture of Toll-like receptor (TLR) agonists present in the bacterial lysate, the modulatory effects were mostly dependent on TLR4 signaling. Our results indicate that the use of simple formulations of TLR-agonists can significantly improve the immune response against critical pediatric respiratory pathogens. PMID:25483455

  8. DNA-crosslinker cisplatin eradicates bacterial persister cells.

    PubMed

    Chowdhury, Nityananda; Wood, Thammajun L; Martínez-Vázquez, Mariano; García-Contreras, Rodolfo; Wood, Thomas K

    2016-09-01

    For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Comparison of DNA preservation methods for environmental bacterial community samples

    USGS Publications Warehouse

    Gray, Michael A.; Pratte, Zoe A.; Kellogg, Christina A.

    2013-01-01

    Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard™, RNAlater®, DMSO–EDTA–salt (DESS), FTA® cards, and FTA Elute® cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA® cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard™, RNAlater®, and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

  10. Structural basis for bacterial transcription-coupled DNA repair.

    PubMed

    Deaconescu, Alexandra M; Chambers, Anna L; Smith, Abigail J; Nickels, Bryce E; Hochschild, Ann; Savery, Nigel J; Darst, Seth A

    2006-02-10

    Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

  11. Bacterial Argonaute samples the transcriptome to identify foreign DNA

    PubMed Central

    Olovnikov, Ivan; Chan, Ken; Sachidanandam, Ravi; Newman, Dianne K.; Aravin, Alexei A.

    2013-01-01

    summary Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15-19 nt RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid– derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids. PMID:24034694

  12. Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

    PubMed Central

    Szeto, Cheuk-Chun; Kwan, Bonnie Ching-Ha; Chow, Kai-Ming; Kwok, Jeffrey Sung-Shing; Lai, Ka-Bik; Cheng, Phyllis Mei-Shan; Pang, Wing-Fai; Ng, Jack Kit-Chung; Chan, Michael Ho-Ming; Lit, Lydia Choi-Wan; Leung, Chi-Bon; Li, Philip Kam-Tao

    2015-01-01

    Background Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients. Methods We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival. Results The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005). Conclusions Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients. PMID:26010741

  13. Stabilising the Herpes Simplex Virus capsid by DNA packaging

    NASA Astrophysics Data System (ADS)

    Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

    2009-03-01

    Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

  14. Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

    PubMed

    Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

    2006-09-18

    A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

  15. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    PubMed

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

  16. Multiple Layers of Chimerism in a Single-Stranded DNA Virus Discovered by Deep Sequencing

    PubMed Central

    Krupovic, Mart; Zhi, Ning; Li, Jungang; Hu, Gangqing; Koonin, Eugene V.; Wong, Susan; Shevchenko, Sofiya; Zhao, Keji; Young, Neal S.

    2015-01-01

    Viruses with single-stranded (ss) DNA genomes infect hosts in all three domains of life and include many medically, ecologically, and economically important pathogens. Recently, a new group of ssDNA viruses with chimeric genomes has been discovered through viral metagenomics. These chimeric viruses combine capsid protein genes and replicative protein genes that, respectively, appear to have been inherited from viruses with positive-strand RNA genomes, such as tombusviruses, and ssDNA genomes, such as circoviruses, nanoviruses or geminiviruses. Here, we describe the genome sequence of a new representative of this virus group and reveal an additional layer of chimerism among ssDNA viruses. We show that not only do these viruses encompass genes for capsid proteins and replicative proteins that have distinct evolutionary histories, but also the replicative genes themselves are chimeras of functional domains inherited from viruses of different families. Our results underscore the importance of horizontal gene transfer in the evolution of ssDNA viruses and the role of genetic recombination in the emergence of novel virus groups. PMID:25840414

  17. DNA phosphorothioation is widespread and quantized in bacterial genomes

    PubMed Central

    Wang, Lianrong; Chen, Shi; Vergin, Kevin L.; Giovannoni, Stephen J.; Chan, Simon W.; DeMott, Michael S.; Taghizadeh, Koli; Cordero, Otto X.; Cutler, Michael; Timberlake, Sonia; Alm, Eric J.; Polz, Martin F.; Pinhassi, Jarone; Deng, Zixin; Dedon, Peter C.

    2011-01-01

    Phosphorothioate (PT) modification of DNA, with sulfur replacing a nonbridging phosphate oxygen, was recently discovered as a product of the dnd genes found in bacteria and archaea. Given our limited understanding of the biological function of PT modifications, including sequence context, genomic frequencies, and relationships to the diversity of dnd gene clusters, we undertook a quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry approach. The results revealed a diversity of unique PT sequence contexts and three discrete genomic frequencies in a wide range of bacteria. Metagenomic analyses of PT modifications revealed unique ecological distributions, and a phylogenetic comparison of dnd genes and PT sequence contexts strongly supports the horizontal transfer of dnd genes. These results are consistent with the involvement of PT modifications in a type of restriction-modification system with wide distribution in prokaryotes. PMID:21285367

  18. A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses

    PubMed Central

    2012-01-01

    Background Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis. Results Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar to those found only in single-stranded RNA viruses. The presence and circular configuration of the complete virus genome was confirmed by inverse PCR amplification from native DNA extracted from lake sediment. The virus genome appears to be the result of a RNA-DNA recombination event between two ostensibly unrelated virus groups. Environmental sequence databases were examined for homologous genes arranged in similar configurations and three similar putative virus genomes from marine environments were identified. This result indicates the existence of a widespread but previously undetected group of viruses. Conclusions This unique viral genome carries implications for theories of virus emergence and evolution, as no mechanism for interviral RNA-DNA recombination has yet been identified, and only scant evidence exists that genetic exchange occurs between such distinct virus lineages. Reviewers This article was reviewed by EK, MK (nominated by PF) and AM. For the full reviews, please go to the Reviewers' comments section. PMID:22515485

  19. Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target

    PubMed Central

    Robinson, Andrew; Causer, Rebecca J; Dixon, Nicholas E

    2012-01-01

    New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials. DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets. PMID:22206257

  20. Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA

    PubMed Central

    Lau, Yu Heng; Stirling, Finn; Kuo, James; Karrenbelt, Michiel A. P.; Chan, Yujia A.; Riesselman, Adam; Horton, Connor A.; Schäfer, Elena; Lips, David; Weinstock, Matthew T.; Gibson, Daniel G.; Way, Jeffrey C.

    2017-01-01

    Abstract The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10–25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification. PMID:28499033

  1. Bacterial ghosts as a novel advanced targeting system for drug and DNA delivery.

    PubMed

    Paukner, Susanne; Stiedl, Thomas; Kudela, Pavol; Bizik, Jozef; Al Laham, Firas; Lubitz, Werner

    2006-01-01

    Although there are powerful drugs against infectious diseases and cancer on the market, delivery systems are needed to decrease serious toxic and noncurative side effects. In order to enhance compliance, several delivery systems such as polymeric micro- and nanoparticles, liposomal systems and erythrocyte ghosts have been developed. Bacterial ghosts representing novel advanced delivery and targeting vehicles suitable for the delivery of hydrophobic or water-soluble drugs, are the main focus of this review. They are useful nonliving carriers, as they can carry different active substances in more than one cellular location separately and simultaneously. Bacterial ghosts combine excellent natural or engineered adhesion properties with versatile carrier functions for drugs, proteins and DNA plasmids or DNA minicircles. The simplicity of both bacterial ghost production and packaging of drugs and/or DNA makes them particularly suitable for the use as a delivery system. Further advantages of bacterial ghost delivery vehicles include high bioavailability and a long shelf life without the need of cold-chain storage due to the possibility to freeze-dry the material.

  2. Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target.

    PubMed

    Robinson, Andrew; Causer, Rebecca J; Dixon, Nicholas E

    2012-03-01

    New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials. DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets.

  3. Improving soil bacterial taxa–area relationships assessment using DNA meta-barcoding

    PubMed Central

    Terrat, S; Dequiedt, S; Horrigue, W; Lelievre, M; Cruaud, C; Saby, N P A; Jolivet, C; Arrouays, D; Maron, P-A; Ranjard, L; Chemidlin Prévost-Bouré, N

    2015-01-01

    The evaluation of the taxa–area relationship (TAR) with molecular fingerprinting data demonstrated the spatial structuration of soil microorganisms and provided insights into the processes shaping their diversity. The increasing use of massive sequencing technologies in biodiversity investigations has now raised the question of the advantages of such technologies over the fingerprinting approach for elucidation of the determinism of soil microbial community assembly in broad-scale biogeographic studies. Our objectives in this study were to compare DNA fingerprinting and meta-barcoding approaches for evaluating soil bacterial TAR and the determinism of soil bacterial community assembly on a broad scale. This comparison was performed on 392 soil samples from four French geographic regions with different levels of environmental heterogeneity. Both molecular approaches demonstrated a TAR with a significant slope but, because of its more sensitive description of soil bacterial community richness, meta-barcoding provided significantly higher and more accurate estimates of turnover rates. Both approaches were useful in evidencing the processes shaping bacterial diversity variations on a broad scale. When different taxonomic resolutions were considered for meta-barcoding data, they significantly influenced the estimation of turnover rates but not the relative importance of each component process. Altogether, DNA meta-barcoding provides a more accurate evaluation of the TAR and may lead to re-examination of the processes shaping soil bacterial community assembly. This should provide new insights into soil microbial ecology in the context of sustainable use of soil resources. PMID:25293875

