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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. Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC).

    PubMed

    Roth, Swaantje J; Höper, Dirk; Beer, Martin; Feineis, Silke; Tischer, B Karsten; Osterrieder, Nikolaus

    2011-01-01

    Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool. PMID:21314965

  3. Robust properties of membrane-embedded connector channel of bacterial virus phi29 DNA packaging motor.

    PubMed

    Jing, Peng; Haque, Farzin; Vonderheide, Anne P; Montemagno, Carlo; Guo, Peixuan

    2010-10-01

    Biological systems contain highly-ordered macromolecular structures with diverse functions, inspiring their utilization in nanotechnology. A motor allows linear dsDNA viruses to package their genome into a preformed procapsid. The central component of the motor is the portal connector that acts as a pathway for the translocation of dsDNA. The elegant design of the connector and its channel motivates its application as an artificial nanopore (Nature Nanotechnology, 4, 765-772). Herein, we demonstrate the robust characteristics of the connector of the bacteriophage phi29 DNA packaging motor by single pore electrophysiological assays. The conductance of each pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by dsDNA are consistent with the dimensions of the channel and dsDNA. Furthermore, the connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for connector quantification. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of DNA passing through the channel. The fingerprint of DNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of DNA transportation, motion, and packaging. PMID:20523933

  4. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  5. Novel ssDNA virus recovered from estuarine Mollusc (Amphibola crenata) whose replication associated protein (Rep) shares similarities with Rep-like sequences of bacterial origin.

    PubMed

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

    2013-05-01

    Over the past couple of years highly diverse novel ssDNA viruses have been discovered. Here, we present the first ssDNA virus, Gastropod-associated circular ssDNA virus (GaCSV), recovered from a mollusc Amphibola crenata Martyn 1784, which is a deposit feeder that grazes micro-organisms and organic detritus on the surface of tidal mudflats. The GaCSV (2351 nt) genome contains two large bidirectionally transcribed ORFs. The smaller ORF (874 nt) has similarities to viral replication-associated protein (Rep) sequences of some bacteria and circoviruses, whereas the larger ORF (955 nt) does not relate to any sequences in public databases and we presume it potentially encodes the capsid protein. Phylogenetic analysis shows that the GaCSV Rep clusters with Rep-like sequences of bacterial origin, highlighting the role of ssDNA viruses in horizontal gene transfer. The occurrence of previously unknown viruses in organisms associated with human pollution is a relatively unexplored field. PMID:23364192

  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.

  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. Cloned Cauliflower Mosaic Virus DNA Infects Turnips (Brassica rapa).

    PubMed

    Howell, S H; Walker, L L; Dudley, R K

    1980-06-13

    Cauliflower mosaic virus DNA cloned in the Sal I site of bacterial plasmid pBR322 infects turnip plants. The cloned viral DNA must be excised from the recombinant plasmid to infect, but need not be circularized and ligated in vitro. The cloned viral DNA lacks site-specific single-strand breaks found in DNA obtained directly from the virus. However, these breaks are reintroduced into the viral genome during multiplication of the virus in the plant host.

  9. 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)

  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; 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. 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. Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel

    PubMed Central

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

    2016-01-01

    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. 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. PMID:26882199

  12. 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.

  13. 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

  14. 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.

  15. [Detection of bacterial DNA using the polymerase chain reaction (PCR)].

    PubMed

    Höfler, G

    1994-01-01

    Enzymatic amplification of DNA using the polymerase chain reaction (PCR) is a very sensitive and rapid way of detecting specific DNA sequences. Bacterial DNA can be detected in a wide variety of samples provided at least partial sequence information is available. For a great number of bacteria PCR detection methods have been published. Most important for the pathologist are mycobacteriae (M. tuberculosis, avium, etc.). Borellia burgdorferi, Listeria monozytogenes and chlamydiae (Ce. trachomatis, C. psittaci). Fresh or fixed paraffin embedded tissues, exfoliated cells, whole blood, serum, sputum, urine, ascites or pleural fluid etc. can be analyzed. The time needed to perform the analysis varies between 5 hours and 2 days mostly depending on the DNA extraction method. Several potential pitfalls have to be avoided. The most common problem is contamination of reagents with target DNA. Amplification of DNA from biological samples may be prevented by enzyme inhibitors (salts, proteins). This problem can at least partially be avoided by changing the DNA purification method. Several additional problems may arise if bacterial DNA has to be amplified. Bacterial walls may have to be disrupted using heat or detergent for accessibility of target DNA. Positive results have to be judged carefully. Unlike the situation in retroviral infections with the virus sometimes present in the absence of disease, in the majority of bacterial infections the presence of bacteria signals manifest disease. A possible exception may be the finding of mycobacterial DNA in sarcoidosis patients who can be treated with steroids without provoking tuberculosis. PCR is especially useful in situations where rapid results are necessary or only fixed tissue is available.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7533972

  16. 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.

  17. 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

  18. A DNA Virus of Drosophila

    PubMed Central

    Unckless, Robert L.

    2011-01-01

    Little is known about the viruses infecting most species. Even in groups as well-studied as Drosophila, only a handful of viruses have been well-characterized. A viral metagenomic approach was used to explore viral diversity in 83 wild-caught Drosophila innubila, a mushroom feeding member of the quinaria group. A single fly that was injected with, and died from, Drosophila C Virus (DCV) was added to the sample as a control. Two-thirds of reads in the infected sample had DCV as the best BLAST hit, suggesting that the protocol developed is highly sensitive. In addition to the DCV hits, several sequences had Oryctes rhinoceros Nudivirus, a double-stranded DNA virus, as a best BLAST hit. The virus associated with these sequences was termed Drosophila innubila Nudivirus (DiNV). PCR screens of natural populations showed that DiNV was both common and widespread taxonomically and geographically. Electron microscopy confirms the presence of virions in fly fecal material similar in structure to other described Nudiviruses. In 2 species, D. innubila and D. falleni, the virus is associated with a severe (∼80–90%) loss of fecundity and significantly decreased lifespan. PMID:22053195

  19. 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.

  20. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. 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

  3. 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

  4. Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity.

    PubMed Central

    Levin, J G; Crouch, R J; Post, K; Hu, S C; McKelvin, D; Zweig, M; Court, D L; Gerwin, B I

    1988-01-01

    The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively. Images PMID:2459414

  5. 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.

  6. 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

  7. 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.

  8. 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. PMID:23536852

  9. Virus-induced secondary bacterial infection: a concise review

    PubMed Central

    Hendaus, Mohamed A; Jomha, Fatima A; Alhammadi, Ahmed H

    2015-01-01

    Respiratory diseases are a very common source of morbidity and mortality among children. Health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. The reason expressed by many clinicians is the trouble to confirm whether the fever is caused by a virus or a bacterium. The aim of this review is to update the current evidence on the virus-induced bacterial infection. We present several clinical as well in vitro studies that support the correlation between virus and secondary bacterial infections. In addition, we discuss the pathophysiology and prevention modes of the virus–bacterium coexistence. A search of the PubMed and MEDLINE databases was carried out for published articles covering bacterial infections associated with respiratory viruses. This review should provide clinicians with a comprehensive idea of the range of bacterial and viral coinfections or secondary infections that could present with viral respiratory illness. PMID:26345407

  10. 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

  11. 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

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

    PubMed

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-01

    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

  13. DNA Tumor Viruses and Cell Metabolism

    PubMed Central

    Mushtaq, Muhammad; Darekar, Suhas

    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. PMID:27034740

  14. 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

  15. 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.

  16. 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.

  17. Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

    PubMed Central

    Rigden, J E; Dry, I B; Krake, L R; Rezaian, M A

    1996-01-01

    Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8816791

  18. The complete DNA sequence of vaccinia virus.

    PubMed

    Goebel, S J; Johnson, G P; Perkus, M E; Davis, S W; Winslow, J P; Paoletti, E

    1990-11-01

    The complete DNA sequence of the genome of vaccinia virus has been determined. The genome consisted of 191,636 bp with a base composition of 66.6% A + T. We have identified 198 "major" protein-coding regions and 65 overlapping "minor" regions, for a total of 263 potential genes. Genes encoded by the virus were located by examination of DNA sequence characteristics and compared with existing vaccinia virus mapping analyses, sequence data, and transcription data. These genes were found to be compactly organized along the genome with relatively few regions of noncoding sequences. Whereas several similarities to proteins of known function were discerned, the function of the majority of proteins encoded by these open reading frames is as yet undetermined.

  19. 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

  20. 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

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

    PubMed Central

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

    2014-01-01

    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. PMID:25372693

  2. 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.

  3. Evolutionary genomics of nucleo-cytoplasmic large DNA viruses.

    PubMed

    Iyer, Lakshminarayan M; Balaji, S; Koonin, Eugene V; Aravind, L

    2006-04-01

    (primarily, endosymbionts and parasites). Amongst the expansions, there are multiple families of predicted virus-specific signaling and regulatory domains. Most NCLDVs have also acquired large arrays of genes related to ubiquitin signaling, and the animal viruses in particular have independently evolved several defenses against apoptosis and immune response, including growth factors and potential inhibitors of cytokine signaling. The mimivirus displays an enormous array of genes of bacterial provenance, including a representative of a new class of predicted papain-like peptidases. It is further demonstrated that a significant number of genes found in NCLDVs also have homologs in bacteriophages, although a vertical relationship between the NCLDVs and a particular bacteriophage group could not be established. On the basis of these observations, two alternative scenarios for the origin of the NCLDVs and other groups of large DNA viruses of eukaryotes are considered. One of these scenarios posits an early assembly of an already large DNA virus precursor from which various large DNA viruses diverged through an ongoing process of displacement of the original genes by xenologous or non-orthologous genes from various sources. The second scenario posits convergent emergence, on multiple occasions, of large DNA viruses from small plasmid-like precursors through independent accretion of similar sets of genes due to strong selective pressures imposed by their life cycles and hosts. PMID:16494962

  4. Influenza virus induces bacterial and nonbacterial otitis media.

    PubMed

    Short, Kirsty R; Diavatopoulos, Dimitri A; Thornton, Ruth; Pedersen, John; Strugnell, Richard A; Wise, Andrew K; Reading, Patrick C; Wijburg, Odilia L

    2011-12-15

    Otitis media (OM) is one of the most common childhood diseases. OM can arise when a viral infection enables bacteria to disseminate from the nasopharynx to the middle ear. Here, we provide the first infant murine model for disease. Mice coinfected with Streptococcus pneumoniae and influenza virus had high bacterial load in the middle ear, middle ear inflammation, and hearing loss. In contrast, mice colonized with S. pneumoniae alone had significantly less bacteria in the ear, minimal hearing loss, and no inflammation. Of interest, infection with influenza virus alone also caused some middle ear inflammation and hearing loss. Overall, this study provides a clinically relevant and easily accessible animal model to study the pathogenesis and prevention of OM. Moreover, we provide, to our knowledge, the first evidence that influenza virus alone causes middle ear inflammation in infant mice. This inflammation may then play an important role in the development of bacterial OM.

  5. Role of bacterial chaperones in DNA replication.

    PubMed

    Konieczny, I; Zylicz, M

    1999-01-01

    Studies on the involvement of chaperone proteins in DNA replication have been limited to a few replication systems, belonging primarily to the prokaryotic world. The insights gained from these studies have substantially contributed to our understanding of the eukaryotic DNA replication process as well. The finding that molecular chaperones can activate some initiation proteins before DNA synthesis has led to the more general suggestion that molecular chaperones can influence the DNA-binding activity of many proteins, including transcriptional factors involved in cell regulatory systems. The DnaK/DnaJ/GrpE molecular chaperone system became a paradigm of our understanding of fundamental processes, such as protein folding, translocation, selective proteolysis and autoregulation of the heat-shock response. Studies on the Clp ATPase family of molecular chaperones will help to define the nature of signals involved in chaperone-dependent proteins' refolding and the degradation of misfolded proteins.

  6. 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.

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

    PubMed Central

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

    2012-01-01

    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. PMID:22743047

  8. 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

  9. 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

  10. Evaluating bacterial pathogen DNA preservation in museum osteological collections

    PubMed Central

    Barnes, Ian; Thomas, Mark G

    2005-01-01

    Reports of bacterial pathogen DNA sequences obtained from archaeological bone specimens raise the possibility of greatly improving our understanding of the history of infectious diseases. However, the survival of pathogen DNA over long time periods is poorly characterized, and scepticism remains about the reliability of these data. In order to explore the survival of bacterial pathogen DNA in bone specimens, we analysed samples from 59 eighteenth and twentieth century individuals known to have been infected with either Mycobacterium tuberculosis or Treponema pallidum. No reproducible evidence of surviving pathogen DNA was obtained, despite the use of extraction and PCR-amplification methods determined to be highly sensitive. These data suggest that previous studies need to be interpreted with caution, and we propose that a much greater emphasis is placed on understanding how pathogen DNA survives in archaeological material, and how its presence can be properly verified and used. PMID:16608682

  11. 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

  12. 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.

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

    PubMed

    Liu, Ting; Sae-Ueng, Udom; Li, Dong; Lander, Gabriel C; Zuo, Xiaobing; Jönsson, 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, resulting locally in less densely packed DNA, reducing DNA-DNA repulsions. This process leads to a significant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.

  14. Horizontal transfer of DNA methylation patterns into bacterial chromosomes

    PubMed Central

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

    2016-01-01

    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. PMID:27084942

  15. A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

    PubMed

    Höfler, Daniela; Nicklas, Werner; Mauter, Petra; Pawlita, Michael; Schmitt, Markus

    2014-01-01

    The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals' physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

  16. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

    PubMed

    Richardson, Tomas T; Harran, Omar; Murray, Heath

    2016-06-16

    DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities. DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms. PMID:27281207

  17. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

  18. 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

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

    PubMed Central

    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. PMID:27668216

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

    PubMed Central

    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.

  1. 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. PMID:27668216

  2. 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

    2013-12-01

    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.

  3. 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

    2013-12-01

    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. PMID:24248361

  4. Bacterial natural transformation by highly fragmented and damaged DNA

    PubMed Central

    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

    2013-01-01

    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. PMID:24248361

  5. 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

  6. 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.

  7. Viral hemorrhagic fevers of animals caused by DNA viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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.

  9. Bacterial sinusitis and otitis media following influenza virus infection in ferrets.

    PubMed

    Peltola, Ville T; Boyd, Kelli L; McAuley, Julie L; Rehg, Jerold E; McCullers, Jonathan A

    2006-05-01

    Streptococcus pneumoniae is the leading cause of otitis media, sinusitis, and pneumonia. Many of these infections result from antecedent influenza virus infections. In this study we sought to determine whether the frequency and character of secondary pneumococcal infections differed depending on the strain of influenza virus that preceded bacterial challenge. In young ferrets infected with influenza virus and then challenged with pneumococcus, influenza viruses of any subtype increased bacterial colonization of the nasopharynx. Nine out of 10 ferrets infected with H3N2 subtype influenza A viruses developed either sinusitis or otitis media, while only 1 out of 11 ferrets infected with either an H1N1 influenza A virus or an influenza B virus did so. These data may partially explain why bacterial complication rates are higher during seasons when H3N2 viruses predominate. This animal model will be useful for further study of the mechanisms that underlie viral-bacterial synergism.

  10. At a crossroads: human DNA tumor viruses and the host DNA damage response.

    PubMed

    Nikitin, Pavel A; Luftig, Micah A

    2011-07-01

    Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection. PMID:21927617

  11. 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.

  12. The nucleotide sequence of cloned wheat dwarf virus DNA

    PubMed Central

    MacDowell, S. W.; Macdonald, H.; Hamilton, W. D. O.; Coutts, R. H. A.; Buck, K. W.

    1985-01-01

    Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (−) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of △G≥-14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication. PMID:15938050

  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. 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

  15. A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

    PubMed Central

    Menissier, J; Laquel, P; Lebeurier, G; Hirth, L

    1984-01-01

    A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa. Images PMID:6514573

  16. A Mechanism for the Inhibition of DNA-PK-Mediated DNA Sensing by a Virus

    PubMed Central

    Peters, Nicholas E.; Ferguson, Brian J.; Mazzon, Michela; Fahy, Aodhnait S.; Krysztofinska, Ewelina; Arribas-Bosacoma, Raquel; Pearl, Laurence H.; Ren, Hongwei; Smith, Geoffrey L.

    2013-01-01

    The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses. PMID:24098118

  17. Distribution of Viruses and Dissolved DNA along a Coastal Trophic Gradient in the Northern Adriatic Sea

    PubMed Central

    Weinbauer, Markus G.; Fuks, Dragica; Peduzzi, Peter

    1993-01-01

    The distribution of viral and other microbial abundances as well as the concentrations of dissolved DNA (D-DNA) along a trophic gradient in the northern Adriatic Sea were determined. Virus abundances, covering a range of 1.2 × 109 to 8.7 × 1010 liter-1 were on average 2.5-fold higher in eutrophic than in mesotrophic stations. A 2.5-fold enrichment was also measured for chlorophyll a concentrations, whereas the densities of bacteria and heterotrophic nanoflagellates were only approximately 1.5-fold higher. The frequency of bacteria containing mature phage increased linearly with bacterial abundance. Assuming that mature phage is only visible during the last 14 to 27% of the latent period (L. M. Proctor, A. Okubo, and J. A. Fuhrman, Microb. Ecol. 25:161-182, 1993), we estimated that between 3.5 and 7.3% of the bacterial population was infected at mesotrophic stations versus between 7.0 and 19.5% at eutrophic stations, indicating that the bacterial mortality due to viral lysis might increase with the degree of eutrophication. The frequency of bacteria with mature phage and the burst size varied significantly with the bacterial morphotype; rod-shape cells, the most abundant morphotype, showed low infection rates but a high burst size. Concentrations of D-DNA varied significantly with season but not with trophic conditions. The estimated percentage of viral DNA on total D-DNA concentrations averaged 17.1% (range, 0.7 to 88.3%). Some kind of interaction between heterotrophic nanoflagellates and viruses is proposed. We conclude (i) that the significance of viruses varies with changing trophic conditions and (ii) that viral activity may play a significant role in food web structure under changing trophic conditions. PMID:16349109

  18. 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.

  19. 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.

  20. 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

  1. New DNA Viruses Identified in Patients with Acute Viral Infection Syndrome

    PubMed Central

    Jones, Morris S.; Kapoor, Amit; Lukashov, Vladimir V.; Simmonds, Peter; Hecht, Frederick; Delwart, Eric

    2005-01-01

    A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing. PMID:15956568

  2. 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

  3. Rescue of mumps virus from cDNA.

    PubMed

    Clarke, D K; Sidhu, M S; Johnson, J E; Udem, S A

    2000-05-01

    A complete DNA copy of the genome of a Jeryl Lynn strain of mumps virus (15,384 nucleotides) was assembled from cDNA fragments such that an exact antigenome RNA could be generated following transcription by T7 RNA polymerase and cleavage by hepatitis delta virus ribozyme. The plasmid containing the genome sequence, together with support plasmids which express mumps virus NP, P, and L proteins under control of the T7 RNA polymerase promoter, were transfected into A549 cells previously infected with recombinant vaccinia virus (MVA-T7) that expressed T7 RNA polymerase. Rescue of infectious virus from the genome cDNA was demonstrated by amplification of mumps virus from transfected-cell cultures and by subsequent consensus sequencing of reverse transcription-PCR products generated from infected-cell RNA to verify the presence of specific nucleotide tags introduced into the genome cDNA clone. The only coding change (position 8502, A to G) in the cDNA clone relative to the consensus sequence of the Jeryl Lynn plaque isolate from which it was derived, resulting in a lysine-to-arginine substitution at amino acid 22 of the L protein, did not prevent rescue of mumps virus, even though an amino acid alignment for the L proteins of paramyxoviruses indicates that lysine is highly conserved at that position. This system may provide the basis of a safe and effective virus vector for the in vivo expression of immunologically and biologically active proteins, peptides, and RNAs.

  4. 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

  5. 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

  6. Titration of recombinant woodchuck hepatitis virus DNA in adult woodchucks.

    PubMed

    Chen, H S; Miller, R H; Hornbuckle, W E; Tennant, B C; Cote, P J; Gerin, J L; Purcell, R H

    1998-02-01

    In vivo transfection of Eastern woodchucks (Marmota monax) with recombinant woodchuck hepatitis virus (WHV) DNA is effective in inducing virus infection for the study of replication, pathogenicity, and oncogenicity of wild-type and mutated WHV. The one drawback to this procedure is the need for preparation of large amounts of WHV DNA. Reduction of the amount of WHV DNA in the transfection protocol necessary to induce infection would save considerable time and resources. Therefore, we conducted a titration of WHV DNA, ranging from 50 micrograms to 50 pg of DNA, in adult woodchucks to determine the minimum infectious dose of recombinant WHV DNA. As little as 50 ng of transfected WHV DNA induced productive infection in adult woodchucks. Thus, transfection with large amounts of recombinant WHV DNA appears to be unnecessary.

  7. 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

  8. 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

  9. [Varicella-zoster virus infection in a child complicated with bacterial pneumonia].

    PubMed

    Wolthers, Benjamin Ole; Thiested, Ebbe; Gyhrs, Anette F

    2015-01-26

    This case report describes how a three-year-old girl referred to our paediatric ward, infected with varicella-zoster virus and a bacterial pneumonia causing atelectasis. The girl did not respond to initial treatment with intravenous antibiotics. Only after a lung drain was inserted she gradually recovered. We briefly describe the incidence of bacterial pneumonia as a complication to varicella-zoster virus and raise the question whether to vaccinate against varicella-zoster virus.

  10. 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

  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. 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

  13. Frog Virus 3 DNA Replication Occurs in Two Stages

    PubMed Central

    Goorha, R.

    1982-01-01

    Viral DNA synthesis in frog virus 3 (FV3)-infected cells occurs both in the nucleus and in the cytoplasm (Goorha et al., Virology 84:32-51, 1978). Relationships between viral DNA molecules synthesized in these two compartments and their role in the virus replication were examined. The data presented here suggest that (i) FV3 DNA replicated in two stages and (ii) nucleus and cytoplasm were the sites of stages 1 and 2 of DNA replication, respectively. Stages 1 and 2 were further distinguished by their temporal appearance during infection and by the sizes of the replicating DNA as determined by sedimentation in neutral sucrose gradients. In stage 1, replicating molecules, between the size of unit and twice the unit length, were produced early in infection (2 h postinfection). In contrast, stage 2 of DNA replication occurred only after 3 h postinfection, and replicating molecules were large concatemers. Results of pulse-chase experiments showed that the concatemeric DNA served as the precursor for the production of mature FV3 DNA. Denaturation of concatemeric DNA with alkali or digestion with S1 nuclease reduced it to less than genome size molecules, indicating the presence of extensive single-stranded regions. Analysis of replicating DNA by equilibrium centrifugation in CsCl gradients after a pulse-chase suggested that these single-stranded regions were subsequently repaired. Based on these and previous data, a scheme of FV3 replication is presented. According to this scheme, FV3 utilizes the nucleus for early transcription and stage 1 of DNA replication. The viral DNA is then transported to the cytoplasm, where it participates in stage 2 DNA replication to form a concatemeric replication complex. The processing of concatemers to produce mature viral DNA and virus assembly also occurs in the cytoplasm. This mode of replication is strikingly different from any other known DNA virus. PMID:7109033

  14. Viral hemorrhagic fevers of animals caused by DNA viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  15. 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.

  16. 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.

  17. The role of respiratory viruses in the etiology of bacterial pneumonia: An ecological perspective.

    PubMed

    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

  18. 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

  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. 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

  1. The circular intracellular form of Epstein-Barr virus DNA is amplified by the virus-associated DNA polymerase.

    PubMed Central

    Shaw, J E

    1985-01-01

    Selective DNA extraction and hybridization procedures were used to estimate the relative number of covalently closed circular viral genomes in cultures of Epstein-Barr virus (EBV)-transformed cells. In virus-producing P3HR-1 cultures that were exposed for 11 days to phosphonoacetic acid or to acyclovir, the content of covalently closed circular EBV DNA was reduced ca. 70% relative to a control culture without drug. The EBV plasmid content of Raji, a virus nonproducer cell line, was not reduced by exposure to these compounds. When P3HR-1 cultures were exposed to 12-O-tetradecanoylphorbol-13-acetate, the number of circular genomes per cell increased. These findings indicate that two enzyme activities synthesize circular EBV DNA and that the virus-associated DNA polymerase synthesizes most of the circular EBV DNA in a virus producer culture. It is suggested that the circular genomes synthesized by the viral enzyme are intermediates in the syntheses of linear virus DNA. PMID:2983082

  2. Examination of Bacterial Inhibition Using a Catalytic DNA

    PubMed Central

    Qu, Long; Ali, M. Monsur; Aguirre, Sergio D.; Liu, Hongxia; Jiang, Yuyang; Li, Yingfu

    2014-01-01

    Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA), RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC) of antibiotics much faster than the conventional optical density (OD) method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research. PMID:25531274

  3. The role of bacterial chaperones in the circulative transmission of plant viruses by insect vectors.

    PubMed

    Kliot, Adi; Ghanim, Murad

    2013-06-01

    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

  4. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. 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.

  7. Taxonomic distribution of large DNA viruses in the sea

    PubMed Central

    Monier, Adam; Claverie, Jean-Michel; Ogata, Hiroyuki

    2008-01-01

    Background Viruses are ubiquitous and the most abundant biological entities in marine environments. Metagenomics studies are increasingly revealing the huge genetic diversity of marine viruses. In this study, we used a new approach - 'phylogenetic mapping' - to obtain a comprehensive picture of the taxonomic distribution of large DNA viruses represented in the Sorcerer II Global Ocean Sampling Expedition metagenomic data set. Results Using DNA polymerase genes as a taxonomic marker, we identified 811 homologous sequences of likely viral origin. As expected, most of these sequences corresponded to phages. Interestingly, the second largest viral group corresponded to that containing mimivirus and three related algal viruses. We also identified several DNA polymerase homologs closely related to Asfarviridae, a viral family poorly represented among isolated viruses and, until now, limited to terrestrial animal hosts. Finally, our approach allowed the identification of a new combination of genes in 'viral-like' sequences. Conclusion Albeit only recently discovered, giant viruses of the Mimiviridae family appear to constitute a diverse, quantitatively important and ubiquitous component of the population of large eukaryotic DNA viruses in the sea. PMID:18598358

  8. 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.

  9. 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. PMID:25697886

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. DNA signature-based approaches for bacterial detection and identification.

    PubMed

    Albuquerque, Pedro; Mendes, Marta V; Santos, Catarina L; Moradas-Ferreira, Pedro; Tavares, Fernando

    2009-06-01

    During the late eighties, environmental microbiologists realized the potential of the polymerase chain reaction (PCR) for the design of innovative approaches to study microbial communities or to detect and identify microorganisms in diverse and complex environments. In contrast to long-established methods of cultivation-based microbial identification, PCR-based techniques allow for the identification of microorganisms regardless of their culturability. A large number of reports have been published that describe PCR-inspired methods, frequently complemented by sequencing or hybridization profiling, to infer taxonomic and clonal microbial diversity or to detect and identify microorganisms using taxa-specific genomic markers. Typing methods have been particularly useful for microbial ecology-driven studies; however, they are not suitable for diagnostic purposes, such as the detection of specific species, strains or clones. Recently, comprehensive reviews have been written describing the panoply of typing methods available and describing their advantages and limitations; however, molecular approaches for bacterial detection and identification were either not considered or only vaguely discussed. This review focuses on DNA-based methods for bacterial detection and identification, highlighting strategies for selecting taxa-specific loci and emphasizing the molecular techniques and emerging technological solutions for increasing the detection specificity and sensitivity. The massive and increasing number of available bacterial sequences in databases, together with already employed bioinformatics tools, hold promise of more reliable, fast and cost-effective methods for bacterial identification in a wide range of samples in coming years. This tendency will foster the validation and certification of these methods and their routine implementation by certified diagnostic laboratories.

  15. Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

    PubMed Central

    Yoo, Wonseok; Lim, Dongbin

    2016-01-01

    A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA. PMID:27103888

  16. 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

  17. 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.

  18. Virus-coded DNA endonuclease from avian retrovirus.

    PubMed Central

    Golomb, M; Grandgenett, D P; Mason, W

    1981-01-01

    Reverse transcriptase from avian retrovirus has a physically associated DNA endonuclease with novel substrate and cofactor requirements. A similar endonuclease activity copurifies with pp32, a protein from viral cores that has been identified with the non-alpha region of the beta subunit of reverse transcriptase. Several temperature-sensitive mutants of avian retrovirus with thermolabile DNA polymerase were tested for thermal sensitivity of their DNA endonuclease activity. Two pol mutants of Rous sarcoma virus, ts335 and ts337, had thermolabile DNA endonuclease; a temperature-resistant revertant of ts335 had a heat-stable DNA endonuclease. DNA endonuclease is therefore a product of the pol gene and an integral part of the reverse transcriptase. A second class of pol mutants, typified by ts568 and ts553, had thermolabile DNA polymerase, but heat-stable DNA endonuclease. PMID:6165835

  19. Negatively supercoiled DNA from plants infected with a single-stranded DNA virus.

    PubMed

    Sunter, G; Coutts, R H; Buck, K W

    1984-02-14

    A method for isolating covalently closed circular double-stranded DNA from plants infected with the geminivirus, tomato golden mosaic virus, is described. Ethidium bromide titration showed this DNA to be negatively supercoiled with a superhelical density of -0.062. The presence of S1 nuclease-sensitive secondary structure in the supercoiled DNA was demonstrated by its conversion to the open circular and linear DNA forms on treatment with this enzyme.

  20. Shared catalysis in virus entry and bacterial cell wall depolymerization

    PubMed Central

    Cohen, Daniel N.; Sham, Yuk Y.; Haugstad, Greg D.; Xiang, Ye; Rossmann, Michael G.; Anderson, Dwight L.; Popham, David L.

    2009-01-01

    Summary Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active site residues and a structural pattern of β-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage φ29. gp13 activity on PG and muropeptides was assayed using high performance liquid chromatography, and gp13 was found to be a D,D-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile bond activation and His247 is oriented to mediate nucleophile generation. This is the first model of a Zn2+-metallopeptidase and its substrate to our knowledge. Residue Asp195 of gp13 was found to be critical for Zn2+-binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle induced X-ray emission spectroscopy showed that the general protein folding and Zn2+-binding was maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model where the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage entry enzymes have a shared chemical mechanism of action. PMID:19361422

  1. Helper-independent transformation by unintegrated Harvey sarcoma virus DNA.

    PubMed

    Lowy, D R; Rands, E; Scolnick, E M

    1978-05-01

    We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins.

  2. 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.

  3. 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

  4. 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. PMID:26884161

  5. Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them.

    PubMed Central

    Frenkel, N; Jacob, R J; Honess, R W; Hayward, G S; Locker, H; Roizman, B

    1975-01-01

    We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious virus yields declined progressively in passages 1 through 10 and gradually increased at passages 11 through 14. The yields correlated with PFU/particle ratios. (ii) In cells infected with virus from passages 6 through 10, there was an overproduction of an early viral polypeptide (no. 4) and a delay in the synthesis of late viral proteins. In addition, the virus in these passages interfered with the replication of a nondefective marker virus. Cells infected with passage 14 virus produced normal amounts of polypeptide 4 and, moreover, this virus showed minimal interfering capacity. (iii) In addition to DNA of density 1.726 g/cm-3, which was the sole component present in viral progeny of passage 0, passages 6 through 14 contained one additional species (p 1.732) and in some instances (passages 6 and 10) also DNA of an intermediate buoyant density. The ratio of p 1.732 to p 1.726 DNA increased to a maximum of 4 in passages 6 through 9 and gradually decreased to 1 in passages 10 through 14. (iv) p 1.732 DNA cannot be differentiated from p 1.726 DNA with respect to size; however, it has no Hin III restriction enzyme cleavage sites and yields only predominantly two kinds of fragments with molecular weights of 5.1 x 10-6 and 5.4 x 10-6 upon digestion with EcoRI enzyme. (v) Partial denaturation profiles of purified p 1.732 DNA from passage 14 revealed the presence of two types of tandemly repeated units corresponding roughly in size to the EcoRI fragments and situated in different molecules. (vi) In addition to the two kinds of p 1.732 molecules consisting of tandem repaeat units of different sizes, other evidence for the diversity of defective DNA molecules emerged from comparisons of specific infectivity and interfering capacity of the progeny from various passages. The data suggest that some of the particles

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. DNA profiling of complex bacterial populations: toxic cyanobacterial blooms.

    PubMed

    Saker, Martin; Moreira, Cristiana; Martins, Joana; Neilan, Brett; Vasconcelos, Vitor Manuel

    2009-11-01

    Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.