  4. Distinct circular single-stranded DNA viruses exist in different soil types.

    PubMed

    Reavy, Brian; Swanson, Maud M; Cock, Peter J A; Dawson, Lorna; Freitag, Thomas E; Singh, Brajesh K; Torrance, Lesley; Mushegian, Arcady R; Taliansky, Michael

    2015-06-15

    The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

  5. Incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients.

    PubMed

    Hishiki, Haruka; Ishiwada, Naruhiko; Fukasawa, Chie; Abe, Katsuaki; Hoshino, Tadashi; Aizawa, Jiro; Ishikawa, Nobuyasu; Kohno, Yoichi

    2011-02-01

    Bacterial coinfection occurs in pediatric bronchopulmonary infections caused by respiratory syncytial virus (RSV), but the incidence is uncertain. Our subjects are 188 pediatric inpatients having RSV bronchopulmonary infection in two hospitals in Chiba Prefecture between 2005 and 2007. On admission, antigen detection kits using nasopharyngeal aspirate were performed to detect RSV infection and washed sputum bacterial culture was performed to detect bacterial infection. Of the 188 pediatric inpatients with RSV bronchopulmonary infection, 95 (50.5%) patients were aged less than 1 year, 57 (30.3%) were aged 1-2 years, and 36 (19.1%) were aged 2 years or more. Thirty-six (19.1%) patients were associated with bronchial asthma attacks. Pathogenic bacteria were predominantly isolated from 43.6% of the patients. The three most frequently isolated bacteria were Haemophilus influenzae (43.9%), Streptococcus pneumoniae (36.6%), and Moraxella catarrhalis (29.3%). We found that 38.9% of H. influenzae strains were β-lactamase-nonproducing ampicillin-resistant strains. All S. pneumoniae strains were penicillin G (PcG) sensitive. However, 21.9% of S. pneumoniae strains showed PcG minimum inhibitory concentration values of 2 μg/ml. RSV bronchopulmonary infections in hospitalized children are often associated with antimicrobial-resistant bacterial infection in their lower airways. These results indicate that we should be aware of bacterial coinfections in the management of pediatric inpatients with RSV bronchopulmonary infection.

  6. Viral eukaryogenesis: was the ancestor of the nucleus a complex DNA virus?

    PubMed

    Bell, P J

    2001-09-01

    In the theory of viral eukaryogenesis I propose here, the eukaryotic nucleus evolved from a complex DNA virus. It is proposed that the virus established a persistent presence in the cytoplasm of a methanogenic mycoplasma and evolved into the eukaryotic nucleus by acquiring a set of essential genes from the host genome and eventually usurping its role. It is proposed that several characteristic features of the eukaryotic nucleus derive from its viral ancestry. These include mRNA capping, linear chromosomes, and separation of transcription from translation. In the model, phagocytosis and other membrane fusion-based processes are derived from viral membrane fusion processes and evolved in concert with the nucleus. The coevolution of phagocytosis and the nucleus rendered much of the host archaeal genome redundant since the protoeukaryote could obtain raw materials and energy by engulfing bacterial syntrophs/prey. This redundancy allowed loss of the archaeal chromosome, generating an organism with eukaryotic features. The evolution of phagocytosis allowed the eukaryotes to be the first organisms to occupy the niche of predator.

  7. DNA from Dust: Comparative Genomics of Large DNA Viruses in Field Surveillance Samples

    PubMed Central

    Pandey, Utsav; Bell, Andrew S.; Renner, Daniel W.; Kennedy, David A.; Shreve, Jacob T.; Cairns, Chris L.; Jones, Matthew J.; Dunn, Patricia A.; Read, Andrew F.

    2016-01-01

    ABSTRACT The intensification of the poultry industry over the last 60 years facilitated the evolution of increased virulence and vaccine breaks in Marek’s disease virus (MDV-1). Full-genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high-quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown. IMPORTANCE Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last half century, we do not yet understand the genetic basis of its pathogenicity. Our knowledge of genome-wide variation between strains of this virus comes exclusively from isolates that have been cultured in the laboratory. MDV-1 isolates tend to lose virulence during repeated cycles of replication in the laboratory, raising concerns about the ability of cultured isolates to accurately

  8. DNA from Dust: Comparative Genomics of Large DNA Viruses in Field Surveillance Samples.

    PubMed

    Pandey, Utsav; Bell, Andrew S; Renner, Daniel W; Kennedy, David A; Shreve, Jacob T; Cairns, Chris L; Jones, Matthew J; Dunn, Patricia A; Read, Andrew F; Szpara, Moriah L

    2016-01-01

    The intensification of the poultry industry over the last 60 years facilitated the evolution of increased virulence and vaccine breaks in Marek's disease virus (MDV-1). Full-genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high-quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown. IMPORTANCE Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last half century, we do not yet understand the genetic basis of its pathogenicity. Our knowledge of genome-wide variation between strains of this virus comes exclusively from isolates that have been cultured in the laboratory. MDV-1 isolates tend to lose virulence during repeated cycles of replication in the laboratory, raising concerns about the ability of cultured isolates to accurately reflect virus in

  9. The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

    NASA Astrophysics Data System (ADS)

    Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-07-01

    Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

  10. Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA

    SciTech Connect

    Dybvig, K.

    1981-01-01

    Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

  11. Conformational Diversity of Single-Stranded DNA from Bacterial Repetitive Extragenic Palindromes: Implications for the DNA Recognition Elements of Transposases

    PubMed Central

    Charnavets, Tatsiana; Nunvar, Jaroslav; Nečasová, Iva; Völker, Jens; Breslauer, Kenneth J; Schneider, Bohdan

    2015-01-01

    Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5′-G and 3′-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015. PMID:25951997

  12. Persistence of DNA sequences of BK virus and JC virus in normal human tissues and in diseased tissues.

    PubMed

    Chesters, P M; Heritage, J; McCance, D J

    1983-04-01

    Available evidence suggests that BK virus (BKV) and JC virus (JCV) persist in the kidneys of healthy individuals after primary infection and may reactivate when the host's immune response is impaired. Data supporting this hypothesis are presented. A previous study had shown BKV to be present in the kidneys of eight (57%) of 14 subjects. In the present study, which extended the investigation to a total of 30 subjects, BKV DNA was found in the renal tissues of 10 (33%) subjects, and JCV DNA was found in the renal tissues of three (10%) subjects. The viral DNA detected appeared not to be integrated with host DNA and to be isolated in foci. Investigation of normal and diseased brain tissue, including tissue from six subjects with multiple sclerosis, failed to reveal the presence of either JCV DNA or BKV DNA.

  13. Novel circular DNA viruses in stool samples of wild-living chimpanzees

    PubMed Central

    Blinkova, Olga; Victoria, Joseph; Li, Yingying; Keele, Brandon F.; Sanz, Crickette; Ndjango, Jean-Bosco N.; Peeters, Martine; Travis, Dominic; Lonsdorf, Elizabeth V.; Wilson, Michael L.; Pusey, Anne E.; Hahn, Beatrice H.; Delwart, Eric L.

    2010-01-01

    Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem–loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (∼1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution. PMID:19759238

  14. Novel circular DNA viruses in stool samples of wild-living chimpanzees.

    PubMed

    Blinkova, Olga; Victoria, Joseph; Li, Yingying; Keele, Brandon F; Sanz, Crickette; Ndjango, Jean-Bosco N; Peeters, Martine; Travis, Dominic; Lonsdorf, Elizabeth V; Wilson, Michael L; Pusey, Anne E; Hahn, Beatrice H; Delwart, Eric L

    2010-01-01

    Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem-loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (approximately 1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution.

  15. Previously unknown and highly divergent ssDNA viruses populate the oceans.

    PubMed

    Labonté, Jessica M; Suttle, Curtis A

    2013-11-01

    Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.

  16. Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

    PubMed

    Sepúlveda, Dagoberto; Lorenzen, Niels

    2016-01-01

    DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.

  17. Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

    PubMed Central

    Sepúlveda, Dagoberto; Lorenzen, Niels

    2016-01-01

    DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms. PMID:27054895

  18. Screening for hepatitis B virus DNA in serum of organ donors and renal transplant recipients.

    PubMed

    Miédougé, M; Rostaing, L; Mansuy, J M; Sandres-Sauné, K; Boudet, F; Izopet, J

    2003-04-01

    In order to determine the impact of screening potential organ donors for hepatitis B virus DNA using a standardized test, the serum of 145 donor candidates was tested. All of the candidates were negative for hepatitis B virus DNA, but the status of one donor was doubtful for hepatitis B virus surface antigen and seven donors tested positive for hepatitis B virus core antibody without hepatitis B virus surface antigen. Nine transplant recipients tested positive for hepatitis B virus surface antibody; they were given kidneys from the donor with a doubtful hepatitis B virus surface antigen result and from four of the seven donors who tested positive for hepatitis B core antibody. Follow-up revealed no case of hepatitis B transmission. In this study, screening for hepatitis B virus DNA was useful and did not lead to donor organ shortage. Patients with hepatitis B virus surface antibodies can safely be given kidneys from donors who are positive for hepatitis B core antibody but negative for hepatitis B virus DNA.

  19. Association of a novel DNA virus with the grapevine vein-clearing and vine decline syndrome.

    PubMed

    Zhang, Yu; Singh, Kashmir; Kaur, Ravneet; Qiu, Wenping

    2011-09-01

    A severe vein-clearing and vine decline syndrome has emerged on grapevines (Vitis vinifera) and hybrid grape cultivars in the Midwest region of the United States. The typical symptoms are translucent vein-clearing on young leaves, short internodes and decline of vine vigor. Known viral pathogens of grapevines were not closely associated with the syndrome. To obtain a comprehensive profile of viruses in a diseased grapevine, small RNAs were enriched and two cDNA libraries were constructed from a symptomatic grapevine and a symptomless grapevine, respectively. Deep sequencing of the two cDNA libraries showed that the most abundant viral small RNAs align with the genomes of viruses in the genus Badnavirus, the family Caulimoviridae. Amplification of the viral DNA by polymerase chain reaction allowed the assembly of the whole genome sequence of a grapevine DNA virus, which shared the highest homology with the Badnavirus sequences. This is the first report of a DNA virus in grapevines. The new DNA virus is closely associated with the vein-clearing symptom, and thus has been given a provisional name Grapevine vein clearing virus (GVCV). GVCV was detected in six grapevine cultivars showing vein-clearing and vine decline syndrome in Missouri, Illinois, and Indiana, suggesting its wide distribution in the Midwest region of the United States. Discovery of DNA viruses in grapevines merits further studies on their epidemics and economic impact on grape production worldwide.

  20. Assessing genetic structure and diversity of airborne bacterial communities by DNA fingerprinting and 16S rDNA clone library

    NASA Astrophysics Data System (ADS)

    Maron, Pierre-Alain; Lejon, David P. H.; Carvalho, Esmeralda; Bizet, Karine; Lemanceau, Philippe; Ranjard, Lionel; Mougel, Christophe

    The density, genetic structure and diversity of airborne bacterial communities were assessed in the outdoor atmosphere. Two air samples were collected on the same location (north of France) at two dates (March 2003 (sample1) and May 2003 (sample 2)). Molecular culture -independent methods were used to characterise airborne bacterial communities regardless of the cell culturability. The automated-ribosomal intergenic spacer analysis (A-RISA) was performed to characterise the community structure in each sample. For both sampling dates, complex A-RISA patterns were observed suggesting a highly diverse community structure, comparable to those found in soil, water or sediment environments. Furthermore, differences in the genetic structure of airborne bacterial communities were observed between samples 1 and 2 suggesting an important variability in time. A clone library of 16S rDNA directly amplified from air DNA of sample 1 was constructed and sequenced to analyse the community composition and diversity. The Proteobacteria group had the greatest representation (60%), with bacteria belonging to the different subdivisions α- (19%), β-(21%), γ-(12%) and δ-(8%). Firmicute and Actinobacteria were also well represented with 14% and 12%, respectively. Most of the identified bacteria are known to be commonly associated with soil or plant environments suggesting that the atmosphere is mainly colonised transiently by microorganisms from local sources, depending on air fluxes.

  1. Bacterial microbiota in small brown planthopper populations with different rice viruses.

    PubMed

    Li, Shuo; Zhou, Changwei; Chen, Guangyi; Zhou, Yijun

    2017-07-01

    The small brown planthopper (SBPH) is an important virus vector, transmitting Rice stripe virus (RSV), and Rice black-streaked dwarf virus (RBSDV). Insect symbionts play an essential role in the insect fitness, however, it is still unclear about their contributions to viral transmission by SBPH. Here, we investigated endosymbiont communities in non-viruliferous, RSV-infected, and RBSDV-infected SBPH populations using Illumina 16S rRNA gene MiSeq sequencing. In total, 281,803 effective sequences of the 16S rRNA gene were generated from different samples. Sequence analysis revealed the percentages of these bacterial groups in different SBPH populations on several taxonomic levels ranging from phyla to genera. The extremely consistent bacterial diversity and abundance indicated that RSV or RBSDV infection did not affect the composition and abundance of symbionts in SBPH. It was notable that Wolbachia was dominant in all populations. The symbiosis between Wolbachia and SBPH might be potentially studied and utilized to control pest SBPH in the future. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection.

    PubMed

    Schliehe, Christopher; Flynn, Elizabeth K; Vilagos, Bojan; Richson, Udochuku; Swaminanthan, Savitha; Bosnjak, Berislav; Bauer, Lisa; Kandasamy, Richard K; Griesshammer, Isabel M; Kosack, Lindsay; Schmitz, Frank; Litvak, Vladimir; Sissons, James; Lercher, Alexander; Bhattacharya, Anannya; Khamina, Kseniya; Trivett, Anna L; Tessarollo, Lino; Mesteri, Ildiko; Hladik, Anastasiya; Merkler, Doron; Kubicek, Stefan; Knapp, Sylvia; Epstein, Michelle M; Symer, David E; Aderem, Alan; Bergthaler, Andreas

    2015-01-01

    Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.

  3. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    PubMed Central

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam

    2016-01-01

    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  4. Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

    PubMed Central

    Bouhamdan, M; Benichou, S; Rey, F; Navarro, J M; Agostini, I; Spire, B; Camonis, J; Slupphaug, G; Vigne, R; Benarous, R; Sire, J

    1996-01-01

    The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr. PMID:8551605

  5. Structure of Bacterial LigD -phosphoesterase Unveils a DNA Repair Superfamily

    SciTech Connect

    Nair, P.; Smith, P; Shuman, S

    2010-01-01

    The DNA ligase D (LigD) 3{prime}-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3{prime} end-healing reactions at DNA double-strand breaks. Here we report the 1.9 {angstrom} crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded {beta} barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis a vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.

  6. Mechanism of foreign DNA selection in a bacterial adaptive immune system.

    PubMed

    Sashital, Dipali G; Wiedenheft, Blake; Doudna, Jennifer A

    2012-06-08

    In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary "nonself" DNA sequences for destruction, while avoiding binding to "self" sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for nonself target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved three base pair motif that is required for nonself target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade.

  7. Cascaded multiple amplification strategy for ultrasensitive detection of HIV/HCV virus DNA.

    PubMed

    Wang, Kun; Fan, Daoqing; Liu, Yaqing; Dong, Shaojun

    2017-01-15

    Ultrasensitive detection of HIV and HCV virus DNA is of great importance for early accurate diagnostics and therapy of HIV virus-infected patients. Herein, to our best knowledge, it is the first to use DNA cascaded multiple amplification strategy for ultrasensitive detection of HIV virus DNA with G-quadruplex-specific fluorescent or colorimetric probes as signal carriers. The developed strategy also exhibited universal applicability for HCV virus DNA detection. After reaction for about 4h, high sensitivity and specificity can be achieved at both fluorescent and colorimetric strategies (limit of detection (LOD) of 10 fM and 0.5pM were reached for fluorescent and colorimetric detection, respectively). And the single-based mismatched DNA even can be distinguished by naked eyes. It is believed that the cascaded multiple amplification strategy presents a huge advance in sensing platform and potential application in future clinical diagnosis.

  8. Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA.

    PubMed Central

    Brandsma, J L; Xiao, W

    1993-01-01

    The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication. Images PMID:8380092

  9. Simplified methods for the construction of RNA and DNA virus infectious clones.

    PubMed

    Nagata, Tatsuya; Inoue-Nagata, Alice Kazuko

    2015-01-01

    Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.

  10. Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial Isolates with Ambiguous Biochemical Profiles

    PubMed Central

    Woo, Patrick C. Y.; Ng, Kenneth H. L.; Lau, Susanna K. P.; Yip, Kam-tong; Fung, Ami M. Y.; Leung, Kit-wah; Tam, Dorothy M. W.; Que, Tak-lun; Yuen, Kwok-yung

    2003-01-01

    Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the “gold standard,” we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S r

  11. Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

    PubMed Central

    Li, Lina; Dimitriadis, Emilios K.; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H.; Garcia, Luis; Kotin, Robert M.

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×109 Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA. PMID:23936358

  12. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    PubMed

    Li, Lina; Dimitriadis, Emilios K; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H; Garcia, Luis; Kotin, Robert M

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  13. Does virus-induced lysis contribute significantly to bacterial mortality in the oxygenated sediment layer of shallow oxbow lakes?

    PubMed

    Fischer, Ulrike R; Wieltschnig, Claudia; Kirschner, Alexander K T; Velimirov, Branko

    2003-09-01

    Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 x 10(9) to 7.2 x 10(9) particles ml of wet sediment(-1) and from undetectable to 22.2 x 10(7) particles ml(-1) h(-1), respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.

  14. Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome

    PubMed Central

    Kailasan Vanaja, Sivapriya; Rathinam, Vijay A. K.; Atianand, Maninjay K.; Kalantari, Parisa; Skehan, Brian; Fitzgerald, Katherine A.; Leong, John M.