  11. Interactions of Dnd proteins involved in bacterial DNA phosphorothioate modification

    PubMed Central

    Xiong, Wei; Zhao, Gong; Yu, Hao; He, Xinyi

    2015-01-01

    DNA phosphorothioation (PT) is the first discovered physiological DNA backbone modification, in which a non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom in Rp (rectus for plane) configuration. PT modification is governed by a highly conserved gene cluster dndA/iscS-dndBCDE that is widespread across bacterial and archaeal species. However, little is known about how these proteins coordinately react with each other to perform oxygen–sulfur swap. We here demonstrated that IscS, DndC, DndD and DndE form a protein complex of which the molecular ratio for four proteins in the complex is approximate 1:1:1:1. DndB here displayed little or weak affinity to the complex and the constructs harboring dndACDE can confer the host in vivo PT modification. Using co-purification and pull down strategy, we demonstrated that the four proteins assemble into a pipeline in collinear to its gene organization, namely, IscS binding to DndC, DndC binding to DndD, and DndD binding to DndE. Moreover, weak interactions between DndE and IscS, DndE and DndC were also identified. PMID:26539172

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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. PMID:21988358

  20. 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.

  1. Biodiversity and distribution of polar freshwater DNA viruses.

    PubMed

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

    2015-06-01

    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

  2. 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.

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

    DOE PAGES

    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 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

  4. 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

  5. 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

  6. 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

  7. The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery.

    PubMed

    Bronkhorst, Alfred W; van Cleef, Koen W R; Vodovar, Nicolas; Ince, Ikbal Agah; Blanc, Hervé; Vlak, Just M; Saleh, Maria-Carla; van Rij, Ronald P

    2012-12-18

    RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses. PMID:23151511

  8. 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

  9. Bacterial DNA Content in the Intestinal Wall from Infants with Necrotizing Enterocolitis

    PubMed Central

    Bucher, Brian T.; McDuffie, Lucas A.; Shaikh, Nurmohammad; Tarr, Phillip I.; Warner, Barbara B.; Hamvas, Aaron; White, Francis V.; Erwin, Christopher R.; Warner, Brad W.

    2011-01-01

    PURPOSE The objectives of our study were to quantify mucosal bacterial DNA within specimens from neonates undergoing small bowel resection for necrotizing enterocolitis (NEC). METHODS We obtained clinical information and pathologic specimens from all infants diagnosed with NEC who underwent surgical treatment at our institution from 1999–2008. Bacterial and human DNA were isolated from paraffin-embedded surgical specimens and real-time PCR was used to amplify bacterial and human genes. Linear regression was used to quantify the amount of human and bacterial DNA in our specimens. RESULTS From a cohort of fifty infants, we identified twenty-three infants who underwent both surgical resection and subsequent intestinal reanastomosis. Thirteen (59%) of the neonates had Bell's Stage III NEC, and nine (41%) had Stage II. There was significantly more bacterial DNA in the resection specimens than in the reanastomosis specimens. This corresponds to a median (IQR) increase of 1.81 (1.11–4.69) fold bacterial DNA in the resection specimen compared to the reanastomosis specimen (p<0.05). CONCLUSION There is more bacterial DNA in infants with acute NEC compared with the same infants after the NEC had clinically resolved. These findings underscore the potential relevance of adherent or invasive bacteria across the bowel wall in the pathogenesis of NEC. PMID:21683193

  10. 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

  11. 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.

  12. 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

    2015-01-01

    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

  13. 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

  14. 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.

  15. 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

    2015-01-01

    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.

  16. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.

  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. Genomic DNA fingerprint analysis of biotype 1 Gardnerella vaginalis from patients with and without bacterial vaginosis.

    PubMed Central

    Wu, S R; Hillier, S L; Nath, K

    1996-01-01

    Of the 20 biotype 1 Gardnerella vaginalis isolates analyzed, 10 from patients with bacterial vaginosis and 10 from patients without bacterial vaginosis, none shared the same DNA fingerprint. However, a 1.18-kb HindIII fragment was common among 18 of the 20 biotype 1 isolates in a restriction fragment length polymorphism analysis with a 7.9-kb G. vaginalis DNA probe. PMID:8748302

  19. Blocking primers reduce co-amplification of plant DNA when studying bacterial endophyte communities.

    PubMed

    Arenz, Brett E; Schlatter, Dan C; Bradeen, James M; Kinkel, Linda L

    2015-10-01

    A blocking primer set based on the technique described by Vestheim and Jarman (2008) was developed to reduce amplification of non-target plant DNA when conducting metagenomic studies on bacterial endophyte communities. Bacterial amplification efficiency was increased 300-fold compared to standard PCR in an Illumina-based study of Sorghastrum nutans leaves.

  20. 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.

  1. 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.

  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. 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

  4. 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

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

    PubMed

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

    2016-02-01

    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

  6. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Quantification of total phosphorothioate in bacterial DNA by a bromoimane-based fluorescent method.

    PubMed

    Xiao, Lu; Xiang, Yu

    2016-06-01

    The discovery of phosphorothioate (PT) modifications in bacterial DNA has challenged our understanding of conserved phosphodiester backbone structure of cellular DNA. This exclusive DNA modification in bacteria is not found in animal cells yet, and its biological function in bacteria is still poorly understood. Quantitative information about the bacterial PT modifications is thus important for the investigation of their possible biological functions. In this study, we have developed a simple fluorescence method for selective quantification of total PTs in bacterial DNA, based on fluorescent labeling of PTs and subsequent release of the labeled fluorophores for absolute quantification. The method was highly selective to PTs and not interfered by the presence of reactive small molecules or proteins. The quantification of PTs in an E. coli DNA sample was successfully achieved using our method and gave a result of about 455 PTs per million DNA nucleotides, while almost no detectable PTs were found in a mammalian calf thymus DNA. With this new method, the content of phosphorothioate in bacterial DNA could be successfully quantified, serving as a simple method suitable for routine use in biological phosphorothioate related studies. PMID:27168171

  9. 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. PMID:26733971

  10. 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

  11. A sequential study of serum bacterial DNA in patients with advanced cirrhosis and ascites.

    PubMed

    Francés, Rubén; Benlloch, Susana; Zapater, Pedro; González, José M; Lozano, Beatriz; Muñoz, Carlos; Pascual, Sonia; Casellas, Juan A; Uceda, Francisco; Palazón, José M; Carnicer, Fernando; Pérez-Mateo, Miguel; Such, José

    2004-02-01

    Bacterial translocation is currently considered the main pathogenic mechanism leading to spontaneous bacterial peritonitis in patients with advanced cirrhosis and ascites. However, to the authors' knowledge there is no information regarding the characteristics of this process in humans. The goals of the current study were to pursue partially identified bacterial DNA in blood (what the authors consider molecular evidence of bacterial translocation) through its relative quantification in a 72-hour study period by using real-time polymerase chain reaction (PCR). A consecutive series of 17 patients with advanced cirrhosis and culture-negative, nonneutrocytic ascites were studied. Therapeutic paracentesis was performed at the time of admission, and blood samples were obtained at baseline and every 8 hours in a 3-day period. Bacterial DNA was detected by a PCR-based method, relatively quantified by real-time PCR, and identified by automated nucleotide sequencing. Seven of 17 patients demonstrated the simultaneous presence of bacterial DNA in blood and ascitic fluid at the time of admission. After therapeutic paracentesis was performed, bacterial DNA persisted in the blood for a minimum of 24 hours, and was reported to last as long as 72 hours in some patients. In addition, different patterns of bacterial DNA appearance and clearance from the blood were identified. The nucleotide sequencing process demonstrated that bacteria detected in the first sample were identical to those noted in subsequent detections over time. In conclusion, bacterial translocation is a single-species, dynamic process that appears to develop in a subgroup of patients with advanced cirrhosis.

  12. Definition of a bacterial type IV secretion pathway for a DNA substrate.

    PubMed

    Cascales, Eric; Christie, Peter J

    2004-05-21

    Bacteria use conjugation systems, a subfamily of the type IV secretion systems, to transfer DNA to recipient cells. Despite 50 years of research, the architecture and mechanism of action of the channel mediating DNA transfer across the bacterial cell envelope remains obscure. By use of a sensitive, quantifiable assay termed transfer DNA immunoprecipitation (TrIP), we identify contacts between a DNA substrate (T-DNA) and 6 of 12 components of the VirB/D4 conjugation system of the phytopathogen Agrobacterium tumefaciens. Our results define the translocation pathway for a DNA substrate through a bacterial conjugation machine, specifying the contributions of each subunit of the secretory apparatus to substrate passage. PMID:15155952

  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. 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.

  15. 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. PMID:25875109

  16. Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

    PubMed

    Marchionni, M A; Roufa, D J

    1981-04-01

    Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

  17. Influenza A virus and secondary bacterial infection in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Influenza A virus (IAV) infection alone causes significant disease characterized by respiratory distress and poor growth in pigs. Endemic strains of IAV in North America pigs consist of the subtypes H1N1, H1N2, and H3N2. These circulating strains contain the triple reassortant internal gene (TRIG) c...

  18. Plasma Levels of Bacterial DNA Correlate with Immune Activation and the Magnitude of Immune Restoration in Persons with Antiretroviral-Treated HIV Infection

    PubMed Central

    Jiang, Wei; Lederman, Michael M.; Hunt, Peter; Sieg, Scott F.; Haley, Kathryn; Rodriguez, Benigno; Landay, Alan; Martin, Jeffrey; Sinclair, Elizabeth; Asher, Ava I.; Deeks, Steven G.; Douek, Daniel C.; Brenchley, Jason M.

    2009-01-01

    The significance of elevated plasma levels of bacterial lipopolysaccharide (LPS) in persons with chronic HIV infection remains undefined. We measured LPS levels by use of limulus lysate assay, and DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) were assessed by quantitative polymerase chain reactions in plasma samples obtained from 242 donors. Plasma levels of 16S rDNA were significantly higher in human immunodeficiency virus (HIV)–infected subjects than in uninfected subjects, and they correlated with LPS levels. Higher levels of 16S rDNA were associated with higher levels of T cell activation and with lower levels of CD4 T cell restoration during antiretroviral therapy. Antiretroviral therapy reduces but does not fully normalize plasma levels of bacterial 16S rDNA, an index of microbial translocation from the gastrointestinal tract. High levels of 16S rDNA during therapy are strongly associated with reduced increases in the CD4+ T lymphocyte count, irrespective of plasma HIV RNA levels. These findings are consistent with the importance of microbial translocation in immunodeficiency and T cell homeostasis in chronic HIV infection. PMID:19265479

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

    PubMed

    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.

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

    PubMed

    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

  1. 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

  2. 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  .

  3. Genomoviridae: a new family of widespread single-stranded DNA viruses.

    PubMed

    Krupovic, Mart; Ghabrial, Said A; Jiang, Daohong; Varsani, Arvind

    2016-09-01

    Here, we introduce a new family of eukaryote-infecting single-stranded (ss) DNA viruses that was created recently by the International Committee on Taxonomy of Viruses (ICTV). The family, named Genomoviridae, contains a single genus, Gemycircularvirus, which currently has one recognized virus species, Sclerotinia gemycircularvirus 1. Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) is currently the sole representative isolate of the family; however, a great number of SsHADV-1-like ssDNA virus genomes has been sequenced from various environmental, plant- and animal-associated samples, indicating that members of family Genomoviridae are widespread and abundant in the environment. PMID:27343045

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

    PubMed Central

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

    2015-01-01

    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 20th century and provides the first evidence of the suitability of bone for exploration of DNA viruses. PMID:26611279

  5. 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.

  6. 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

  7. Ty virus-like particles, DNA vaccines and Modified Vaccinia Virus Ankara; comparisons and combinations.

    PubMed

    Gilbert, S C; Schneider, J; Plebanski, M; Hannan, C M; Blanchard, T J; Smith, G L; Hill, A V

    1999-03-01

    Three types of vaccine, all expressing the same antigen from Plasmodium berghei, or a CD8+ T cell epitope from that antigen, were compared for their ability to induce CD8+ T cell responses in mice. Higher levels of lysis and numbers of IFN-gamma secreting T cells were primed with Ty virus-like particles and Modified Vaccinia Virus Ankara (MVA) than with DNA vaccines, but none of the vaccines were able to protect immunised mice from infectious challenge even after repeated doses. However, when the immune response was primed with one type of vaccine (Ty-VLPs or DNA) and boosted with another (MVA) complete protection against infection was achieved. Protection correlated with very high levels of IFN-gamma secreting T cells and lysis. This method of vaccination uses delivery systems and routes that can be used in humans and could provide a generally applicable regime for the induction of high levels of CD8+ T cells.

  8. 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

  9. 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.

  10. 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.

  11. 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

    2016-03-01

    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. PMID:26962942

  12. 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

    2016-03-01

    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.

  13. Release of Bacterial DNA by Marine Nanoflagellates, an Intermediate Step in Phosphorus Regeneration

    PubMed Central

    Turk, Valentina; Rehnstam, Ann-Sofi; Lundberg, Erik; Hagström, Åke

    1992-01-01

    The concentrations of dissolved DNA and nanoflagellates were found to covary during a study of diel dynamics of the microbial food web in the Adriatic Sea. This observation was further investigated in a continuous seawater culture when nanoflagellates were fed bacteria grown in filtered seawater. Analysis of dissolved organic phosphorus and dissolved DNA showed a sixfold increase of dissolved DNA in the presence of the nanoflagellates (Ochromonas sp.). The amount of DNA released suggested that the majority of the consumed bacterial DNA was ejected. Phagotrophic nanoflagellates thus represent an important source of origin for dissolved DNA. The rate of breakdown of dissolved DNA and release of inorganic phosphorus in the pelagic ecosystem is suggested to be dependent on the ambient phosphate pool. In the P-limited northern Adriatic Sea, rapid degradation of the labelled DNA could be demonstrated, whereas the N-limited southern California bight water showed a much lower rate. Phosphorus originating from dissolved DNA was shown to be transferred mainly to organisms in the <3-μm-size fractions. On the basis of the C/P ratios, we suggest that a significant fraction of the phosphorus demand by the autotrophs may be sustained by the released DNA during stratified conditions. Thus, the nucleic acid-rich bacterial biomass grazed by protozoa plays an important role in the biogeochemical cycling of phosphorus in the marine environment. PMID:16348813

  14. Regulation of Bacterial DNA Packaging in Early Stationary Phase by Competitive DNA Binding of Dps and IHF

    PubMed Central

    Lee, Sin Yi; Lim, Ci Ji; Dröge, Peter; Yan, Jie

    2015-01-01

    The bacterial nucleoid, a bacterial genome packed by nucleoid binding proteins, forms the physical basis for cellular processes such as gene transcription and DNA replication. Bacteria need to dynamically modulate their nucleoid structures at different growth phases and in response to environmental changes. At the nutrients deficient stationary phase, DNA-binding proteins from starved cells (Dps) and Integration host factors (IHF) are the two most abundant nucleoid associated proteins in E. coli. Yet, it remains unclear how the nucleoid architecture is controlled by the interplay between these two proteins, as well as the nucleoid’s response to environmental changes. This question is addressed here using single DNA manipulation approach. Our results reveal that the two proteins are differentially selected for DNA binding, which can be tuned by changing environmental factors over physiological ranges including KCl (50–300 mM), MgCl2 (0–10 mM), pH (6.5–8.5) and temperature (23–37 °C). Increasing pH and MgCl2 concentrations switch from Dps-binding to IHF-binding. Stable Dps-DNA and IHF-DNA complexes are insensitive to temperature changes for the range tested. The environment dependent selection between IHF and Dps results in different physical organizations of DNA. Overall, our findings provide important insights into E. coli nucleoid architecture. PMID:26657062

  15. 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. PMID:25979733

  16. 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.

  17. 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. PMID:24462907

  18. 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

  19. 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

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

    PubMed

    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

  1. 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

  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. 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…

  4. Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites.

    PubMed

    Such, José; Francés, Rubén; Muñoz, Carlos; Zapater, Pedro; Casellas, Juan A; Cifuentes, Ana; Rodríguez-Valera, Francisco; Pascual, Sonia; Sola-Vera, Javier; Carnicer, Fernando; Uceda, Francisco; Palazón, José M; Pérez-Mateo, Miguel

    2002-07-01

    The current pathogenic theory of spontaneous bacterial peritonitis (SBP) in patients with cirrhosis and ascites suggests that repeated episodes of bacterial translocation (BT) from intestinal lumen to mesenteric lymph nodes followed by systemic seeding are the key steps for the final development of infectious events. However, most of the episodes of systemic bacterial circulation remain undetected. Therefore, we investigated the hypothetical presence of bacteria in blood and/or ascitic fluid (AF) from patients with cirrhosis and sterile (culture negative) AF by means of bacterial DNA (bactDNA) detection and identification. Twenty-eight consecutively admitted patients with cirrhosis and presence of AF were included in the study. BactDNA was detected using a polymerase chain reaction (PCR)-based method. The corresponding bacteria were identified by nucleotide sequencing of purified PCR products. BactDNA was detected simultaneously in blood and AF in 9 patients (32.1%). DNA sequencing allowed the identification of Escherichia coli (n = 7) and Staphylococcus aureus (n = 2). In all cases, the similarity between the sequence found in AF and blood indicated that the bactDNA present in both locations originated from a single clone (single translocation event). Child-Pugh score and basic hemodynamic, clinical, endoscopic, and biochemical characteristics were similar among patients with or without the presence of bactDNA. In conclusion, we have detected bactDNA in serum and AF in 32% of all patients studied, and this likely represents single clone episodes of translocation and systemic seeding. E. coli is the most frequently identified bacteria.