    2014-01-01

    Enterohemorrhagic Escherichia coli (EHEC) is an extracellular pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The proinflammatory cytokine, interleukin-1β, has been linked to hemolytic uremic syndrome. Here we identify the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome as an essential mediator of EHEC-induced IL-1β. Whereas EHEC-specific virulence factors were dispensable for NLRP3 activation, bacterial nucleic acids such as RNA:DNA hybrids and RNA gained cytosolic access and mediated inflammasome-dependent responses. Consistent with a direct role for RNA:DNA hybrids in inflammasome activation, delivery of synthetic EHEC RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, and introduction of RNase H, which degrades such hybrids, into infected cells specifically inhibited inflammasome activation. Notably, an E. coli rnhA mutant, which is incapable of producing RNase H and thus harbors increased levels of RNA:DNA hybrid, induced elevated levels of NLRP3-dependent caspase-1 activation and IL-1β maturation. Collectively, these findings identify RNA:DNA hybrids of bacterial origin as a unique microbial trigger of the NLRP3 inflammasome. PMID:24828532

  15. Base-modified NAD and AMP derivatives and their activity against bacterial DNA ligases.

    PubMed

    Pergolizzi, Giulia; Cominetti, Marco M D; Butt, Julea N; Field, Robert A; Bowater, Richard P; Wagner, Gerd K

    2015-06-14

    We report the chemical synthesis and conformational analysis of a collection of 2-, 6- and 8-substituted derivatives of β-NAD(+) and AMP, and their biochemical evaluation against NAD(+)-dependent DNA ligases from Escherichia coli and Mycobacterium tuberculosis. Bacterial DNA ligases are validated anti-microbial targets, and new strategies for their inhibition are therefore of considerable scientific and practical interest. Our study includes several pairs of β-NAD(+) and AMP derivatives with the same substitution pattern at the adenine base. This has enabled the first direct comparison of co-substrate and inhibitor behaviour against bacterial DNA ligases. Our results suggest that an additional substituent in position 6 or 8 of the adenine base in β-NAD(+) is detrimental for activity as either co-substrate or inhibitor. In contrast, substituents in position 2 are not only tolerated, but appear to give rise to a new mode of inhibition, which targets the conformational changes these DNA ligases undergo during catalysis. Using a molecular modelling approach, we highlight that these findings have important implications for our understanding of ligase mechanism and inhibition, and may provide a promising starting point for the rational design of a new class of inhibitors against NAD(+)-dependent DNA ligases.

  16. Analysis of bacterial DNA patterns--an approach for controlling biotechnological processes.

    PubMed

    Müller, S; Babel, W

    2003-12-01

    Optimisation of biotechnological processes catalysed by microbial cells requires detailed information about operational limits of the single cells. Their performance is correlated with distinct physiological states. We related these states to cell cycle events, which were found to proceed extremely diversely in different bacterial strains. Characteristic DNA patterns were found flow cytometrically, depending on the type of strain, substrates and growth conditions involved; this information can be used for the development of control strategies of bioprocesses, although some skill is required. Four bacterial strains (the Gram-negative strains Acinetobacter calcoaceticus 69-V, Ralstonia eutropha JMP 134, Ochrobactrum anthropi K2-14 and the Gram-positive strain Rhodococcus erythropolis K2-3) were grown in mono- and mixed cultures on different substrates, and analysed regarding their proliferation behaviour. The resulting DNA distribution patterns provided three types of valuable information. First, correlation of proliferation activity with the appearance of a major part of cells within the C(2) stage of the cell cycle is a strain-specific feature. Second, bacteria usually maintain more than one chromosome under limiting growth conditions: DNA replication is completed in such cases, but cell division fails. Third, high growth rates are associated with uncoupled DNA synthesis. Its general initiation might be genetically determined in the first place, but it is promoted by optimal growth conditions and the presence of substrates that can be metabolised at high rates, thereby allowing substantial amounts of carbon, other nutrients and energy to be used exclusively for DNA synthesis.

  17. (Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems): Final report

    SciTech Connect

    Friedberg, E.C.

    1987-08-01

    This study sought to exploit the use of uv radiation as a source of genomic damage. We explored the molecular mechanism of the repair of DNA damage at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian cells. Not only have observations obtained in one biological system suggested specific experimental approaches in others, but we have also learned that some biochemical pathways for DNA repair are unique to specific organisms. Our studies are summarized in terms of 4 major areas of research activity that span the past 16 years. 86 refs.

  18. The action of the bacterial toxin, microcin B17, on DNA gyrase.

    PubMed

    Parks, William M; Bottrill, Andrew R; Pierrat, Olivier A; Durrant, Marcus C; Maxwell, Anthony

    2007-04-01

    Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.

  19. NAD homeostasis in the bacterial response to DNA/RNA damage.

    PubMed

    Sorci, Leonardo; Ruggieri, Silverio; Raffaelli, Nadia

    2014-11-01

    In mammals, NAD represents a nodal point for metabolic regulation, and its availability is critical to genome stability. Several NAD-consuming enzymes are induced in various stress conditions and the consequent NAD decline is generally accompanied by the activation of NAD biosynthetic pathways to guarantee NAD homeostasis. In the bacterial world a similar scenario has only recently begun to surface. Here we review the current knowledge on the involvement of NAD homeostasis in bacterial stress response mechanisms. In particular, we focus on the participation of both NAD-consuming enzymes (DNA ligase, mono(ADP-ribosyl) transferase, sirtuins, and RNA 2'-phosphotransferase) and NAD biosynthetic enzymes (both de novo, and recycling enzymes) in the response to DNA/RNA damage. As further supporting evidence for such a link, a genomic context analysis is presented showing several conserved associations between NAD homeostasis and stress responsive genes.

  20. Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

    PubMed

    Tillett, D; Burns, B P; Neilan, B A

    2000-03-01

    A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.

  1. Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing.

    PubMed

    Karas, Bogumil J; Jablanovic, Jelena; Irvine, Edward; Sun, Lijie; Ma, Li; Weyman, Philip D; Gibson, Daniel G; Glass, John I; Venter, J Craig; Hutchison, Clyde A; Smith, Hamilton O; Suzuki, Yo

    2014-04-01

    Direct cell-to-cell transfer of genomes from bacteria to yeast facilitates genome engineering for bacteria that are not amenable to genetic manipulation by allowing instead for the utilization of the powerful yeast genetic tools. Here we describe a protocol for transferring whole genomes from bacterial cells to yeast spheroplasts without any DNA purification process. The method is dependent on the treatment of the bacterial and yeast cellular mixture with PEG, which induces cell fusion, engulfment, aggregation or lysis. Over 80% of the bacterial genomes transferred in this way are complete, on the basis of structural and functional tests. Excluding the time required for preparing starting cultures and for incubating cells to form final colonies, the protocol can be completed in 3 h.

  2. Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA.

    PubMed

    Roth, M G; Compans, R W; Giusti, L; Davis, A R; Nayak, D P; Gething, M J; Sambrook, J

    1983-06-01

    Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.

  3. Higher Prevalence of Epstein–Barr Virus DNA in Deeper Periodontal Pockets of Chronic Periodontitis in Japanese Patients

    PubMed Central

    Kato, Ayako; Imai, Kenichi; Ochiai, Kuniyasu; Ogata, Yorimasa

    2013-01-01

    Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans. Although traditional microbiological research on periodontitis has focused on putative bacteria such as Porphyromonas gingivalis, the herpes virus is proposed to be involved in the pathogenesis of periodontitis because bacterial etiology alone does not adequately explain various clinical aspects. In this study, we established for the first time, more Epstein–Barr virus (EBV) DNA is found deeper in periodontal pockets of chronic periodontitis in Japanese patients. Subgingival samples were collected from 85 patients with chronic periodontitis having two periodontal sites with probing depths (PD) of ≤3 mm (shallow) or ≥5 mm (deep) and were subjected to a nested polymerase chain reaction. EBV DNA was more frequently detected in patients with deeper PD sites (66%) than in those with shallow PD sites (48%) or healthy controls (45%). Coexistence of EBV DNA and P. gingivalis was significantly higher in patients with deeper PD sites (40%) than in those with shallow PD sites (14%) or healthy controls (13%). Although no difference in clinical index for periodontitis, the odds ratio of EBV DNA in patients with deeper PD sites was 2.36, which was 2.07-fold higher than that in those with shallow PD sites. Interestingly, the odds of acquiring chronic periodontitis (PD ≥5 mm) were higher in the presence of both EBV DNA and P. gingivalis compared with either EBV DNA or P. gingivalis only. In addition, we also observed that EBV-encoded small RNA (EBER) in positive cells of human gingival tissues. These results would suggest that EBV DNA may serve as a pathogenic factor leading to chronic periodontitis among Japanese patients. PMID:23991022

  4. A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

    PubMed Central

    León, Esther; Navarro-Avilés, Gloria; Santiveri, Clara M.; Flores-Flores, Cesar; Rico, Manuel; González, Carlos; Murillo, Francisco J.; Elías-Arnanz, Montserrat; Jiménez, María Angeles; Padmanabhan, S.

    2010-01-01

    Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel β-sheet fold resembling SH3 domains, protein–protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein–protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription. PMID:20410074

  5. Physical mapping and molecular cloning of mung bean yellow mosaic virus DNA.

    PubMed

    Morinaga, T; Ikegami, M; Miura, K

    1990-01-01

    Viral single-stranded DNA of mung bean yellow mosaic virus (MYMV) was converted to the double-stranded state in vitro, and physical mapping was carried out. The genome of MYMV was found to consist of two major components (designated as DNA 1 and DNA 2). In addition, some minor components were detected. Molecular cloning of the major components was carried out, using in vitro double-stranded DNA and replicative intermediate DNAs. DNA 1 is about 2.72 and DNA 2 about 2.67 kilobase pairs. No similarities were observed when the two restriction maps of DNA 1 and 2 were compared.

  6. The role of enzymology in a structure-based drug discovery program: bacterial DNA gyrase.

    PubMed

    Cunningham, Mark L

    2012-01-01

    The capability to accurately, rapidly, and reproducibly determine the affinity of a ligand for a target protein or enzyme is a vital component for a successful structure-based drug design effort. In order to successfully drive a structure-based drug design (SBDD) project forward, multiple distinct assays, each with particular strengths and weaknesses, need to be employed. Using bacterial DNA gyrase as an example, a range of assays are described that will fully support an SBDD program.

  7. Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series.

    PubMed

    Stokes, Suzanne S; Gowravaram, Madhusudhan; Huynh, Hoan; Lu, Min; Mullen, George B; Chen, Brendan; Albert, Robert; O'Shea, Thomas J; Rooney, Michael T; Hu, Haiqing; Newman, Joseph V; Mills, Scott D

    2012-01-01

    Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.

  8. Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

    PubMed Central

    Boregowda, Rajeev; Sohn, Ji A.; Ledesma, Felipe Cortes; Caldecott, Keith W.; Seeger, Christoph; Hu, Jianming

    2015-01-01

    Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5’ end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5’ DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5’ DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5’ end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo. PMID:26079492

  9. Shared Roles of Halobacteriovorax and Viruses in Bacterial Mortality: The Environment Dictates the Winner

    NASA Astrophysics Data System (ADS)

    Chen, H.; Laws, E. A.; Gulig, P. A.; Berhane, T. K.; Martin, J. L.; Williams, H.

    2016-02-01

    Bacteriophages (phages) are considered to be a major contributor to bacterial mortality. Although recent evidence shows a similar role for the predatory bacterium, Halobacteriovorax (HBx), this organism has been largely ignored. We designed controlled laboratory microcosm studies to examine and compare the predation of a virus and an HBx strain on Vibrio vulnificus (Vv), under a range of environmental conditions. Predator-prey models were used to simulate the results and interpolated using Matlab software. The results show that although the HBx and virus both preyed on Vv, the magnitudes of their respective responses were different and were largely driven by environmental conditions. In low nutrient seawater, HBx was highly active in preying on Vv, resulting in a 4.4 log reduction of prey within 40 hours, whereas phage contributed little to bacterial mortality. However, when nutrients were added to the seawater, phage was the more active predator. At moderate levels of nutrient concentrations (DNB 1:10 and DNB 1:100) both predators were active. Both virus and HBx grew well at salt concentrations ranging from 9 to 30 ppt. Phage reproduction was optimized at 30 ppt and also occurred at higher levels at 40 and 45 ppt. HBx, on the other hand, grew best at 9 ppt and did not grow at 40 and 45 ppt. At temperatures between 15 and 37˚C both predators grew well. The impact of predation on Vv was positively correlated with temperature. The collective results of this study suggest that both HBx and phages can play significant roles in bacterial mortality and hence in shaping microbial communities and cycling nutrients. However, whether HBx or phages play the larger role in any circumstance may be orchestrated by environmental conditions. These results warrant reconsideration of the roles of different biological agents and the environment in bacteria mortality.

  10. High-sensitivity detection of fruit tree viruses using bacterial magnetic particles.

    PubMed

    Chen, Ji-Feng; Li, Ying; Wang, Zhen-Fang; Li, Ji-Lun; Jiang, Wei; Li, Shao-Hua

    2009-04-01

    Prunus necrotic ring spot virus (PNRSV) and grapevine fanleaf virus (GFLV) were detected by fluoroimmunoassay using bacterial magnetic particles (BMPs), and a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). For the fluoroimmunoassay, fluorescein isothiocyanate labeled anti-PNRSV antibody or anti-GFLV antibody was conjugated onto BMPs of Magnetospirillum gryphiswaldense MSR-1. With this method, a very low minimum antigen concentration (1 x 10(6) dilution of the original sample concentration) could be detected. Using DAS-ELISA, the minimum antigen detection concentration was the original sample concentration. Thus, comparing these two methods, a BMP-based method could increase the sensitivity up to six orders of magnitude (10(6)) higher than an ELISA-based method of detection PNRSV and GFLV.

  11. Autoclave method for rapid preparation of bacterial PCR-template DNA.

    PubMed

    Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S

    2004-02-01

    An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

  12. Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

    PubMed

    Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

    2014-05-01

    Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

  13. Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis.

    PubMed

    Deutch, Susanna; Pedersen, Lisbeth N; Pødenphant, Lone; Olesen, Rikke; Schmidt, Michael B; Møller, Jens K; Ostergaard, Lars

    2006-01-01

    Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.

  14. Detection of periodontal bacterial DNA in serum and synovial fluid in refractory rheumatoid arthritis patients.

    PubMed

    Martinez-Martinez, Rita E; Abud-Mendoza, Carlos; Patiño-Marin, Nuria; Rizo-Rodríguez, Juan C; Little, James W; Loyola-Rodríguez, Juan Pablo

    2009-12-01

    To identify periodontal bacterial DNA (PBDNA) by PCR in subgingival dental plaque (SDP), serum and synovial fluid (SF) of rheumatoid arthritis (RA) with periodontal disease (PD) patients and to explore the possible PBDNA transport pathways from mouth to joints. This cross-sectional prolective study involved 19 subjects with RA and PD. Informed consent, health and dental questionnaires were obtained. SDP, SF and serum samples were obtained, and leucocytes were isolated from blood. DNA was extracted and PCR assays to detect main PD species were carried out. Cultures on agar plates and broth, from each sample, were performed. Hundred percentage of patients showed PBDNA in SDP and SF and 83.5% in serum. Prevotella intermedia (89.4% and 73.6%) and Porphyromonas gingivalis (57.8% and 42.1%) were the species most frequently detected in SDP and SF, respectively. In SDP, 4.05 different bacterial species were found followed by 1.19 in serum and 2.26 in SF. Culture onto agar plates and broth did not show any bacterial growth, leucocytes were not positive to PBDNA by PCR. This study suggests that PBDNA could have a role on the RA aetiology. The possible pathway of transport of PBDNA from mouth to joints could be via the free form of DNA.

  15. Exposure to West Nile Virus Increases Bacterial Diversity and Immune Gene Expression in Culex pipiens

    PubMed Central

    Zink, Steven D.; Van Slyke, Greta A.; Palumbo, Michael J.; Kramer, Laura D.; Ciota, Alexander T.

    2015-01-01

    Complex interactions between microbial residents of mosquitoes and arboviruses are likely to influence many aspects of vectorial capacity and could potentially have profound effects on patterns of arbovirus transmission. Such interactions have not been well studied for West Nile virus (WNV; Flaviviridae, Flavivirus) and Culex spp. mosquitoes. We utilized next-generation sequencing of 16S ribosomal RNA bacterial genes derived from Culex pipiens Linnaeus following WNV exposure and/or infection and compared bacterial populations and broad immune responses to unexposed mosquitoes. Our results demonstrate that WNV infection increases the diversity of bacterial populations and is associated with up-regulation of classical invertebrate immune pathways including RNA interference (RNAi), Toll, and Jak-STAT (Janus kinase-Signal Transducer and Activator of Transcription). In addition, WNV exposure alone, without the establishment of infection, results in similar alterations to microbial and immune signatures, although to a lesser extent. Multiple bacterial genera were found in greater abundance in WNV-exposed and/or infected mosquitoes, yet the most consistent and notable was the genus Serratia. PMID:26516902

  16. Update on New Antivirals under Development for the Treatment of Double-stranded DNA Virus Infections

    PubMed Central

    Dropulic, Lesia K.; Cohen, Jeffrey I.

    2012-01-01

    All of the currently available antiviral agents used to treat double-stranded (ds) DNA viruses inhibit the same target, the viral DNA polymerase, with the exception of interferon-α. With increasing reports of the development of resistance of herpes simplex virus, cytomegalovirus, and hepatitis B virus to some of these drugs, new antiviral agents are needed for these infections. Additionally, no drugs are approved to treat several DNA virus infections including those caused by adenovirus, smallpox, molluscum contagiosum, and BK virus. We report on the status of 10 new antiviral drugs for the treatment of dsDNA viruses. CMX-001 has broad activity against dsDNA viruses; 3 helicase-primase inhibitors, marabavir, and FV-100 have activity against certain herpesviruses; ST-246 inhibits poxviruses; GS-9191 inhibits papillomaviruses; and clevudine and emtricitabine are active against hepatitis B virus. Most of these drugs have completed at least phase I trials in humans and several are in additional clinical trials. PMID:20881959

  17. Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection.

    PubMed

    Luo, Jun; Cui, Xiuji; Gao, Lu; Hu, Jianming

    2017-06-21

    The hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC to CCC DNA conversion, two 3' exonucleases Exo I and Exo III, in combination were used to degrade all DNA strands with a free 3' end, which would nevertheless preserve closed circular DNA, either single-stranded (SS) or double-stranded (DS). Indeed, a RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA or cM-RC DNA) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5' end of the plus strand in RC DNA, suggesting that minus strand closing can occur before plus strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and factors involved.IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the molecular basis of viral persistence, by serving as the viral transcriptional template. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC to CCC DNA, thus providing

  18. Incident Herpes Simplex Virus Type 2 Infection Increases the Risk of Subsequent Episodes of Bacterial Vaginosis

    PubMed Central

    Masese, Linnet; Baeten, Jared M.; Richardson, Barbra A.; Bukusi, Elizabeth; John-Stewart, Grace; Jaoko, Walter; Shafi, Juma; Kiarie, James; McClelland, R. Scott

    2014-01-01

    Herpes simplex virus type 2 (HSV-2) infected women have a higher prevalence of bacterial vaginosis (BV) compared to HSV-2-seronegative women. To explore the temporal association between these conditions, we evaluated the frequency of BV episodes before and after HSV-2 acquisition in a prospective study of 406 HSV-2/HIV-1-seronegative Kenyan women, of whom 164 acquired HSV-2. Incident HSV-2 was associated with increased likelihood of BV (adjusted OR, 1.28; 95% CI, 1.05–1.56; P = .01). Our findings strengthen the evidence for a causal link between genital HSV-2 infection and disruption of the vaginal microbiota. PMID:24273042

  19. [Mechanisms of Chlamydia trachomatis and herpes simplex virus persistence during viral-bacterial infection].