  5. 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. PMID:24900632

  6. Fragment-Based Discovery of 6-Azaindazoles As Inhibitors of Bacterial DNA Ligase

    PubMed Central

    2013-01-01

    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. PMID:24900632

  7. 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.

  8. 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.

  9. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. DNA methylation pattern of CALCA in preterm neonates with bacterial sepsis as a putative epigenetic biomarker.

    PubMed

    Tendl, Kristina A; Schulz, Stefan M F; Mechtler, Thomas P; Bohn, Adele; Metz, Thomas; Greber-Platzer, Susanne; Kasper, David C; Herkner, Kurt R; Item, Chike B

    2013-12-01

    Diagnosis of bacterial sepsis in preterm neonates can be difficult when using serum markers that rely on physiological changes because these changes may not necessarily be the result of bacterial infections alone. This retrospective investigation explores the potential use of the DNA methylation pattern of CpG sites in the promoter region of the calcitonin-related polypeptide α (CALCA) gene as an epigenetic biomarker for bacterial sepsis in preterm newborns. Four novel changes in the DNA methylation of eight CpG sites were detected in this gene and are present only in neonates with bacterial sepsis: (1) partial methylation at -769 CpG in gram-negative or gram-positive early onset sepsis (EOS) and late onset sepsis (LOS) episodes; (2) demethylation of 8 CpGs in gram-negative EOS followed by LOS (ELS) and in gram-negative EOS; (3) demethylation of 7 CpGs in gram-positive ELS and gram-positive EOS; (4) -771 C:G>T:A; 5' de novo -778 CpG mutation on both alleles in EOS. These changes were not detected in birth weight and gestational age matched controls or in newborns with isolated infections. Our results indicate that the DNA methylation pattern of the promoter region of the CALCA gene varies in different types of bacterial preterm sepsis, thus suggesting a potential use as an epigenetic biomarker. A prospective confirmation of these results is essential.

  11. 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

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... 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.”...

  12. 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

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... 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.”...

  13. 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

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... 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.”...

  14. 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

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... 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.”...

  15. Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

    PubMed

    Brutlag, D; Fry, K; Nelson, T; Hung, P

    1977-03-01

    Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes. PMID:403010

  16. Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine

    PubMed Central

    BAO, LIDAO; WEI, GUOMIN; GAN, HONGMEI; REN, XIANHUA; MA, RUILIAN; WANG, YI; LV, HAIJUN

    2016-01-01

    In the present study a eukaryotic expression vector of varicella zoster virus (VZV) glycoprotein E (gE) was constructed and enabled to express in COS7 cells. Furthermore, a specific immune response against the VZV gE eukaryotic expression plasmid was induced in BALB/c mice. The VZV gE gene was amplified using polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector, pcDNA3.1. The recombinant vector was subsequently transfected into COS7 cells using a liposome transfection reagent. The recombinant protein was instantaneously expressed by the transfected cells, as detected by immunohistochemistry, and the recombinant pcDNA-VZV gE plasmid was subsequently used to immunize mice. Tissue expression levels were analyzed by reverse transcription-PCR. In addition, the levels of serum antibodies and spleen lymphocyte proliferation activity were investigated. The amplified target gene included the full-length gE gene (~2.7 kb), and the recombinant expression vector induced gE expression in COS7 cells. In addition, the expression plasmid induced sustained expression in vivo following immunization of mice. Furthermore, the plasmid was capable of inducing specific antibody production and effectively stimulating T cell proliferation. Effective humoral and cellular immunity was triggered in the mice immunized with the VZV gE eukaryotic expression vector. The results of the present study laid the foundation for future research into a VZV DNA vaccine. PMID:27168804

  17. 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.

  18. Construction and gene expression analysis of a single-stranded DNA minivector based on an inverted terminal repeat of adeno-associated virus.

    PubMed

    Ping, Han; Liu, Xiaomei; Zhu, Dongqin; Li, Taiming; Zhang, Chun

    2015-04-01

    The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid.

  19. Application of silica magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan; Chen, Chuh-Yean

    2006-10-01

    The aim of this study was to develop a simple and rapid method for purification of ultrapure plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical precipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. Silica-magnetite nanocomposites were prepared by the method of acid hydrolysis of tetraethoxysilane (TEOS) to coat the silica onto magnetite nanoparticles. DNA was adsorbed to the support under high salt conditions, and recovered directly in water for immediate downstream application, without the need for precipitation. We demonstrated that a useful plasmid, pRSETB-EGFP, encoding for the green fluorescent protein with T7 promoter, could be amplified in Escherichia coli of DE3 strain. Up to approximately 43 μg of high-purity ( A260/ A280 ratio=1.75) plasmid DNA was isolated from 3 ml of an overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and polymerase chain reaction (PCR) amplification with success. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmid takes less than 8 min. The silica-magnetite nanocomposites deliver significant time-savings, overall higher yields, lower RNA contamination, and better PCR amplification compared to commercial available silica-based and other methods.

  20. Effects of Bacterial Microflora of the Lower Digestive Tract of Free-Range Waterfowl on Influenza Virus Activation ▿

    PubMed Central

    King, Marcus D.; Guentzel, M. Neal; Arulanandam, Bernard P.; Bodour, Adria A.; Brahmakshatriya, Vinayak; Lupiani, Blanca; Chambers, James P.

    2011-01-01

    Proteolytic cleavage activation of influenza virus hemagglutinin (HA0) is required for cell entry via receptor-mediated endocytosis. Despite numerous studies describing bacterial protease-mediated influenza A viral activation in mammals, very little is known about the role of intestinal bacterial flora of birds in hemagglutinin cleavage/activation. Therefore, the cloaca of wild waterfowl was examined for (i) representative bacterial types and (ii) their ability to cleave in a “trypsin-like” manner the precursor viral hemagglutinin molecule (HA0). Using radiolabeled HA0, bacterial secretion-mediated trypsin-like conversion of HA0 to HA1 and HA2 peptide products was observed to various degrees in 42 of 44 bacterial isolates suggestive of influenza virus activation in the cloaca of wild waterfowl. However, treatment of uncleaved virus with all bacterial isolates gave rise to substantially reduced emergent virus progeny compared with what was expected. Examination of two isolates exhibiting pronounced trypsin-like conversion of HA0 to HA1 and HA2 peptide products and low infectivity revealed lipase activity to be present. Because influenza virus possesses a complex lipid envelope, the presence of lipid hydrolase activity could in part account for the observed less-than-expected level of viable progeny. A thorough characterization of respective isolate protease HA0 hydrolysis products as well as other resident activities (i.e., lipase) is ongoing such that the role of these respective contributors in virus activation/inactivation can be firmly established. PMID:21531837

  1. 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

  2. Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota

    PubMed Central

    Gill, Christina; Blow, Frances; Darby, Alistair C.

    2016-01-01

    Background 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. Results 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. Conclusions 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

  3. 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

  4. A Novel AT-Rich DNA Recognition Mechanism for Bacterial Xenogeneic Silencer MvaT

    PubMed Central

    Ding, Pengfei; McFarland, Kirsty A.; Jin, Shujuan; Tong, Grace; Duan, Bo; Yang, Ally; Hughes, Timothy R.; Liu, Jun; Dove, Simon L.; Navarre, William Wiley; Xia, Bin

    2015-01-01

    Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an “AT-pincer” motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA. PMID:26068099

  5. 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.

  6. Bacterial conjugation protein MobA mediates integration of complex DNA structures into plant cells.

    PubMed

    Bravo-Angel, A M; Gloeckler, V; Hohn, B; Tinland, B

    1999-09-01

    Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end. Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins. PMID:10482518

  7. 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.

  8. 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.

  9. 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. PMID:26914280

  10. Transcriptional mapping of the DNA polymerase gene of vaccinia virus

    SciTech Connect

    Traktman, P.; Sridhar, P.; Condit, R.C.; Roberts, B.E.

    1984-01-01

    Vaccinia virus DNA polymerase, a single-subunit enzyme of 110,000 molecular weight, is induced early after infection. Genetic analysis suggests that the gene encoding the enzyme maps within a 15-kilobase HindIII fragment located 45 kilobases from the left-hand end of the genome. The authors identified the in vitro translation product with these propeties and mapped the transcript by hybrid selection, RNA filter hybridization, and S1 nuclease mapping. Two mRNAs from this region, 3.4 and 3.9 kilobases in size, could be translated in vitro to yield a 110K polypeptide. The two RNAs shared a common 5' terminus and had staggered 3' ends. Sequences mapping entirely within the gene were shown to be biologically active in rescuing mutants with temperature-sensitive or drug-resistant polymerase activity to the wild-type phenotype.

  11. Molecular Identification of Bacterial DNA in the Chorioretinal Scars of Chronic Granulomatous Disease

    PubMed Central

    Wang, Yujuan; Marciano, Beatriz E.; Shen, Defen; Bishop, Rachel J.; Park, Stanley; Holland, Steven M.; Chan, Chi-Chao

    2013-01-01

    Purpose Chronic granulomatous disease (CGD) is an inherited disorder characterized by defects in phagocyte-derived nicotinamide adenine dinucleotide phosphate oxidase. It is typically diagnosed in childhood and leads to severe, recurrent bacterial or fungal infections. Chorioretinal lesions are the most common ocular manifestation. We sought to determine whether there are infectious agents in CGD-associated chorioretinopathy. Methods Medical records and ocular histopathology from CGD cases from January 1983 to January 2012 at the National Institutes of Health were retrospectively reviewed. Chorioretinal cells from normal and lesional tissues of the same eye were microdissected. Primers for Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia sp., and a panbacterial 16S ribosomal DNA were used for polymerase chain reaction. Results Seventeen CGD patients had ocular tissues (16 autopsied cases and 1 chorioretinal biopsy) examined. Of these 17, 8 demonstrated CGD-associated chorioretinal lesions in at least one eye on histopathology. Of these 8, 7 showed amplification of 16S ribosomal DNA within the lesion; of these 7, two also amplified S. epidermidis and one P. aeruginosa. One had no bacterial DNA amplified. Importantly, no microbial DNA was amplified from the normal, non-lesional ocular tissues of these 8 cases. Furthermore, only 1 of the 9 eyes without chorioretinopathy had amplified Burkholderia DNA, that patient had a history of Burkholderia infection. Conclusions We detected bacterial DNA in 7 of 8 (88 %) cases with CGD-associated chorioretinopathy and only in 1 normal ocular tissue of 17 CGD cases. Bacterial infection may play a role in the pathogenesis of CGD-associated chorioretinal lesions. PMID:23636897

  12. 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

  13. 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.

  14. Previously unknown and highly divergent ssDNA viruses populate the oceans

    PubMed Central

    Labonté, Jessica M; Suttle, Curtis A

    2013-01-01

    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. PMID:23842650

  15. Formation of Hepatitis B Virus Covalently Closed Circular DNA: Removal of Genome-Linked Protein▿

    PubMed Central

    Gao, Weifan; Hu, Jianming

    2007-01-01

    Hepatitis B virus (HBV) contains a small, partially double-stranded, relaxed circular (RC) DNA genome. RC DNA needs to be converted to covalently closed circular (CCC) DNA, which serves as the template for all viral RNA transcription. As a first step toward understanding how CCC DNA is formed, we analyzed the viral and host factors that may be involved in CCC DNA formation, using transient and stable DNA transfections of HBV and the related avian hepadnavirus, duck hepatitis B virus (DHBV). Our results show that HBV CCC DNA formed in hepatoma cells was derived predominantly from RC DNA with a precise junction sequence. In contrast to that of DHBV, HBV CCC DNA formation in cultured cells was accompanied by the accumulation of a RC DNA species from which the covalently attached viral reverse transcriptase (RT) protein was removed (protein-free or PF-RC DNA). Furthermore, whereas envelope deficiency led to increased CCC DNA formation in DHBV, it resulted mainly in increased PF-RC, but not CCC, DNA in HBV, suggesting that the envelope protein(s) may negatively regulate a step in CCC DNA formation that precedes deproteination in both HBV and DHBV. Interestingly, PF-RC DNA, in contrast to RT-linked RC DNA, contained, almost exclusively, mature plus-strand DNA, suggesting that the RT protein was removed preferentially from mature RC DNA. PMID:17409153

  16. C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair

    PubMed Central

    McGovern, Stephen; Baconnais, Sonia; Roblin, Pierre; Nicolas, Pierre; Drevet, Pascal; Simonson, Héloïse; Piétrement, Olivier; Charbonnier, Jean-Baptiste; Le Cam, Eric; Noirot, Philippe; Lecointe, François

    2016-01-01

    Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region. PMID:26961308

  17. 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. PMID:16370937

  18. 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

  19. 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.

  20. 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

  1. Extent of Transcription of Mouse Sarcoma-Leukemia Virus by RNA-Directed DNA Polymerase

    PubMed Central

    Tavitian, A.; Hamelin, R.; Tchen, P.; Olofsson, B.; Boiron, M.

    1974-01-01

    The DNA product obtained from the endogenous RNA-directed DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) reaction of the Moloney sarcoma:leukemia viruses produced by the 78 A-1 cell line was analyzed and characterized. The extent of transcription of viral 70S RNA was measured by RNA·DNA hybridization (32P-viral RNA-3H product DNA). No double-stranded DNA was obtained. The product consisted of 95-99% single-stranded DNA with an average length of 200 nucleotides. In contrast to the results reported with avian and other RNA oncogenic viruses, it was found that the entire 70S viral RNA genome was transcribed into DNA pieces and that a small excess of the product DNA was sufficient to anneal the 70S RNA and render it totally resistant to single-stranded-specific enzyme digestion. PMID:4132533

  2. Synthesis of a non-radioactive hepatitis B virus DNA probe from human serum by the polymerase chain reaction.

    PubMed

    Rodríguez-Frías, F; Arranz, J A; Buti, M; Esteban, R; Jardí, R

    1994-05-01

    A method for synthesizing probes for detecting hepatitis B virus DNA in serum was developed. It uses DNA extracted from the serum of an hepatitis B virus carrier as target, and digoxigenin-11-dUTP incorporated in DNA sequences during the polymerase chain reaction as tracer. Using a spot hybridization assay, the sensitivity and specificity of the digoxigenin-labelled DNA probe were compared with two standard hepatitis B virus DNA probes, synthesized with cloned hepatitis B virus DNA and labelled either with digoxigenin or 32P by random priming. Data obtained from the three methods showed an excellent correlation. Thus, hepatitis B virus DNA extracted from human serum and labelled by polymerase chain reaction may be considered a suitable alternative to cloned DNA. It provides reliable probes and eliminates the need for facilities and personnel dedicated to the manipulation of clones. These advantages will allow a wider application of hepatitis B virus DNA testing in clinical practice.

  3. DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions.

    PubMed

    Grimsley, N; Hohn, B; Ramos, C; Kado, C; Rogowsky, P

    1989-06-01

    DNA transfer from Agrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyle-donous plant Zea mays, was analysed using the recently developed technique of agroinfection. Agroinfection of Z. mays with maize streak virus using strains of A. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterial virC genes. In contrast, agroinfection of the control host Brassica rapa with cauliflower mosaic virus was less dependent on the virC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intact virA, B, D and G functions were absolutely necessary, mutants in virE were attenuated. Agroinfection of maize was effective in the absence of an exogenously supplied vir gene inducer, and indeed wounded Z. mays tissues were found to produce substance(s) which induced the expression of A. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function of Agrobacterium vir genes.

  4. Epstein-barr virus coinfection in cerebrospinal fluid is associated with increased mortality in Malawian adults with bacterial meningitis.

    PubMed

    Kelly, Matthew J; Benjamin, Laura A; Cartwright, Katharine; Ajdukiewicz, Katherine M B; Cohen, Danielle B; Menyere, Mavis; Galbraith, Sareen; Guiver, Malcolm; Neuhann, Florian; Solomon, Tom; Lalloo, David G; Heyderman, Robert S

    2012-01-01

    Mortality from adult bacterial meningitis exceeds 50% in sub-Saharan Africa. We postulated that-particularly in individuals infected with human immunodeficiency virus (HIV)-herpes simplex virus, varicella zoster virus, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) in the cerebrospinal fluid (CSF) contribute to poor outcome. CSF from 149 Malawian adults with bacterial meningitis and 39 controls were analyzed using polymerase chain reaction. EBV was detected in 79 of 149 bacterial meningitis patients. Mortality (54%) was associated with higher CSF EBV load when adjusted for HIV (P = .01). CMV was detected in 11 of 115 HIV-infected patients, 8 of whom died. The mechanisms by which EBV and CMV contribute to poor outcome require further investigation.