    PubMed

    Bekhalo, V A; Sysoliatina, E V; Nagurskaia, E V

    2009-01-01

    Possible mechanisms of persistence on the example of Chlamydia trachomatis in conditions of herpes simplex virus type 2 (HSV-2) superinfection in vitro and in vivo are described. Emergence of persisting forms of Chlamydia as well as factors influencing on this process are considered. Contemporary views on pathogenesis of viral-bacterial infection with HSV-2 and C. trachomatis as well as interactions of the agents with local immunity factors are described. It was suggested that there are signaling pathways through which HSV-2 changes life cycle of Chlamydia.

  20. Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

    PubMed Central

    Robinson, H L; Zinkus, D M

    1990-01-01

    Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the absence of the antibody was present in a broad smear of apparently incomplete viral sequences. In cultures grown in the presence or absence of the antibody, the 9.6-kilobase linear duplex of viral DNA appeared to undergo integration within 24 h of its appearance. These results demonstrate that T cells accumulate unintegrated human immunodeficiency virus type 1 DNA as a result of multiple virions entering cells. Images PMID:2398529

  1. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    NASA Astrophysics Data System (ADS)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  2. Abundant DNase I-sensitive bacterial DNA in healthy porcine lungs and its implications for the lung microbiome.

    PubMed

    Pezzulo, Alejandro A; Kelly, Patrick H; Nassar, Boulos S; Rutland, Cedric J; Gansemer, Nicholas D; Dohrn, Cassie L; Costello, Andrew J; Stoltz, David A; Zabner, Joseph

    2013-10-01

    Human lungs are constantly exposed to bacteria in the environment, yet the prevailing dogma is that healthy lungs are sterile. DNA sequencing-based studies of pulmonary bacterial diversity challenge this notion. However, DNA-based microbial analysis currently fails to distinguish between DNA from live bacteria and that from bacteria that have been killed by lung immune mechanisms, potentially causing overestimation of bacterial abundance and diversity. We investigated whether bacterial DNA recovered from lungs represents live or dead bacteria in bronchoalveolar lavage (BAL) fluid and lung samples in young healthy pigs. Live bacterial DNA was DNase I resistant and became DNase I sensitive upon human antimicrobial-mediated killing in vitro. We determined live and total bacterial DNA loads in porcine BAL fluid and lung tissue by comparing DNase I-treated versus untreated samples. In contrast to the case for BAL fluid, we were unable to culture bacteria from most lung homogenates. Surprisingly, total bacterial DNA was abundant in both BAL fluid and lung homogenates. In BAL fluid, 63% was DNase I sensitive. In 6 out of 11 lung homogenates, all bacterial DNA was DNase I sensitive, suggesting a predominance of dead bacteria; in the remaining homogenates, 94% was DNase I sensitive, and bacterial diversity determined by 16S rRNA gene sequencing was similar in DNase I-treated and untreated samples. Healthy pig lungs are mostly sterile yet contain abundant DNase I-sensitive DNA from inhaled and aspirated bacteria killed by pulmonary host defense mechanisms. This approach and conceptual framework will improve analysis of the lung microbiome in disease.

  3. Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

    PubMed

    Kamboj, Aman; Saini, Mohini; Rajan, Lekshmi S; Patel, Chhabi Lal; Chaturvedi, V K; Gupta, Praveen K

    2015-12-15

    To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

  4. Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.

    PubMed

    Liang, Te; Liu, Xiaoxiao; Cui, Shulin; Qu, Shenghua; Wang, Dan; Liu, Ning; Wang, Fumin; Ning, Kang; Zhang, Bing; Zhang, Dabing

    2016-02-02

    Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. THE PROTEASOME REGULATES BACTERIAL CpG DNA-INDUCED SIGNALING PATHWAYS IN MURINE MACROPHAGES

    PubMed Central

    Gao, Jian Jun; Shen, Jing; Kolbert, Christopher; Raghavakaimal, Sreekumar; Papasian, Christopher J.; Qureshi, Asaf A.; Vogel, Stefanie N.; Morrison, David C.; Qureshi, Nilofer

    2010-01-01

    Our previous work has provided strong evidence that the proteasome is central to the vast majority of genes induced in mouse macrophages in response to lipopolysaccharide (LPS) stimulation. In the studies presented here, we evaluated the role of the macrophage proteasome in response to a second microbial product CpG DNA (unmethylated bacterial DNA). For these studies, we applied Affymetrix microarray analysis of RNA derived from murine macrophages stimulated with CpG DNA in the presence or absence of proteasome inhibitor, lactacystin. The results of these studies revealed that similar to LPS, a vast majority of those macrophage genes regulated by CpG DNA are also under the control of the proteasome at 4 h. In contrast to LPS stimulation, however, many of these genes were induced much later than 4 h, at 18 h, in response to CpG DNA. Lactacystin treatment of macrophages completely blocked the CpG DNA-induced gene expression of TNF-α and other genes involved in production of inflammatory mediators. These data strongly support the conclusion that, similar to LPS, the macrophage proteasome is a key regulator of CpG DNA-induced signaling pathways. PMID:20160661

  6. Extraction of DNA from soil for analysis of bacterial diversity in transgenic and nontransgenic papaya sites.

    PubMed

    Sheu, Ceshing; Wu, Chung-Yi; Chen, Shu-Chuan; Lo, Chi-Chu

    2008-12-24

    The influence of transgenic crops on the soil diversity of microorganisms is one of the major risk assessments being conducted in Taiwan since 2007, and a reliable soil DNA extraction method for denaturing gradient gel electrophoresis (DGGE) is required. Six soils of different type, organic matter content, cation exchange capacity, and pH were tested, and four previously reported soil DNA extraction methods were applied to these soils. Soil DNA extracts by Zhou's CS method plus QIAquick gel was recommended in our laboratory for DGGE to monitor the microbial diversity in soil. There were some differences on the bacterial diversity based on DGGE patterns at the beginning of planting, and the difference decreased after six months. The results also indicated that clay content (10.8-25.0%) and pH (4.4-6.9) of different soil samples we tested did not affect the DNA extraction efficiencies, but positive correlations were found between the organic matter content (1.2-3.9%) of soils and the DNA yields in Widmer's GS method (r = 0.93, p = 0.005) and the MoBio UC method (r = 0.92, p = 0.007). Coefficient of determinations between organic matter content and DNA yield were higher than those between clay content, CEC, and pH, indicating that organic matter content was more correlated with DNA yield than that clay content, CEC, and pH in our soil samples tested.

  7. Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

    PubMed

    Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

    2017-02-01

    DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr (+) ][Ntf 2(-) ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

  8. The structure of a thermophilic archaeal virus shows a dsDNA viral capsid type that spans all domains of life

    SciTech Connect

    G. Rice; L. Tang; K. Stedman; F. Roberto; J. Spuhler; E. Gillitzer; J. E. Johnson; T. Douglas; M. Young

    2004-05-01

    Of the three domains of life (Eukarya, Bacteria, and Archaea), the least understood is Archaea and its associated viruses. Many Archaea are extremophiles, with species that are capable of growth at some of the highest temperatures and extremes of pH of all known organisms. Phylogenetic rRNA-encoding DNA analysis places many of the hyperthermophilic Archaea (species with an optimum growth >80°C) at the base of the universal tree of life, suggesting that thermophiles were among the first forms of life on earth. Very few viruses have been identified from Archaea as compared to Bacteria and Eukarya. We report here the structure of a hyperthermophilic virus isolated from an archaeal host found in hot springs in Yellowstone National Park. The sequence of the circular double-stranded DNA viral genome shows that it shares little similarity to other known genes in viruses or other organisms. By comparing the tertiary and quaternary structures of the coat protein of this virus with those of a bacterial and an animal virus, we find conformational relationships among all three, suggesting that some viruses may have a common ancestor that precedes the division into three domains of life >3 billion years ago.