  5. DNA thermodynamic stability and supercoil dynamics determine the gene expression program during the bacterial growth cycle.

    PubMed

    Sobetzko, Patrick; Glinkowska, Monika; Travers, Andrew; Muskhelishvili, Georgi

    2013-07-01

    The chromosomal DNA polymer constituting the cellular genetic material is primarily a device for coding information. Whilst the gene sequences comprise the digital (discontinuous) linear code, physiological alterations of the DNA superhelical density generate in addition analog (continuous) three-dimensional information essential for regulation of both chromosome compaction and gene expression. Insight into the relationship between the DNA analog information and the digital linear code is of fundamental importance for understanding genetic regulation. Our previous study in the model organism Escherichia coli suggested that the chromosomal gene order and a spatiotemporal gradient of DNA superhelicity associated with DNA replication determine the growth phase-dependent gene transcription. In this study we reveal a general gradient of DNA thermodynamic stability correlated with the polarity of chromosomal replication and manifest in the spatiotemporal pattern of gene transcription during the bacterial growth cycle. Furthermore, by integrating the physical and dynamic features of the transcribed sequences with their functional content we identify spatiotemporal domains of gene expression encompassing different functions. We thus provide both an insight into the organisational principle of the bacterial growth program and a novel holistic methodology for exploring chromosomal dynamics.

  6. Identification of Bacterial DNA Markers for the Detection of Human Fecal Pollution in Water▿ †

    PubMed Central

    Shanks, Orin C.; Domingo, Jorge W. Santo; Lu, Jingrang; Kelty, Catherine A.; Graham, James E.

    2007-01-01

    We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds. PMID:17209067

  7. 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. PMID:25287508

  8. 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.

  9. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction.

    PubMed

    Wilson, K H; Blitchington, R B; Greene, R C

    1990-09-01

    The sequence of small-subunit rRNA varies in an orderly manner across phylogenetic lines and contains segments that are conserved at the species, genus, or kingdom level. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, we amplified bacterial 16S ribosomal DNA sequences with the polymerase chain reaction. Priming sites were located at the extreme 5' end, the extreme 3' end, and the center of 16S ribosomal DNA. The isolates tested with these primers included members of the genera Staphylococcus, Coxiella, Rickettsia, Clostridium, Neisseria, Mycobacterium, Bilophila, Eubacterium, Fusobacterium, and Lactobacillus and the family Enterobacteriaceae. Initially, the yields from the reactions were erratic because the primers were self-complementary at the 3' ends. Revised primers that were not self-complementary gave more reproducible results. With the latter primers, 0.4 pg of Escherichia coli DNA consistently gave a visible band after amplification. This method should be useful for increasing the amounts of bacterial 16S ribosomal DNA sequences for the purposes of sequencing and probing. It should have a broad range of applications, including the detection and identification of known pathogens that are difficult to culture. This approach may make it possible to identify new, nonculturable bacterial pathogens.

  10. 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.

  11. 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. PMID:12957915

  12. 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

  13. Lyophilization to improve the sensitivity of qPCR for bacterial DNA detection in serum: the Q fever paradigm.

    PubMed

    Edouard, Sophie; Raoult, Didier

    2016-06-01

    Quantitative real-time PCR (qPCR) on serum provides significant added value to the diagnosis of Q fever, mainly at the acute stage of the disease in seronegative patients and in patients with endocarditis. We evaluated the benefits of Coxiella burnetii DNA concentration in serum by lyophilization to improve qPCR sensitivity. The detection limit of qPCR was determined by comparing six 10-fold dilutions of serum (calibrated with 104 bacteria ml-1) with and without lyophilization. We also tested, after lyophilization, 73 sera from patients with acute Q fever and 10 sera from patients with endocarditis for which specific qPCR for C. burnetii performed under our usual conditions remained negative. Lyophilization of DNA was found to improve sensitivity of the qPCR; the limit of detection of C. burnetii DNA by qPCR was 100-fold lower in lyophilized sera (1 bacterium ml-1) than in non-lyophilized sera (102 bacteria ml-1). Among the 73 sera from patients with acute Q fever, 26 (36 %) were positive after lyophilization, demonstrating a sensitivity gain of 44 % for early negative sera and 30 % for positive sera compared to our usual qPCR conditions. Sensitivity was also higher in sera from patients with endocarditis for which 8/10 (80 %) were positive after lyophilization. Our results serve as a proof of concept that lyophilization increases the sensitivity of qPCR in serum by concentrating bacterial DNA. This technique may be applied for the earlier diagnosis of other fastidious bacteria or viruses and extended to other clinical samples. PMID:27008653

  14. Extensive domain shuffling in transcription regulators of DNA viruses and implications for the origin of fungal APSES transcription factors

    PubMed Central

    Iyer, Lakshminarayan M; Koonin, Eugene V; Aravind, L

    2002-01-01

    Background Viral DNA-binding proteins have served as good models to study the biochemistry of transcription regulation and chromatin dynamics. Computational analysis of viral DNA-binding regulatory proteins and identification of their previously undetected homologs encoded by cellular genomes might lead to a better understanding of their function and evolution in both viral and cellular systems. Results The phyletic range and the conserved DNA-binding domains of the viral regulatory proteins of the poxvirus D6R/N1R and baculoviral Bro protein families have not been previously defined. Using computational analysis, we show that the amino-terminal module of the D6R/N1R proteins defines a novel, conserved DNA-binding domain (the KilA-N domain) that is found in a wide range of proteins of large bacterial and eukaryotic DNA viruses. The KilA-N domain is suggested to be homologous to the fungal DNA-binding APSES domain. We provide evidence for the KilA-N and APSES domains sharing a common fold with the nucleic acid-binding modules of the LAGLIDADG nucleases and the amino-terminal domains of the tRNA endonuclease. The amino-terminal module of the Bro proteins is another, distinct DNA-binding domain (the Bro-N domain) that is present in proteins whose domain architectures parallel those of the KilA-N domain-containing proteins. A detailed analysis of the KilA-N and Bro-N domains and the associated domains points to extensive domain shuffling and lineage-specific gene family expansion within DNA virus genomes. Conclusions We define a large class of novel viral DNA-binding proteins and their cellular homologs and identify their domain architectures. On the basis of phyletic pattern analysis we present evidence for a probable viral origin of the fungus-specific cell-cycle regulatory transcription factors containing the APSES DNA-binding domain. We also demonstrate the extensive role of lineage-specific gene expansion and domain shuffling, within a limited set of approximately

  15. Sample preparation module for bacterial lysis and isolation of DNA from human urine

    PubMed Central

    Gillers, Sara; Zhang, Jane Y.; Singh, Satish; Klapperich, Catherine M.

    2015-01-01

    Silica impregnated polymer monolithic columns may provide a simple method for lysing and extracting DNA from bacteria inside of microfluidic chips. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 101–105 colony-forming units/mL (CFU/mL) to simulate patient samples. We then performed on-chip lysis and DNA extraction. The bacterial DNA was amplified using real-time PCR demonstrating lysis and isolation down to 101 CFU/mL. Results were comparable to a commercial kit at higher concen trations and performed better at recovering DNA at lower concentrations. PMID:19130239

  16. Structure of bacterial LigD 3′-phosphoesterase unveils a DNA repair superfamily

    PubMed Central

    Nair, Pravin A.; Smith, Paul; Shuman, Stewart

    2010-01-01

    The DNA ligase D (LigD) 3′-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3′ end-healing reactions at DNA double-strand breaks. Here we report the 1.9 Å 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 β 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 à vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily. PMID:20616014

  17. 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.

  18. 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.

  19. 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.

  20. The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses.

    PubMed

    Khare, Sonal; Ratsimandresy, Rojo A; de Almeida, Lúcia; Cuda, Carla M; Rellick, Stephanie L; Misharin, Alexander V; Wallin, Melissa C; Gangopadhyay, Anu; Forte, Eleonora; Gottwein, Eva; Perlman, Harris; Reed, John C; Greaves, David R; Dorfleutner, Andrea; Stehlik, Christian

    2014-04-01

    The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages. PMID:24531343

  1. 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.

  2. 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. PMID:26478540

  3. Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin.

    PubMed

    Arnold, Anna R; Zhou, Andy; Barton, Jacqueline K

    2016-09-01

    Dps proteins are bacterial ferritins that protect DNA from oxidative stress and have been implicated in bacterial survival and virulence. In addition to direct oxidation of the Dps iron sites by diffusing oxidants, oxidation from a distance via DNA charge transport (CT), where electrons and electron holes are rapidly transported through the base-pair π-stack, could represent an efficient DNA protection mechanism utilized by Dps. Here, we spectroscopically characterize the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation using an intercalating ruthenium photooxidant and the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, consistent with guanine radical intermediates facilitating Dps oxidation. We have also explored possible protein electron transfer (ET) intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the iron-binding ferroxidase site (W52 in E. coli Dps). In EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, a W52A Dps mutant was significantly deficient compared to WT Dps in forming the characteristic EPR signal at g = 4.3, consistent with W52 acting as an ET hopping intermediate. This effect is mirrored in vivo in E. coli survival in response to hydrogen peroxide, where mutation of W52 leads to decreased survival under oxidative stress.

  4. Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin.

    PubMed

    Arnold, Anna R; Zhou, Andy; Barton, Jacqueline K

    2016-09-01

    Dps proteins are bacterial ferritins that protect DNA from oxidative stress and have been implicated in bacterial survival and virulence. In addition to direct oxidation of the Dps iron sites by diffusing oxidants, oxidation from a distance via DNA charge transport (CT), where electrons and electron holes are rapidly transported through the base-pair π-stack, could represent an efficient DNA protection mechanism utilized by Dps. Here, we spectroscopically characterize the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation using an intercalating ruthenium photooxidant and the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, consistent with guanine radical intermediates facilitating Dps oxidation. We have also explored possible protein electron transfer (ET) intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the iron-binding ferroxidase site (W52 in E. coli Dps). In EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, a W52A Dps mutant was significantly deficient compared to WT Dps in forming the characteristic EPR signal at g = 4.3, consistent with W52 acting as an ET hopping intermediate. This effect is mirrored in vivo in E. coli survival in response to hydrogen peroxide, where mutation of W52 leads to decreased survival under oxidative stress. PMID:27571139

  5. 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.

  6. Exposure to West Nile Virus Increases Bacterial Diversity and Immune Gene Expression in Culex pipiens.

    PubMed

    Zink, Steven D; Van Slyke, Greta A; Palumbo, Michael J; Kramer, Laura D; Ciota, Alexander T

    2015-10-01

    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 inWNV-exposed and/or infected mosquitoes, yet the most consistent and notable was the genus Serratia. PMID:26516902

  7. 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

  8. Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis.

    PubMed

    Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

    2004-03-01

    Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.

  9. Isolation and Characterization of a Protein That Stimulates DNA Synthesis from Avian Myeloblastosis Virus*

    PubMed Central

    Leis, Jonathan P.; Hurwitz, Jerard

    1972-01-01

    A protein has been isolated from avian myeloblastosis virus that stimulates the rate and yield of DNA synthesis primed by viral RNA with purified viral polymerase. It specifically affects the viral polymerase and does not stimulate other DNA polymerases under the conditions tested. The viral polymerase, in conjunction with this protein, transcribes extended single-stranded regions of DNA, and permits the enzyme to initiate synthesis from single-strand breaks in DNA. PMID:4340754

  10. 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.

  11. Conjugative DNA Transfer Induces the Bacterial SOS Response and Promotes Antibiotic Resistance Development through Integron Activation

    PubMed Central

    Baharoglu, Zeynep; Bikard, David; Mazel, Didier

    2010-01-01

    Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria. PMID:20975940

  12. 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

  13. 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.

  14. A field guide to eukaryotic circular single-stranded DNA viruses: insights gained from metagenomics.

    PubMed

    Rosario, Karyna; Duffy, Siobain; Breitbart, Mya

    2012-10-01

    Despite their small size and limited protein-coding capacity, the rapid evolution rates of single-stranded DNA (ssDNA) viruses have led to their emergence as serious plant and animal pathogens. Recently, metagenomics has revealed an unprecedented diversity of ssDNA viruses, expanding their known environmental distributions and host ranges. This review summarizes and contrasts the basic characteristics of known circular ssDNA viral groups, providing a resource for analyzing the wealth of ssDNA viral sequences identified through metagenomics. Since ssDNA viruses are largely identified based on conserved rolling circle replication proteins, this review highlights distinguishing motifs and catalytic residues important for replication. Genomes identified through metagenomics have demonstrated unique ssDNA viral genome architectures and revealed characteristics that blur the boundaries between previously well-defined groups. Metagenomic discovery of ssDNA viruses has created both a challenge to current taxonomic classification schemes and an opportunity to revisit hypotheses regarding the evolutionary history of these viruses.

  15. Abundant DNase I-Sensitive Bacterial DNA in Healthy Porcine Lungs and Its Implications for the Lung Microbiome

    PubMed Central

    Pezzulo, Alejandro A.; Kelly, Patrick H.; Nassar, Boulos S.; Rutland, Cedric J.; Gansemer, Nicholas D.; Dohrn, Cassie L.; Costello, Andrew J.; Stoltz, David A.

    2013-01-01

    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. PMID:23872563

  16. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Detection, purification, and characterization of two species of covalently closed circular proviral DNA molecules of bovine leukemia virus.

    PubMed Central

    Kashmiri, S V; Mehdi, R; Ferrer, J F

    1983-01-01

    Cocultivation of uninfected and bovine leukemia virus-producing bat cells yielded, in addition to the unintegrated linear DNA duplex, DNA molecules that migrated as 4.4- and 4.8-kilobase-pair DNA fragments in gel electrophoresis. These DNA molecules were purified by acid-phenol extraction and cleaved with restriction endonucleases EcoRI, and HindIII, which have one recognition site each on the bovine leukemia virus proviral DNA. Such cleavage generated DNA molecules of approximately 10.0 and 9.4 kilobase pairs, thus indicating the existence of two species of covalently closed circular molecules of bovine leukemia virus proviral DNA. Images PMID:6300454

  19. 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

  20. 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

    2015-12-01

    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.

  1. 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

    2015-12-01

    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. PMID:26186890

  2. Identification of a Novel Circular DNA Virus in New Zealand Fur Seal (Arctocephalus forsteri) Fecal Matter.

    PubMed

    Sikorski, Alyssa; Dayaram, Anisha; Varsani, Arvind

    2013-08-08

    Fur seal feces-associated circular DNA virus (FSfaCV) is a novel virus isolated from the fecal matter of New Zealand fur seals. FSfaCV has two main open reading frames in its 2,925-nucleotide (nt) genome. The replication-associated protein (Rep) of FSfaCV has similarity to Rep-like sequences in the Giardia intestinalis genome.

  3. 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

  4. Scientists Map DNA of Zika Virus from Semen

    MedlinePlus

    ... 2016 (HealthDay News) -- The first complete genetic "blueprint" -- genome -- of a sample of Zika virus derived from ... new study, published Oct. 13 in the journal Genome Announcements . "Isolating Zika virus from semen has been ...

  5. Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

    PubMed Central

    Merryweather, A; Barth, P T; Wilkins, B M

    1986-01-01

    The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid. PMID:3522540

  6. 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.

  7. DNA microarrays: experimental issues, data analysis, and application to bacterial systems.

    PubMed

    Dharmadi, Yandi; Gonzalez, Ramon

    2004-01-01

    DNA microarrays are currently used to study the transcriptional response of many organisms to genetic and environmental perturbations. Although there is much room for improvement of this technology, its potential has been clearly demonstrated in the past 5 years. The general consensus is that the bottleneck is now located in the processing and analysis of transcriptome data and its use for purposes other than the quantification of changes in gene expression levels. In this article we discuss technological aspects of DNA microarrays, statistical and biological issues pertinent to the design of microarray experiments, and statistical tools for microarray data analysis. A review on applications of DNA microarrays in the study of bacterial systems is presented. Special attention is given to studies in the following areas: (1) bacterial response to environmental changes; (2) gene identification, genome organization, and transcriptional regulation; and (3) genetic and metabolic engineering. Soon, the use of DNA microarray technologies in conjunction with other genome/system-wide analyses (e.g., proteomics, metabolomics, fluxomics, phenomics, etc.) will provide a better assessment of genotype-phenotype relationships in bacteria, which serve as a basis for understanding similar processes in more complex organisms.

  8. Annual changes of bacterial mortality due to viruses and protists in an oligotrophic coastal environment (NW Mediterranean).

    PubMed

    Boras, Julia A; Sala, M Montserrat; Vázquez-Domínguez, Evaristo; Weinbauer, Markus G; Vaqué, Dolors

    2009-05-01

    The impact of viruses and protists on bacterioplankton mortality was examined monthly during 2 years (May 2005-April 2007) in an oligotrophic coastal environment (NW Mediterranean Sea). We expected that in such type of system, (i) bacterial losses would be caused mainly by protists, and (ii) lysogeny would be an important type of virus-host interaction. During the study period, viruses and grazers together were responsible for 50.6 +/- 40.1% day(-1) of bacterial standing stock losses (BSS) and 59.7 +/- 44.0% day(-1) of bacterial production losses (BP). Over the first year (May 2005-April 2006), protists were the principal cause of bacterial mortality, removing 29.9 +/- 20.4% day(-1) of BSS and 33.9 +/- 24.3% day(-1) of BP, whereas viral lysis removed 13.5 +/- 17.0% day(-1) of BSS and 12.3 +/- 12.3% day(-1) of BP. During the second year (May 2006-April 2007), viruses caused comparable bacterial losses (29.2 +/- 14.8% day(-1) of BSS and 40.9 +/- 20.7% day(-1) of BP) to protists (28.6 +/- 25.5% day(-1) of BSS and 32.4 +/- 20.0% day(-1) of BP). In 37% of cases higher losses of BP due to viruses than due to protists were found. Lysogenic infection was detected in 11 of 24 samplings. Contrary to our expectations, lytic infections dominated over the two years, and viruses resulted to be a significant source of bacterial mortality in this oligotrophic site.

  9. Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

    PubMed Central

    Nagy, E

    1992-01-01

    A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1316798

  10. Origin and evolution of polydnaviruses by symbiogenesis of insect DNA viruses in endoparasitic wasps.

    PubMed

    Federici, B A; Bigot, Y

    2003-05-01

    During oviposition, many endoparasitic wasps inject virus-like particles into their insect hosts that enable these parasitoids to evade or directly suppress their hosts' immune system, especially encapsulation by hemocytes. These particles are defined as virions that belong to viruses of the two genera that comprise the family Polydnaviridae, bracoviruses (genus Bracovirus) transmitted by braconid wasps, and ichnoviruses (genus Ichnovirus) transmitted by ichneumonid wasps. Structurally, bracovirus virions resemble nudivirus and baculovirus virions (family Baculoviridae), and ichnovirus virions resemble those of ascoviruses (family Ascoviridae). Whereas nudiviruses, baculoviruses and ascoviruses replicate their DNA and produce progeny virions, polydnavirus DNA is integrated into and replicated from the wasp genome, which also directs virion synthesis. The structural similarity of polydnavirus virions to those of viruses that attack the wasps' lepidopteran hosts, along with polydnavirus transmission and replication biology, suggest that these viruses evolved from insect DNA viruses by symbiogenesis, the same process by which mitochondia and chloroplasts evolved from bacteria. Molecular evidence supporting this hypothesis comes from similarities among structural proteins of ascoviruses and the Campoletis sonorensis ichnovirus. Implications of this hypothesis are that polydnaviruses evolved from viruses, but are no longer viruses, and that DNA packaged into polydnavirus virions is not viral genomic DNA per se, but rather wasp genomic DNA consisting primarily of wasp genes and non-coding DNA. Thus, we suggest that a better understanding of polydnaviruses would result by viewing these not as viruses, but rather as a wasp organelle system that evolved to shuttle wasp genes and proteins into hosts to evade and suppress their immune response. PMID:12770621

  11. Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow▿

    PubMed Central

    Tomaru, Yuji; Takao, Yoshitake; Suzuki, Hidekazu; Nagumo, Tamotsu; Koike, Kanae; Nagasaki, Keizo

    2011-01-01

    Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 104 infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus. PMID:21666026

  12. Isolation and characterization of a single-stranded DNA virus infecting Chaetoceros lorenzianus Grunow.

    PubMed

    Tomaru, Yuji; Takao, Yoshitake; Suzuki, Hidekazu; Nagumo, Tamotsu; Koike, Kanae; Nagasaki, Keizo

    2011-08-01

    Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 10(4) infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus.

  13. Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

    PubMed Central

    Galula, Jedhan U.; Shen, Wen-Fan; Chuang, Shih-Te

    2014-01-01

    ABSTRACT Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine

  14. Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses.

    PubMed

    Kilianski, Andy; Roth, Pierce A; Liem, Alvin T; Hill, Jessica M; Willis, Kristen L; Rossmaier, Rebecca D; Marinich, Andrew V; Maughan, Michele N; Karavis, Mark A; Kuhn, Jens H; Honko, Anna N; Rosenzweig, C Nicole

    2016-08-01

    Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization. PMID:27191483

  15. Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

    SciTech Connect

    Sparger, Ellen E. Dubie, Robert A.; Shacklett, Barbara L.; Cole, Kelly S.; Chang, W.L.; Luciw, Paul A.

    2008-05-10

    Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-{gamma} enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus.

  16. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus

    PubMed Central

    Qi, Yonghe; Gao, Zhenchao; Peng, Bo; Yan, Huan; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

    2016-01-01

    Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. PMID:27783675

  17. Seasonal Dynamics of the Airborne Bacterial Community and Selected Viruses in a Children's Daycare Center.

    PubMed

    Prussin, Aaron J; Vikram, Amit; Bibby, Kyle J; Marr, Linsey C

    2016-01-01

    Children's daycare centers appear to be hubs of respiratory infectious disease transmission, yet there is only limited information about the airborne microbial communities that are present in daycare centers. We have investigated the microbial community of the air in a daycare center, including seasonal dynamics in the bacterial community and the presence of specific viral pathogens. We collected filters from the heating, ventilation, and air conditioning (HVAC) system of a daycare center every two weeks over the course of a year. Amplifying and sequencing the 16S rRNA gene revealed that the air was dominated by Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes that are commonly associated with the human skin flora. Clear seasonal differences in the microbial community were not evident; however, the community structure differed when the daycare center was closed and unoccupied for a 13-day period. These results suggest that human occupancy, rather than the environment, is the major driver in shaping the microbial community structure in the air of the daycare center. Using PCR for targeted viruses, we detected a seasonal pattern in the presence of respiratory syncytial virus that included the period of typical occurrence of the disease related to the virus; however, we did not detect the presence of adenovirus or rotavirus at any time. PMID:26942410

  18. Blowing Off Steam: Virus Inhibition of cGAS DNA Sensing.

    PubMed

    Diner, Benjamin A; Cristea, Ileana M

    2015-09-01

    Detection of viral DNA is essential for eliciting mammalian innate immunity. However, viruses have acquired effective mechanisms for blocking host defense. Indeed, in this issue of Cell Host & Microbe, Wu et al. (2015) discover a herpesviral strategy for inhibiting the prominent host sensor of viral DNA, cGAS. PMID:26355212

  19. Bacteriophage orphan DNA methyltransferases: insights from their bacterial origin, function, and occurrence.

    PubMed

    Murphy, James; Mahony, Jennifer; Ainsworth, Stuart; Nauta, Arjen; van Sinderen, Douwe

    2013-12-01

    Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function. PMID:24123737

  20. Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products▿

    PubMed Central

    Cao, Boyang; Li, Rongrong; Xiong, Songjin; Yao, Fangfang; Liu, Xiangqian; Wang, Min; Feng, Lu; Wang, Lei

    2011-01-01

    We established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes of Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica and the 16S-23S rRNA gene internal transcribed spacer (ITS) region of Proteus mirabilis and Proteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays. PMID:21965411

  1. Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

    NASA Astrophysics Data System (ADS)

    Brandt, Petra; Gerdts, Gunnar; Boersma, Maarten; Wiltshire, Karen H.; Wichels, Antje

    2010-12-01

    Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp . and Temora sp . from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.

  2. Bacteriophage Orphan DNA Methyltransferases: Insights from Their Bacterial Origin, Function, and Occurrence

    PubMed Central

    Murphy, James; Mahony, Jennifer; Ainsworth, Stuart; Nauta, Arjen

    2013-01-01

    Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function. PMID:24123737

  3. Bacterial DNA of Ocean and Land on the Surface of the International Space Station.

    NASA Astrophysics Data System (ADS)

    Grebennikova, Tatiana

    A.V. Syroeshkin2, T.V. Grebennikova1, E.V. Shubralova3, V.A. Shuvalov3, O.S. Tsygankov4, V.B. Lapshin2 1D. I. Ivanovsky Virology Institute, Moscow, Russia 2 Academician E. K. Fedorov Institute of Applied Geophysics, Moscow, Russia 3S.P. Korolev Rocket and Space Corporation «Energia» Korolev, Russia 4Central Research Institute of Machine Building, Korolev, Russia Existence of biological molecules as markers of microorganisms in the space environment has always attracted attention of researchers. There is great attention to the search for extraterrestrial life forms [Nicholson W.L. 2009, Kawaguchi Y. et al 2013], and as well as the coping mechanisms of living organisms in the interplanetary space [Hotchin J. et al 1965, Baranov V.M. 2009, Horneck G. et al 2010]. Experiments on American and Japanese segments of the International Space Station (ISS) over the different nature of resistance during prolonged stay in space were conducted [Scalzi G et al 2012, Wassmann M. et al 2012]. As a result of these experiments confirmed the possibility of preserving the viability of organisms in an open space for a long time. Consequence, became interested in the transfer of living matter from the stratosphere to near-Earth space [Smith D.J. 2013]. We hypothesized that viable forms, or at least, intact DNA can be transferred to the orbit of the ISS with the ascending branch of the global electric circuit. Samples of cosmic dust collected from the surface of the window of the ISS during the exit of an astronaut in space. Samples (washes with material of tampons and tampons) which were in vacuo, were analyzed for the presence of bacterial DNA by nested PCR using primers specific DNA genus Mycobacterium, the DNA of the strain of the genus Bacillus anthracis and DNA encoding the bacterial 16S ribosomal RNA after transportation of the samples to Earth. The results of amplification, followed by sequencing and phylogenetic analysis showed the presence in samples of cosmic dust DNA

  4. Infectious cDNA clones of the DA strain of Theiler's murine encephalomyelitis virus.

    PubMed Central

    Roos, R P; Stein, S; Ohara, Y; Fu, J L; Semler, B L

    1989-01-01

    The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis viruses cause a persistent demyelinating infection, whereas the GDVII strain and other GDVII subgroup strains cause an acute lethal polioencephalomyelitis. We generated an infectious DA cDNA clone inserted into a transcription vector. Virus derived from transfection of transcripts produced a demyelinating disease indistinguishable from that of wild-type virus. The infectious clone provides a critical reagent for the production of interstrain recombinant viruses to help identify genetic loci responsible for the biological activities of the strains. Images PMID:2555569

  5. A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems

    PubMed Central

    Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.

    2011-01-01

    scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins. PMID:21306995

  6. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus.

    PubMed

    Ramanan, Vyas; Shlomai, Amir; Cox, David B T; Schwartz, Robert E; Michailidis, Eleftherios; Bhatta, Ankit; Scott, David A; Zhang, Feng; Rice, Charles M; Bhatia, Sangeeta N

    2015-01-01

    Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients. PMID:26035283

  7. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

    PubMed Central

    Ramanan, Vyas; Shlomai, Amir; Cox, David B.T.; Schwartz, Robert E.; Michailidis, Eleftherios; Bhatta, Ankit; Scott, David A.; Zhang, Feng; Rice, Charles M.; Bhatia, Sangeeta N.

    2015-01-01

    Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients. PMID:26035283

  8. Opportunistic DNA Recombination With Epstein-Barr Virus at Sites of Control Region Rearrangements Mediating JC Virus Neurovirulence.

    PubMed

    Wortman, Margaret J; Lundberg, Patric S; Dagdanova, Ayuna V; Venkataraman, Pranav; Daniel, Dianne C; Johnson, Edward M

    2016-05-01

    We document a unique DNA recombination between polyomavirus JC (JC virus [JCV]) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression, JCV can infect the brain, causing a demyelinating disease, progressive multifocal leukoencephalopathy. Rearrangements in the archetype noncoding control region are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be coinfected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed patients with multiple sclerosis, those susceptible to progressive multifocal leukoencephalopathy. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical noncoding control region sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types.

  9. The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

    ERIC Educational Resources Information Center

    Falconer, A. C.; Hayes, L. J.

    1986-01-01

    Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

  10. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

    SciTech Connect

    Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

    1986-07-15

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.

  11. In planta cloning of geminiviral DNA: the true Sida micrantha mosaic virus.

    PubMed

    Jeske, Holger; Gotthardt, Diether; Kober, Sigrid

    2010-02-01

    The circular single-stranded DNAs of geminiviruses are multiplied efficiently and preferentially by rolling circle amplification (RCA), and can be diagnosed readily by restriction fragment length polymorphism (RFLP) and direct sequencing of the RCA product. Two strategies are described for cloning geminiviruses from plants harboring mixed infections by using RCA and RFLP with plant-derived nucleic acids without the need for bacterial amplification. By combining both these approaches, the true Sida micrantha mosaic virus was identified. The advantages of maintaining the quasispecies nature of a virus during in planta cloning is discussed with respect to reliable virus identification and resistance breeding.

  12. Use of a Sindbis virus DNA-based expression vector for induction of protective immunity against pseudorabies virus in pigs.

    PubMed

    Dufour, Vinciane; De Boisséson, Claire

    2003-06-20

    Injection of plasmid DNA encoding pseudorabies virus (PRV) glycoproteins into pig muscle has been shown to result in protective immunity against lethal infection. Nevertheless, such DNA vaccines are still less efficient than some attenuated or killed live vaccines. One way to increase DNA vaccine efficacy is to improve the vectorisation system at the molecular level, thereby enhancing the rate of in vivo-produced immunogen protein and consequently specific acquired immunity. The present study compared the effectiveness of the protein expression system depending on Sindbis virus (SIN) replicase [J. Virol. 70 (1996) 508] with that of more classical pcDNA3 plasmid. Pigs were vaccinated twice at 3-week interval with a mixture of three pcDNA3 plasmids expressing gB, gC and gD (designated as PRV-pcDNA3) or a mixture of three SIN plasmids expressing the same glycoproteins (PRV-pSINCP), and were challenged with a highly virulent PRV strain. The two DNA vaccines induced PRV-specific T cell-mediated immune response characterized by very low levels of IFN-gamma mRNA in PBMC after in vitro antigen-specific stimulation. Very low levels of neutralizing antibodies (NAb) were also obtained in sera following DNA injection(s). A second DNA injection did not boost immune responses. After a lethal challenge, high levels of IFN-gamma mRNA and high NAb response were induced in all DNA-vaccinated pigs, regardless of the vector used. Therefore, the two eukaryotic expression systems showed comparable efficacy in inducing antiviral immunity and clinical protection against PRV in pigs. This suggests that SIN DNA-based vector immunizing potential may differ according to antigen and/or host. PMID:12814698

  13. Comparison of bacterial DNA profiles of footwear insoles and soles of feet for the forensic discrimination of footwear owners.

    PubMed

    Goga, Haruhisa

    2012-09-01

    It is crucial to identify the owner of unattended footwear left at a crime scene. However, retrieving enough DNA for DNA profiling from the owner's foot skin (plantar skin) cells from inside the footwear is often unsuccessful. This is sometimes because footwear that is used on a daily basis contains an abundance of bacteria that degrade DNA. Further, numerous other factors related to the inside of the shoe, such as high humidity and temperature, can encourage bacterial growth inside the footwear and enhance DNA degradation. This project sought to determine if bacteria from inside footwear could be used for footwear trace evidence. The plantar skins and insoles of shoes of volunteers were swabbed for bacteria, and their bacterial community profiles were compared using bacterial 16S rRNA terminal restriction fragment length polymorphism analysis. Sufficient bacteria were recovered from both footwear insoles and the plantar skins of the volunteers. The profiling identified that each volunteer's plantar skins harbored unique bacterial communities, as did the individuals' footwear insoles. In most cases, a significant similarity in the bacterial community was identified for the matched foot/insole swabs from each volunteer, as compared with those profiles from different volunteers. These observations indicate the probability to discriminate the owner of footwear by comparing the microbial DNA fingerprint from inside footwear with that of the skin from the soles of the feet of the suspected owner. This novel strategy will offer auxiliary forensic footwear evidence for human DNA identification, although further investigations into this technique are required.

  14. Response of bacterial metabolic activity to riverine dissolved organic carbon and exogenous viruses in estuarine and coastal waters: implications for CO2 emission.