  9. Effect of grazers and viruses on bacterial community structure and production in two contrasting trophic lakes

    PubMed Central

    2011-01-01

    Background Over the last 30 years, extensive studies have revealed the crucial roles played by microbes in aquatic ecosystems. It has been shown that bacteria, viruses and protozoan grazers are dominant in terms of abundance and biomass. The frequent interactions between these microbiological compartments are responsible for strong trophic links from dissolved organic matter to higher trophic levels, via heterotrophic bacteria, which form the basis for the important biogeochemical roles of microbial food webs in aquatic ecosystems. To gain a better understanding of the interactions between bacteria, viruses and flagellates in lacustrine ecosystems, we investigated the effect of protistan bacterivory on bacterial abundance, production and structure [determined by 16S rRNA PCR-DGGE], and viral abundance and activity of two lakes of contrasting trophic status. Four experiments were conducted in the oligotrophic Lake Annecy and the mesotrophic Lake Bourget over two seasons (early spring vs. summer) using a fractionation approach. In situ dark vs. light incubations were performed to consider the effects of the different treatments in the presence and absence of phototrophic activity. Results The presence of grazers (i.e. < 5-μm small eukaryotes) affected viral production positively in all experiments, and the stimulation of viral production (compared to the treatment with no eukaryotic predators) was more variable between lakes than between seasons, with the highest value having been recorded in the mesotrophic lake (+30%). Viral lysis and grazing activities acted additively to sustain high bacterial production in all experiments. Nevertheless, the stimulation of bacterial production was more variable between seasons than between lakes, with the highest values obtained in summer (+33.5% and +37.5% in Lakes Bourget and Annecy, respectively). The presence of both predators (nanoflagellates and viruses) did not seem to have a clear influence upon bacterial community

  10. Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past.

    PubMed

    Tijssen, Peter; Pénzes, Judit J; Yu, Qian; Pham, Hanh T; Bergoin, Max

    2016-10-01

    A wide spectrum of invertebrates is susceptible to various single-stranded DNA viruses. Their relative simplicity of replication and dependence on actively dividing cells makes them highly pathogenic for many invertebrates (Hexapoda, Decapoda, etc.). We present their taxonomical classification and describe the evolutionary relationships between various groups of invertebrate-infecting viruses, their high degree of recombination, and their relationship to viruses infecting mammals or other vertebrates. They share characteristics of the viruses within the various families, including structure of the virus particle, genome properties, and gene expression strategy. Copyright © 2016. Published by Elsevier Inc.

  11. Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

    NASA Astrophysics Data System (ADS)

    Chung, Da-Jung; Kim, Ki-Chul; Choi, Seong-Ho

    2011-09-01

    An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin-biotin conjugation of a biotinylated probe DNA, 5'-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3', and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5'-TTC GAC CTC GGT TAG AAG ACT CAT-3' and two-base mismatched DNA, 5'-TTC GAC AGC GGT TAT AAG ACT CAT-3'.

  12. Cloacal aerobic bacterial flora and absence of viruses in free-living slow worms (Anguis fragilis), grass snakes (Natrix natrix) and European Adders (Vipera berus) from Germany.

    PubMed

    Schmidt, Volker; Mock, Ronja; Burgkhardt, Eileen; Junghanns, Anja; Ortlieb, Falk; Szabo, Istvan; Marschang, Rachel; Blindow, Irmgard; Krautwald-Junghanns, Maria-Elisabeth

    2014-12-01

    Disease problems caused by viral or bacterial pathogens are common in reptiles kept in captivity. There is no information available on the incidence of viral pathogens or the physiological cloacal bacterial flora of common free-living reptiles in Germany. Therefore, 56 free-living reptiles including 23 European adders (Vipera berus), 12 grass snakes (Natrix natrix) and 21 slow worms (Anguis fragilis) were investigated on the island Hiddensee in northeastern Germany. Pharyngeal and cloacal swabs were taken immediately after capture. Bacteriological examination was performed from the cloacal swabs to study the aerobic cloacal flora. Molecular biological examination included amplification of DNA or RNA from adeno-, rana- and ferlaviruses as well as culturing on Russell's viper heart cells for virus isolation. Salmonella spp. were isolated from European adders but not from the other reptiles examined. The minimal inhibitory concentration was determined from the isolated Salmonella spp. However, some potentially human pathogenic bacteria, such as Proteus vulgaris, Aeromonas hydrophila, Klebsiella pneumoniae and Escherichia coli were isolated. Viruses were not detected in any of the examined reptiles. To the authors' best knowledge, the present study is the first survey of viral pathogens in free-living snakes and slow worms in Germany and the first survey of cloacal aerobic bacterial flora of slow worms.

  13. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  14. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    SciTech Connect

    Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A.; Mason, William S.; Litwin, Samuel; Jilbert, Allison R.

    2013-11-15

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.

  15. Activation of the immune system by bacterial CpG-DNA

    PubMed Central

    Häcker, Georg; Redecke, Vanessa; Häcker, Hans

    2002-01-01

    The past decade has seen a remarkable process of refocusing in immunology. Cells of the innate immune system, especially macrophages and dendritic cells, have been at the centre of this process. These cells had been regarded by some scientists as non-specific, sometimes perhaps even confined to the menial job of serving T cells by scavenging antigen and presenting it to the sophisticated adaptive immune system. Only over the last few years has it become unequivocally clear that cells of the innate immunity hold, by variation of context and mode of antigen presentation, the power of shaping an adaptive immune response. The innate immune response, in turn, is to a significant degree the result of stimulation by so-called pathogen-associated molecular patterns (PAMPs). One compound with high stimulatory potential for the innate immune system is bacterial DNA. Here we will review recent evidence that bacterial DNA should be ranked with other PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid. We will further review our present knowledge of DNA recognition and DNA-dependent signal transduction in cells of the immune system. PMID:11918685

  16. In vivo single-molecule imaging of bacterial DNA replication, transcription, and repair.

    PubMed

    Stracy, Mathew; Uphoff, Stephan; Garza de Leon, Federico; Kapanidis, Achillefs N

    2014-10-01

    In vivo single-molecule experiments offer new perspectives on the behaviour of DNA binding proteins, from the molecular level to the length scale of whole bacterial cells. With technological advances in instrumentation and data analysis, fluorescence microscopy can detect single molecules in live cells, opening the doors to directly follow individual proteins binding to DNA in real time. In this review, we describe key technical considerations for implementing in vivo single-molecule fluorescence microscopy. We discuss how single-molecule tracking and quantitative super-resolution microscopy can be adapted to extract DNA binding kinetics, spatial distributions, and copy numbers of proteins, as well as stoichiometries of protein complexes. We highlight experiments which have exploited these techniques to answer important questions in the field of bacterial gene regulation and transcription, as well as chromosome replication, organisation and repair. Together, these studies demonstrate how single-molecule imaging is transforming our understanding of DNA-binding proteins in cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

    PubMed

    Juhas, Mario; Ajioka, James W

    2016-06-01

    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation-and Beyond?

    PubMed

    Schreiner, Sabrina; Nassal, Michael

    2017-05-22

    Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system.

  19. A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

    PubMed Central

    Schreiner, Sabrina; Nassal, Michael

    2017-01-01

    Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system. PMID:28531167

  20. Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication.

    PubMed

    Jiang, Changying; Hwang, Ying T; Randell, John C W; Coen, Donald M; Hwang, Charles B C

    2007-04-01

    The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.

  1. DNA-Dependent RNA Polymerase Detects Hidden Giant Viruses in Published Databanks

    PubMed Central

    Sharma, Vikas; Colson, Philippe; Giorgi, Roch; Pontarotti, Pierre; Raoult, Didier

    2014-01-01

    Environmental metagenomic studies show that there is a “dark matter,” composed of sequences not linked to any known organism, as determined mainly using ribosomal DNA (rDNA) sequences, which therefore ignore giant viruses. DNA-dependent RNA polymerase (RNAP) genes are universal in microbes and conserved in giant viruses and may replace rDNA for identifying microbes. We found while reconstructing RNAP subunit 2 (RNAP2) phylogeny that a giant virus sequenced together with the genome of a large eukaryote, Hydra magnipapillata, has been overlooked. To explore the dark matter, we used viral RNAP2 and reconstructed putative ancestral RNAP2, which were significantly superior in detecting distant clades than current sequences, and we revealed two additional unknown mimiviruses, misclassified as an euryarchaeote and an oomycete plant pathogen, and detected unknown putative viral clades. We suggest using RNAP systematically to decipher the black matter and identify giant viruses. PMID:24929085

  2. The DNA Damage Response Induced by Infection with Human Cytomegalovirus and Other Viruses

    PubMed Central

    E, Xiaofei; Kowalik, Timothy F.

    2014-01-01

    Viruses use different strategies to overcome the host defense system. Recent studies have shown that viruses can induce DNA damage response (DDR). Many of these viruses use DDR signaling to benefit their replication, while other viruses block or inactivate DDR signaling. This review focuses on the effects of DDR and DNA repair on human cytomegalovirus (HCMV) replication. Here, we review the DDR induced by HCMV infection and its similarities and differences to DDR induced by other viruses. As DDR signaling pathways are critical for the replication of many viruses, blocking these pathways may represent novel therapeutic opportunities for the treatment of certain infectious diseases. Lastly, future perspectives in the field are discussed. PMID:24859341

  3. Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus.

    PubMed

    Lee, Chih-Chun; Kim, Bong-Suk; Wu, Ching Ching; Lin, Tsang Long

    2015-01-01

    Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-protein-expressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 × 10(2) times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p < 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-α, IFN-β, IRF-1, IRF-10, IL-1β, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-γ, IL-12(p40), IL-18, GM-CSF, GATA-3

  4. Making Ends Meet: Repairing Breaks in Bacterial DNA by Non-Homologous End-Joining

    PubMed Central

    Bowater, Richard; Doherty, Aidan J

    2006-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages. PMID:16518468

  5. Archaeal Haloarcula californiae Icosahedral Virus 1 Highlights Conserved Elements in Icosahedral Membrane-Containing DNA Viruses from Extreme Environments.