    PubMed

    Xu, Jie; Sun, Mingming; Shi, Zhen; Harrison, Paul J; Liu, Hongbin

    2014-01-01

    A cross-transplant experiment between estuarine water and seawater was conducted to examine the response of bacterial metabolic activity to riverine dissolved organic carbon (DOC) input under virus-rich and virus-free conditions, as well as to exogenous viruses. Riverine DOC input increased bacterial production significantly, but not bacterial respiration (BR) because of its high lability. The bioavailable riverine DOC influenced bulk bacterial respiration in two contrasting ways; it enhanced the bulk BR by stimulating bacterial growth, but simultaneously reduced the cell-specific BR due to its high lability. As a result, there was little stimulation of the bulk BR by riverine DOC. This might be partly responsible for lower CO2 degassing fluxes in estuaries receiving high sewage-DOC that is highly labile. Viruses restricted microbial decomposition of riverine DOC dramatically by repressing the growth of metabolically active bacteria. Bacterial carbon demand in the presence of viruses only accounted for 7-12% of that in the absence of viruses. Consequently, a large fraction of riverine DOC was likely transported offshore to the shelf. In addition, marine bacteria and estuarine bacteria responded distinctly to exogenous viruses. Marine viruses were able to infect estuarine bacteria, but not as efficiently as estuarine viruses, while estuarine viruses infected marine bacteria as efficiently as marine viruses. We speculate that the rapid changes in the viral community due to freshwater input destroyed the existing bacteria-virus relationship, which would change the bacterial community composition and affect the bacterial metabolic activity and carbon cycling in this estuary.

  15. An improved assay for the detection of alterations in bacterial DNA supercoiling in vivo.

    PubMed

    Abu Mraheil, M; Heisig, A; Heisig, P

    2013-07-01

    Due to the increasing prevalence of antibiotic resistance and the yet low output of the genomics-based drug discovery approach novel strategies are urgently needed to detect new antibiotics. One such strategy uses known ubiquitous targets like DNA topoisomerases. However, to detect inhibitors of these enzymes by an in vitro assay time-consuming isolation of enzymes and DNA followed by electrophoretic separation of topoisomers are required. Instead, this study aimed at developing an in vivo assay for the detection of alterations in DNA supercoiling indicative of topoisomerase inhibition by a reporter gene assay. A pair of plasmids was developed which carry the reporter gene luc for firefly luciferase under control of either promoter ptopA (pPHB90) or pgyrA (pPHB91), whose activities are reciprocally affected by alterations of the supercoiling degree. Each plasmid is individually transferred into E. coli cells. The quotient of the luciferase activities determined using cells with either plasmid was taken as relative measure of the global supercoiling degree Qsc (quotient of supercoiling). Using isogenic reference strains with known alterations of the global DNA supercoiling degree due to mutations in either gyrB or topA, the reporter gene system was able to detect both a decrease and an increase of the negative supercoiling degree compared to the isogenic parent strain. Treating cells with known inhibitors of DNA gyrase, like fluoroquinolones, novobiocin as well as simocyclinone D8 from Streptomyces antibioticus which has been identified as an inhibitor of DNA gyrase in vitro, also caused decreases of the Qsc value in vivo. The suitability of this reporter gene system to screen for anti-topoisomerase I and II compounds from various natural sources like plant extracts by sensing alterations of the DNA supercoiling was demonstrated and offers a new application to identify novel compounds active against bacterial topoisomerases I and gyrase.

  16. Infectious linear DNA sequences replicating in simian virus 40-infected cells.

    PubMed Central

    Gruss, P; Sauer, G

    1977-01-01

    A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions. Images PMID:189087

  17. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen

    PubMed Central

    Weynberg, Karen D.; Voolstra, Christian R.; Neave, Matthew J.; Buerger, Patrick; van Oppen, Madeleine J. H.

    2015-01-01

    Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements. PMID:26644037

  18. Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen.

    PubMed

    Kang, Su-Min; Rhee, Jin-Kyu; Kim, Eui-Joong; Han, Kwang-Hyub; Oh, Jong-Won

    2003-09-26

    Cell surface expression of protein has been widely used to display enzymes and antigens. Here we show that Pseudomonas syringae ice nucleation protein with a deletion of internal repeating domain (INC) can be used in Escherichia coli to display peptide in a conformationally active form on the outside of the folded protein by fusing to the C-terminus of INC. Diagnostic potential of this technology was demonstrated by effective mapping of antigenic epitopes derived from hepatitis C virus (HCV) core protein. Amino acids 1-38 and 26-53 of HCV core protein were found to react more sensitively in a native conformation with the HCV patient sera than commercial diagnostic antigen, c22p (amino acids 10-53) by display-ELISA. These results demonstrate that the bacterial cell surface display using INC is useful for peptide presentation and thus epitope mapping of antigen. PMID:14553932

  19. Advances and challenges in the development of therapeutic DNA vaccines against hepatitis B virus infection.

    PubMed

    Cova, Lucyna

    2014-01-01

    Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Because very weak and functionally impaired virus-specific immune responses play a key role in the persistence of HBV infection, the stimulation of these responses appears to be of particular importance for virus clearance. In this regard DNA-based vaccination has emerged as novel, promising therapeutic approach for chronic hepatitis B. This review provides an update of preclinical studies in animal models (mouse, chimpanzee, duck, woodchuck), which evaluated the ability of DNA vaccines targeting hepadnaviral proteins to induce potent and sustained immune responses in naïve animals and to enhance virus clearance and break immune tolerance in chronic virus-carriers. Different strategies have been developed and evaluated in these models to optimize DNA vaccine including genetic adjuvants, combination with antiviral drugs, prime-boost regimens and plasmid delivery. The delivery of DNA by in vivo electroporation appears to be of particular interest for increase of vaccine potency in both small and large animal models. Based on the promising results generated in preclinical studies, first clinical trials of DNA vaccines have been initiated, although effective therapy of chronic hepatitis B awaits further improvements in vaccine efficacy.

  20. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

    SciTech Connect

    Heilbronn, R.; zur Hausen, H. )

    1989-09-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of sic HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensable for SV40 DNA amplification. The results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

  1. Plasma Epstein–Barr virus and Hepatitis B virus in non-Hodgkin lymphomas: Two lymphotropic, potentially oncogenic, latently occurring DNA viruses

    PubMed Central

    Sinha, Mahua; Rao, Clementina Rama; Premalata, C. S.; Shafiulla, Mohammed; Lakshmaiah, K. C.; Jacob, Linu Abraham; Babu, Govind K.; Viveka, B. K.; Appaji, L.; Subramanyam, Jayshree R.

    2016-01-01

    Context: There is a need to study potential infective etiologies in lymphomas. Lymphocyte-transforming viruses can directly infect lymphocytes, disrupt normal cell functions, and promote cell division. Epstein–Barr virus (EBV) is known to be associated with several lymphomas, especially Hodgkin lymphomas (HLs). And recently, the lymphocyte-transforming role of hepatitis B virus (HBV) has been emphasized. Aims: The aim of this study was to elucidate the association of two potentially oncogenic, widely prevalent latent DNA viruses, EBV and HBV, in non-HL (NHL). Settings and Design: In this prospective study, we estimated plasma EBV and HBV DNA in NHL patients. Materials and Methods: Peripheral blood was obtained from newly diagnosed, treatment na ïve, histologically confirmed NHL patients. Plasma EBV DNA was quantified by real-time polymerase chain reaction (PCR) targeting Epstein–Barr Nucleic acid 1 while the plasma HBV DNA was detected using nested PCR targeting HBX gene. In a small subset of patients, follow-up plasma samples post-anticancer chemotherapy were available and retested for viral DNA. Results: Of the 110 NHL patients, ~79% were B-cell NHL and ~21% were T-cell NHL. Plasma EBV-DNA was detected in 10% NHLs with a higher EBV association in Burkitt lymphoma (33.3%) than other subtypes. Pretherapy HBV DNA was detected in 21% NHLs; most of them being diffuse large B-cell lymphoma (DLBCL). Moreover, 42% of DLBCL patients had HBV DNA in plasma. Since all patients were HBV surface antigen seronegative at diagnosis, baseline plasma HBV-DNAemia before chemotherapy was indicative of occult hepatitis B infection. Conclusions: Our findings indicate a significant association of HBV with newly diagnosed DLBCL.

  2. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

    We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

  3. Temperate membrane-containing halophilic archaeal virus SNJ1 has a circular dsDNA genome identical to that of plasmid pHH205.

    PubMed

    Zhang, Ziqian; Liu, Ying; Wang, Shuai; Yang, Di; Cheng, Yichen; Hu, Jiani; Chen, Jin; Mei, Yunjun; Shen, Ping; Bamford, Dennis H; Chen, Xiangdong

    2012-12-20

    A temperate haloarchaeal virus, SNJ1, was induced from the lysogenic host, Natrinema sp. J7-1, with mitomycin C, and the virus produced plaques on lawns of Natrinema sp. J7-2. Optimization of the induction conditions allowed us to increase the titer from ~10(4) PFU/ml to ~10(11) PFU/ml. Single-step growth curves exhibited a burst size of ~100 PFU/cell. The genome of SNJ1 was observed to be a circular, double-stranded DNA (dsDNA) molecule (16,341 bp). Surprisingly, the sequence of SNJ1 was identical to that of a previously described plasmid, pHH205, indicating that this plasmid is the provirus of SNJ1. Several structural protein-encoding genes were identified in the viral genome. In addition, the comparison of putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses, as well as the presence of lipid constituents from the host phospholipid pool, strongly suggest that SNJ1 belongs to the PRD1-type lineage of dsDNA viruses, which have an internal membrane. PMID:22784791

  4. Virus-derived small RNAs in the penaeid shrimp Fenneropenaeus chinensis during acute infection of the DNA virus WSSV

    PubMed Central

    Liu, Chengzhang; Li, Fuhua; Sun, Yumiao; Zhang, Xiaojun; Yuan, Jianbo; Yang, Hui; Xiang, Jianhai

    2016-01-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are two classes of small RNAs (sRNAs) that are critical for virus-host interplay via the RNA interference (RNAi) pathway. One virus-derived siRNA and numerous miRNAs has been reported for the double-stranded DNA virus white spot syndrome virus (WSSV), however, the expression profiles of these different types of sRNAs have not been assessed. Here, by sequencing the sRNAs and mRNAs of WSSV-infected Chinese shrimp (Fenneropenaeus chinensis), we found that the viral transcripts were universally targeted by WSSV-derived siRNAs, supporting a pivotal role for RNAi in the anti-viral immunity of shrimp. The genesis of WSSV-derived siRNAs was associated with long RNA structures. Moreover, by separating miRNAs from siRNAs, 12 WSSV miRNAs were identified. Investigation of conserved viral miRNA targets in different host species indicated the involvement of viral miRNAs in host immune responses. Collectively, our data provide new insights into the role of the RNAi pathway in the interplay between DNA viruses and crustaceans. PMID:27349643

  5. Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses

    PubMed Central

    Son, Kyung-No; Liang, Zhiguo

    2015-01-01

    ABSTRACT Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. IMPORTANCE An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds

  6. Contribution of virus-induced lysis and protozoan grazing to benthic bacterial mortality estimated simultaneously in microcosms.

    PubMed

    Fischer, Ulrike R; Wieltschnig, Claudia; Kirschner, Alexander K T; Velimirov, Branko

    2006-08-01

    In contrast to the water column, the fate of bacterial production in freshwater sediments is still a matter of debate. Thus, the importance of virus-induced lysis and protozoan grazing of bacteria was investigated for the first time simultaneously in a silty sediment layer of a mesotrophic oxbow lake. Microcosms were installed in the laboratory in order to study the dynamics of these processes over 15 days. All microbial and physicochemical parameters showed acceptable resemblance to field data observed during a concomitant in situ study, and similar conclusions can be drawn with respect to the quantitative impact of viruses and protozoa on the bacterial compartment. Viral decay rates ranged from undetectable to 0.078 h(-1) (average, 0.033 h(-1)), and the control of bacterial production from below the detection limit to 36% (average, 12%). The contribution of virus-induced lysis of bacteria to the dissolved organic matter pool as well as to benthic bacterial nutrition was low. Ingestion rates of protozoan grazers ranged from undetectable to 24.7 bacteria per heterotrophic nanoflagellate (HNF) per hour (average, 4.8 bacteria HNF(-1) h(-1)) and from undetectable to 73.3 bacteria per ciliate per hour (average, 11.2 bacteria ciliate(-1) h(-1)). Heterotrophic nanoflagellate and ciliates together cropped up to 5% (average, 1%) of bacterial production. The viral impact on bacteria prevailed over protozoan grazing by a factor of 2.5-19.9 (average, 9.5). In sum, these factors together removed up to 36% (average, 12%) of bacterial production. The high number of correlations between viral and protozoan parameters is discussed in view of a possible relationship between virus removal and the presence of protozoan grazers. PMID:16872403

  7. DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L. ) larvae

    SciTech Connect

    Keating, S.T.; Burand, J.P.; Elkinton, J.S. )

    1989-11-01

    Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. The hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.

  8. Comparison of DNA extraction methods in analysis of salivary bacterial communities.

    PubMed

    Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; François, Patrice; Schrenzel, Jacques

    2013-01-01

    Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

  9. Efficient extraction of virus DNA by NucliSens Extractor allows sensitive detection of hepatitis B virus by PCR.

    PubMed

    Gobbers, E; Oosterlaken, T A; van Bussel, M J; Melsert, R; Kroes, A C; Claas, E C

    2001-12-01

    The NucliSens Extractor is an automated nucleic acid isolation system based on guanidinium thiocyanate (GuSCN)-silica extraction technology. The system has been validated for the isolation of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNAs from human samples in combination with nucleic acid sequence-based amplification- and reverse transcription-PCR-based methods. We evaluated the extractor for hepatitis B virus (HBV) DNA extraction from human samples using a noncommercial HBV DNA PCR. Several sample pretreatment procedures in combination with the extractor were compared with the Qiagen extraction method, and the impact of the sample volume used in the extraction on the sensitivity was investigated. Heating of the lysed sample prior to extractor isolation and the use of a large sample volume resulted in highly sensitive detection of HBV DNA. Incubation of a 1-ml sample in GuSCN at 80 degrees C (10 min) and at 37 degrees C (30 min) allowed detection of 4 and 40 HBV genome equivalents/ml, respectively, in standard dilution panels. Sample lysis in GuSCN at room temperature and proteinase K treatment prior to use of the extractor were less efficient procedures. All clinical samples that were PCR positive after Qiagen extraction and/or that were HBsAg positive were also PCR positive after extractor isolation. HBV DNA, HCV RNA, and HIV type 1 RNA were efficiently coextracted from a single sample, allowing reliable detection of viral genomes.

  10. Archaeal Haloarcula californiae Icosahedral Virus 1 Highlights Conserved Elements in Icosahedral Membrane-Containing DNA Viruses from Extreme Environments

    PubMed Central

    Demina, Tatiana A.; Pietilä, Maija K.; Svirskaitė, Julija; Ravantti, Janne J.; Atanasova, Nina S.; Bamford, Dennis H.

    2016-01-01

    ABSTRACT 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. PMID:27435460

  11. Fast responses of bacterial membranes to virus adsorption: a fluorescence study.

    PubMed Central

    Bayer, M E; Bayer, M H

    1981-01-01

    After collision with their host cells, virus particles may remain mobile on cell surfaces until they become attached at firm binding sites. We propose that a virion will arrive within a typical median time at such a site, generating a membrane signal such as an increased membrane fluorescence in cells labeled with the voltage-sensitive dyes 8-anilino-1-naphthalene-sulfonate (Mg-salt) (ANS), N-phenylnaphthylamine (NPA), or 3,3'-dipentyl-2,2'-oxacarbocyanine (di-O-C5[3]). We found that the time span between virus adsorption and fluorescence response varies widely among phages and also depends on bacterial strain, metabolic state, and type of dye. di-O-C5[3]-labeled cells react within 1 sec to uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells labeled with ANS and NPA react to CCCP in 4-6 sec. Bacteriophages T4, T5, chi, and BF23, added to ANS-labeled cells, change the fluorescence in 9-15 sec. T-even ghosts cause a response at identical times. Baseplate-defective phage mutant T412- and isolated adsorption organelles of smaller viruses fail to cause an effect. di-O-C5[3]-labeled cells respond to T4 at a multiplicity of infection greater than or equal to 40 within 1 sec. A longer time (8 sec) is required at lower multiplicities. The receptor-degrading phages epsilon 15, epsilon 34, c 341, and K29 need the longest time (1 min for ANS) to cause a fluorescence increase. We suggest that the delayed fluorescence response is concomitant with the surface "walk" of the virion, which is terminated at an injection site. T4 tail sheath contraction coincides with the onset of the membrane fluorescence response. PMID:7029540

  12. Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication.