    PubMed

    Demina, Tatiana A; Pietilä, Maija K; Svirskaitė, Julija; Ravantti, Janne J; Atanasova, Nina S; Bamford, Dennis H; Oksanen, Hanna M

    2016-07-19

    Despite their high genomic diversity, all known viruses are structurally constrained to a limited number of virion morphotypes. One morphotype of viruses infecting bacteria, archaea, and eukaryotes is the tailless icosahedral morphotype with an internal membrane. Although it is considered an abundant morphotype in extreme environments, only seven such archaeal viruses are known. Here, we introduce Haloarcula californiae icosahedral virus 1 (HCIV-1), a halophilic euryarchaeal virus originating from salt crystals. HCIV-1 also retains its infectivity under low-salinity conditions, showing that it is able to adapt to environmental changes. The release of progeny virions resulting from cell lysis was evidenced by reduced cellular oxygen consumption, leakage of intracellular ATP, and binding of an indicator ion to ruptured cell membranes. The virion contains at least 12 different protein species, lipids selectively acquired from the host cell membrane, and a 31,314-bp-long linear double-stranded DNA (dsDNA). The overall genome organization and sequence show high similarity to the genomes of archaeal viruses in the Sphaerolipoviridae family. Phylogenetic analysis based on the major conserved components needed for virion assembly-the major capsid proteins and the packaging ATPase-placed HCIV-1 along with the alphasphaerolipoviruses in a distinct, well-supported clade. On the basis of its virion morphology and sequence similarities, most notably, those of its core virion components, we propose that HCIV-1 is a member of the PRD1-adenovirus structure-based lineage together with other sphaerolipoviruses. This addition to the lineage reinforces the notion of the ancient evolutionary links observed between the viruses and further highlights the limits of the choices found in nature for formation of a virion. Under conditions of extreme salinity, the majority of the organisms present are archaea, which encounter substantial selective pressure, being constantly attacked by

  6. Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication.

    PubMed

    Mitchell, Jonathan K; Friesen, Paul D

    2012-12-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.

  7. Repair of DNA damage in shuttle vectors, virus, and chromosomal DNAs may depend on their biological imprinting--a 'Pygmalion' effect.

    PubMed

    Cleaver, J E; Vuksanovic, L; Player, A N; Lutze, L H

    1989-01-01

    We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their 'cultural background', i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.

  8. Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin.

    PubMed Central

    Justewicz, D M; Morin, M J; Robinson, H L; Webster, R G

    1995-01-01

    Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells. PMID:7494280

  9. Lethal fragmentation of bacterial chromosomes mediated by DNA gyrase and quinolones.

    PubMed

    Malik, Muhammad; Zhao, Xilin; Drlica, Karl

    2006-08-01

    When DNA gyrase is trapped on bacterial chromosomes by quinolone antibacterials, reversible complexes form that contain DNA ends constrained by protein. Two subsequent processes lead to rapid cell death. One requires ongoing protein synthesis; the other does not. The prototype quinolone, nalidixic acid, kills wild-type Escherichia coli only by the first pathway; fluoroquinolones kill by both. Both lethal processes correlated with irreversible chromosome fragmentation, detected by sedimentation and viscosity of DNA from quinolone-treated cells. However, only fluoroquinolones fragmented purified nucleoids when incubated with gyrase purified from wild-type cells. A GyrA amino acid substitution (A67S) expected to perturb a GyrA-GyrA dimer interface allowed nalidixic acid to fragment chromosomes and kill cells in the absence of protein synthesis; moreover, it made a non-inducible lexA mutant hypersusceptible to nalidixic acid, a property restricted to fluoroquinolones with wild-type cells. The GyrA variation also facilitated immunoprecipitation of DNA fragments by GyrA antiserum following nalidixic acid treatment of cells. The ability of changes in both gyrase and quinolone structure to enhance protein synthesis-independent lethality and chromosome fragmentation is explained by drug-mediated destabilization of gyrase-DNA complexes. Instability of type II topoisomerase-DNA complexes may be a general phenomenon that can be exploited to kill cells.

  10. An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses

    PubMed Central

    Carpentier, Kathryn S.

    2016-01-01

    To establish productive infections, viruses must counteract numerous cellular defenses that are poised to recognize viruses as nonself and to activate antiviral pathways. The opposing goals of host and viral factors lead to evolutionary arms races that can be illuminated by evolutionary and computational methods and tested in experimental models. Here we illustrate how this perspective has been contributing to our understanding of the interactions of the protein kinase R pathway with large DNA viruses. PMID:26792736

  11. A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

    USDA-ARS?s Scientific Manuscript database

    Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. While investigating virus-invertebrate host interactions we found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), were unable to infect certain Lepido...

  12. Update on new antivirals under development for the treatment of double-stranded DNA virus infections.

    PubMed

    Dropulic, L K; Cohen, J I

    2010-11-01

    All the currently available antiviral agents used in the treatment of double-stranded (ds) DNA viruses, with the exception of interferon-α, inhibit the same target, the viral DNA polymerase. With increasing reports of the development of resistance of herpes simplex virus (HSV), cytomegalovirus (CMV), and hepatitis B virus (HBV) to some of these drugs, new antiviral agents are needed to treat these infections. Additionally, no drugs have been approved to treat several DNA virus infections, including those caused by adenovirus, smallpox, molluscum contagiosum, and BK virus. We report the status of 10 new antiviral drugs for the treatment of dsDNA viruses. CMX-001 has broad activity against dsDNA viruses; 3 helicase-primase inhibitors, maribavir, and FV-100 have activity against certain herpesviruses; ST-246 inhibits poxviruses; GS-9191 inhibits papillomaviruses; and clevudine and emtricitabine are active against HBV. Most of these drugs have completed at least phase I trials in humans, and many are in additional clinical trials.

  13. Characterization of Circular ssDNA Viruses within the Echinoderm Nanobiome

    NASA Astrophysics Data System (ADS)

    Jackson, E.; Bistolas, K. S.; Hewson, I.

    2016-02-01

    Viral metagenomics has revealed a great diversity and presence of circular single-stranded(ss) DNA viruses most similar to the viral family Circoviridae in various environments both ambient and host. These viruses are an emerging paradigm in viral discovery amongst aquatic invertebrates mainly from the sub-phlya Crustacea and to a lesser extent the phylum Echinodermata. This parasite-host relationship is furthered here with the discovery of circo-like viruses extracted from the tissue of members from the family Holothuroidea (sea cucumbers). Verification and presence of these viruses within the tissue of the host was confirmed through rigorous genome architecture screening and PCR amplification of the rep gene from unamplified viral DNA extracts. Phylogenetic analysis of the rep gene reveals high similarity to circular ssDNA viruses from environmental metagenomic surveys of marine habitats. The significance of these findings is changing the perception and understanding of circular ssDNA viruses by broadening the known host range and blurring certain defining characteristics established by their pathogenic counterparts. Aside from discover and characterization, the potential ecological impacts of ssDNA viruses upon their host remains relatively unknown and further investigations should aim to determine the pathology, route of infection, and ecological implications of viral infection.

  14. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

    PubMed Central

    2011-01-01

    Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses) via horizontal gene transfer (HGT). It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA) viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep)-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts. PMID:21943216

  15. A novel, highly divergent ssDNA virus identified in Brazil infecting apple, pear and grapevine.

    PubMed

    Basso, Marcos Fernando; da Silva, José Cleydson Ferreira; Fajardo, Thor Vinícius Martins; Fontes, Elizabeth Pacheco Batista; Zerbini, Francisco Murilo

    2015-12-02

    Fruit trees of temperate and tropical climates are of great economical importance worldwide and several viruses have been reported affecting their productivity and longevity. Fruit trees of different Brazilian regions displaying virus-like symptoms were evaluated for infection by circular DNA viruses. Seventy-four fruit trees were sampled and a novel, highly divergent, monopartite circular ssDNA virus was cloned from apple, pear and grapevine trees. Forty-five complete viral genomes were sequenced, with a size of approx. 3.4 kb and organized into five ORFs. Deduced amino acid sequences showed identities in the range of 38% with unclassified circular ssDNA viruses, nanoviruses and alphasatellites (putative Replication-associated protein, Rep), and begomo-, curto- and mastreviruses (putative coat protein, CP, and movement protein, MP). A large intergenic region contains a short palindromic sequence capable of forming a hairpin-like structure with the loop sequence TAGTATTAC, identical to the conserved nonanucleotide of circoviruses, nanoviruses and alphasatellites. Recombination events were not detected and phylogenetic analysis showed a relationship with circo-, nano- and geminiviruses. PCR confirmed the presence of this novel ssDNA virus in field plants. Infectivity tests using the cloned viral genome confirmed its ability to infect apple and pear tree seedlings, but not Nicotiana benthamiana. The name "Temperate fruit decay-associated virus" (TFDaV) is proposed for this novel virus. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. The FACT complex promotes avian leukosis virus DNA integration.

    PubMed

    Winans, Shelby; Larue, Ross C; Abraham, Carly M; Shkriabai, Nikolozi; Skopp, Amelie; Winkler, Duane; Kvaratskhelia, Mamuka; Beemon, Karen L

    2017-01-25

    All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. Here, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro Likewise, in infected cells, the FACT complex promotes ALV integration activity with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-LTR circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.

  17. A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

    PubMed Central

    Hoover, T A; Vodkin, M H; Williams, J C

    1992-01-01

    A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a