    PubMed Central

    Pfüller, R; Hammerschmidt, W

    1996-01-01

    During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells. It is not known, however, what the early events in viral DNA replication that yield these concatemers are. In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed. As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA). A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus. This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction. The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids. Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication. PMID:8648674

  13. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof

    PubMed Central

    Nolden, T.; Pfaff, F.; Nemitz, S.; Freuling, C. M.; Höper, D.; Müller, T.; Finke, Stefan

    2016-01-01

    Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache’s reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level. PMID:27046474

  14. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    PubMed

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment.

  15. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    PubMed

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment. PMID:26812543

  16. Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses.

    PubMed

    Pride, David T; Salzman, Julia; Relman, David A

    2012-09-01

    Explorations of human microbiota have provided substantial insight into microbial community composition; however, little is known about interactions between various microbial components in human ecosystems. In response to the powerful impact of viral predation, bacteria have acquired potent defences, including an adaptive immune response based on the clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas system. To improve our understanding of the interactions between bacteria and their viruses in humans, we analysed 13 977 streptococcal CRISPR sequences and compared them with 2 588 172 virome reads in the saliva of four human subjects over 17 months. We found a diverse array of viruses and CRISPR spacers, many of which were specific to each subject and time point. There were numerous viral sequences matching CRISPR spacers; these matches were highly specific for salivary viruses. We determined that spacers and viruses coexist at the same time, which suggests that streptococcal CRISPR/Cas systems are under constant pressure from salivary viruses. CRISPRs in some subjects were just as likely to match viral sequences from other subjects as they were to match viruses from the same subject. Because interactions between bacteria and viruses help to determine the structure of bacterial communities, CRISPR-virus analyses are likely to provide insight into the forces shaping the human microbiome.

  17. Molecular characterisation of a novel cassava associated circular ssDNA virus.

    PubMed

    Dayaram, Anisha; Opong, Allen; Jäschke, Anja; Hadfield, James; Baschiera, Marianna; Dobson, Renwick C J; Offei, Samuel K; Shepherd, Dionne N; Martin, Darren P; Varsani, Arvind

    2012-06-01

    The application of sequence non-specific rolling circle amplification of circular single stranded (ss) DNA molecules to viral metagenomics has facilitated the discovery in various ecosystems of what is probably a diverse array of novel ssDNA viruses. Here we describe a putative novel ssDNA virus (at a genome level), cassava associated circular DNA virus (CasCV), isolated from cassava leaf samples infected with the fungi Collectotrichum and Plectosphaerella. CasCV has a circular ambisense genome and shares significant genome similarities with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), Mosquito VEM virus SDBVL and Meles meles faecal virus (MmFV). The CasCV genome (2220 nt) has three large open reading frames. While it is probable that one of these encodes a capsid protein, the other two probably express a replication associated protein (Rep) following the removal of an intron such as that found in the Rep encoding genes of some geminiviruses. This Rep would contain four conserved rolling circle replication (RCR) related motifs that have previously been identified in geminivirus, circovirus and nanovirus Reps. Given both that the CasCV Rep and CP share 62.7% and 39.8% amino acid identity respectively with the Rep and CP of SsHADV-1, and that CasCV was discovered associated with cassava infecting fungi, we suggest that CasCV should be classified within the mycovirus taxonomic family. However, host range studies using infectious clones will be required to demonstrate the novel virus' likely origin and actual host species.

  18. Identification of a Novel Circular DNA Virus in New Zealand Fur Seal (Arctocephalus forsteri) Fecal Matter

    PubMed Central

    Sikorski, Alyssa; Dayaram, Anisha

    2013-01-01

    Fur seal feces-associated circular DNA virus (FSfaCV) is a novel virus isolated from the fecal matter of New Zealand fur seals. FSfaCV has two main open reading frames in its 2,925-nucleotide (nt) genome. The replication-associated protein (Rep) of FSfaCV has similarity to Rep-like sequences in the Giardia intestinalis genome. PMID:23929471

  19. Identification of a Novel Circular DNA Virus in New Zealand Fur Seal (Arctocephalus forsteri) Fecal Matter.

    PubMed

    Sikorski, Alyssa; Dayaram, Anisha; Varsani, Arvind

    2013-01-01

    Fur seal feces-associated circular DNA virus (FSfaCV) is a novel virus isolated from the fecal matter of New Zealand fur seals. FSfaCV has two main open reading frames in its 2,925-nucleotide (nt) genome. The replication-associated protein (Rep) of FSfaCV has similarity to Rep-like sequences in the Giardia intestinalis genome. PMID:23929471

  20. [Quantitative determination of the infectivity of nuclear polyhedrosis virus DNA on honeycomb moth (Galleria mellonella) larvae].

    PubMed

    Gorbunova, E E; Makarova, N I; Shchelkunova, G A

    1980-01-01

    The possibility of using honeycomb moth larvae for titration of nuclear polyhedrosis virus (NPV) infectious DNA and determinations of transfection effectiveness was studied. Honeycomb moth larvae were shown to be a sensitive system for NPV DNA titration. DEAE-dextran used as a protector increased NPV DNA infectivity 1000-fold, LD50 in this instance being 2 X 10(8) molecules per larva. The method of NPV DNA infectivity determinations by the number of larvae with polyhedreae in the fatty tissue is more sensitive than infectivity determinations by the number of dead larvae and permits titrations of low DNA concentrations. The curve of DNA titration in the presence of DEAE-dextran by the number of larvae with polyhedrae in the fatty tissue allows to quantitate native DNA within the range of 0.01 to 5 micrograms/ml.

  1. Implication of the Bacterial Endosymbiont Rickettsia spp. in Interactions of the Whitefly Bemisia tabaci with Tomato yellow leaf curl virus

    PubMed Central

    Kliot, Adi; Cilia, Michelle; Czosnek, Henryk

    2014-01-01

    ABSTRACT Numerous animal and plant viruses are transmitted by arthropod vectors in a persistent, circulative manner. Tomato yellow leaf curl virus (TYLCV) is transmitted by the sweet potato whitefly Bemisia tabaci. We report here that infection with Rickettsia spp., a facultative endosymbiont of whiteflies, altered TYLCV-B. tabaci interactions. A B. tabaci strain infected with Rickettsia acquired more TYLCV from infected plants, retained the virus longer, and exhibited nearly double the transmission efficiency compared to an uninfected B. tabaci strain with the same genetic background. Temporal and spatial antagonistic relationships were discovered between Rickettsia and TYLCV within the whitefly. In different time course experiments, the levels of virus and Rickettsia within the insect were inversely correlated. Fluorescence in situ hybridization analysis of Rickettsia-infected midguts provided evidence for niche exclusion between Rickettsia and TYLCV. In particular, high levels of the bacterium in the midgut resulted in higher virus concentrations in the filter chamber, a favored site for virus translocation along the transmission pathway, whereas low levels of Rickettsia in the midgut resulted in an even distribution of the virus. Taken together, these results indicate that Rickettsia, by infecting the midgut, increases TYLCV transmission efficacy, adding further insights into the complex association between persistent plant viruses, their insect vectors, and microorganism tenants that reside within these insects. IMPORTANCE Interest in bacterial endosymbionts in arthropods and many aspects of their host biology in agricultural and human health systems has been increasing. A recent and relevant studied example is the influence of Wolbachia on dengue virus transmission by mosquitoes. In parallel with our recently studied whitefly-Rickettsia-TYLCV system, other studies have shown that dengue virus levels in the mosquito vector are inversely correlated with

  2. Absence of bacterial DNA in culture-negative urine from cats with and without lower urinary tract disease.

    PubMed

    Lund, Heidi Sjetne; Skogtun, Gaute; Sørum, Henning; Eggertsdóttir, Anna Vigdís

    2015-10-01

    A diagnosis of bacterial cystitis commonly relies on a positive microbiological culture demonstrating the presence of a significant number of colony-forming units/ml urine, as urine within the upper urinary tract, bladder and proximal urethra generally is considered sterile. Recent studies from human and veterinary medicine indicate the presence of non-culturable bacteria in culture-negative urine samples. The aim of the present study was to determine the occurrence of bacterial DNA in culture-negative urine samples from cats with signs of feline lower urinary tract disease (FLUTD) and healthy control cats by 16S ribosomal DNA PCR and subsequent sequencing. The study sample included 38 culture-negative urine samples from cats with FLUTD and 43 culture-negative samples from control cats. Eight culture-positive urine samples from cats with FLUTD were included as external positive controls in addition to negative reaction controls. Of possible methodological limitations, degradation of DNA due to storage, the use of non-sedimented urine for DNA isolation and lack of internal positive reaction controls should be mentioned. The positive controls were recognised, but occurrence of bacterial DNA in culture-negative urine from cats with or without signs of lower urinary tract disease was not demonstrated. However, considering the possible methodological limitations, the presence of bacterial DNA in the urine of culture-negative FLUTD cats cannot be excluded based on the present results alone. Therefore, a prospective study reducing the possibility of degradation of DNA due to storage, in combination with modifications enhancing the chance of detecting even lower levels of bacterial DNA in culture-negative samples, seems warranted.

  3. Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).

    PubMed

    Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla

    2015-05-01

    Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most

  4. Effects of different methods of DNA extraction for activated sludge on the subsequent analysis of bacterial community profiles.

    PubMed

    Sun, Lianpeng; Ouyang, Xiong; Tang, Yueheng; Yang, Ying; Luo, Ying

    2012-02-01

    The effect of different DNA extraction protocols on activated sludge DNA yield and bacterial community composition was evaluated by temperature gradient gel electrophoresis (TGGE). Nine different procedures to extract DNA were compared-sonication (30s), sonication (40s), sonication (50s), freezing-thawing, bead milling, sodium dodecyl sulfate (SDS)-lysozyme, SDS-proteinase K, SDS-lysozyme-proteinase, and a commercial extraction kit. It was found that the TGGE profiles and the DNA band numbers made significant differences via various extraction methods. The yield and purity of DNA extracted by sonication and other physical methods were not satisfactory, while the DNA purity extracted by SDS and other chemical-biological methods were better. Crude DNA extracts isolated by sonication and other physical methods passed the polymerase chain reaction, despite the absence of purification and acquired affluent DNA bands in TGGE. The affluence of bands in TGGE was not consistent with the yield and purification of DNA, but was correlative with extraction protocols. To analyze the activated sludge bacterial community by TGGE fingerprint, it is necessary to make a synthesis of the TGGE fingerprint profiles of chemical and physical DNA extraction methods to overcome the representative bias.

  5. Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

    NASA Astrophysics Data System (ADS)

    Krupovic, Mart; Koonin, Eugene V.

    2014-06-01

    Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

  6. Application of motif-based tools on evolutionary analysis of multipartite single-stranded DNA viruses.

    PubMed

    Wang, Hsiang-Iu; Chang, Chih-Hung; Lin, Po-Heng; Fu, Hui-Chuan; Tang, Chuanyi; Yeh, Hsin-Hung

    2013-01-01

    Multipartite viruses contain more than one distinctive genome component, and the origin of multipartite viruses has been suggested to evolve from a non-segmented wild-type virus. To explore whether recombination also plays a role in the evolution of the genomes of multipartite viruses, we developed a systematic approach that employs motif-finding tools to detect conserved motifs from divergent genomic regions and applies statistical approaches to select high-confidence motifs. The information that this approach provides helps us understand the evolution of viruses. In this study, we compared our motif-based strategy with current alignment-based recombination-detecting methods and applied our methods to the analysis of multipartite single-stranded plant DNA viruses, including bipartite begomoviruses, Banana bunchy top virus (BBTV) (consisting of 6 genome components) and Faba bean necrotic yellows virus (FBNYV) (consisting of 8 genome components). Our analysis revealed that recombination occurred between genome components in some begomoviruses, BBTV and FBNYV. Our data also show that several unusual recombination events have contributed to the evolution of BBTV genome components. We believe that similar approaches can be applied to resolve the evolutionary history of other viruses.

  7. Application of Motif-Based Tools on Evolutionary Analysis of Multipartite Single-Stranded DNA Viruses

    PubMed Central

    Wang, Hsiang-Iu; Chang, Chih-Hung; Lin, Po-Heng; Fu, Hui-Chuan; Tang, ChuanYi; Yeh, Hsin-Hung

    2013-01-01

    Multipartite viruses contain more than one distinctive genome component, and the origin of multipartite viruses has been suggested to evolve from a non-segmented wild-type virus. To explore whether recombination also plays a role in the evolution of the genomes of multipartite viruses, we developed a systematic approach that employs motif-finding tools to detect conserved motifs from divergent genomic regions and applies statistical approaches to select high-confidence motifs. The information that this approach provides helps us understand the evolution of viruses. In this study, we compared our motif-based strategy with current alignment-based recombination-detecting methods and applied our methods to the analysis of multipartite single-stranded plant DNA viruses, including bipartite begomoviruses, Banana bunchy top virus (BBTV) (consisting of 6 genome components) and Faba bean necrotic yellows virus (FBNYV) (consisting of 8 genome components). Our analysis revealed that recombination occurred between genome components in some begomoviruses, BBTV and FBNYV. Our data also show that several unusual recombination events have contributed to the evolution of BBTV genome components. We believe that similar approaches can be applied to resolve the evolutionary history of other viruses. PMID:23936517

  8. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    PubMed

    Huang, Yong; Xing, Na; Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  9. RNA-dependent DNA polymerase activity of RNA tumor virus. VI. Processive mode of action of avian myeloblastosis virus polymerase.

    PubMed Central

    Leis, J P

    1976-01-01

    Purified avian myeloblastosis virus (AMV) polymerase consisting of alpha,beta subunits has been shown to act processively in catalyzing DNA synthesis primed with 34S AMV RNA oligo(dT), poly(A)-poly(dT), and poly(I)-poly(dC). DNA transcripts prepared with 34S AMV RNA-oligo(dT)14 and AMV polymerase (alphabeta) have been shown to have a molecular weight of 1.05 X 10(6), or approximately one-third the size of the 34S RNA genome. Polymerase subunit alpha acts nonprocessively with the above templates. PMID:61286

  10. Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing

    PubMed Central

    Lagacé, L.; Pitre, M.; Jacques, M.; Roy, D.

    2004-01-01

    The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the γ- and β-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). γ-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control. PMID:15066796

  11. Tsv-N1: A Novel DNA Algal Virus that Infects Tetraselmis striata

    PubMed Central

    Pagarete, António; Grébert, Théophile; Stepanova, Olga; Sandaa, Ruth-Anne; Bratbak, Gunnar

    2015-01-01

    Numbering in excess of 10 million per milliliter of water, it is now undisputed that aquatic viruses are one of the major factors shaping the ecology and evolution of Earth’s microbial world. Nonetheless, environmental viral diversity and roles remain poorly understood. Here we report the first thorough characterization of a virus (designated TsV) that infects the coastal marine microalga Tetraselmis striata. Unlike previously known microalgae-infecting viruses, TsV is a small (60 nm) DNA virus, with a 31 kb genome. From a range of eight different strains belonging to the Chlamydomonadaceae family, TsV was only able to infect T. striata. Gene expression dynamics revealed an up-regulation of viral transcripts already 1 h post-infection (p.i.). First clear signs of infection were observed 24 h p.i., with the appearance of viral factories inside the nucleus. TsV assembly was exclusively nuclear. TsV-N1 genome revealed very different from previously known algae viruses (Phycodnaviridae). Putative function and/or homology could be resolved for only 9 of the 33 ORFs encoded. Among those was a surprising DNA polymerase type Delta (only found in Eukaryotes), and two genes with closest homology to genes from human parasites of the urogenital tract. These results support the idea that the diversity of microalgae viruses goes far beyond the Phycodnaviridae family and leave the door open for future studies on implications of microalgae viruses for human health. PMID:26193304

  12. Tsv-N1: A Novel DNA Algal Virus that Infects Tetraselmis striata.

    PubMed

    Pagarete, António; Grébert, Théophile; Stepanova, Olga; Sandaa, Ruth-Anne; Bratbak, Gunnar

    2015-07-01

    Numbering in excess of 10 million per milliliter of water, it is now undisputed that aquatic viruses are one of the major factors shaping the ecology and evolution of Earth's microbial world. Nonetheless, environmental viral diversity and roles remain poorly understood. Here we report the first thorough characterization of a virus (designated TsV) that infects the coastal marine microalga Tetraselmis striata. Unlike previously known microalgae-infecting viruses, TsV is a small (60 nm) DNA virus, with a 31 kb genome. From a range of eight different strains belonging to the Chlamydomonadaceae family, TsV was only able to infect T. striata. Gene expression dynamics revealed an up-regulation of viral transcripts already 1 h post-infection (p.i.). First clear signs of infection were observed 24 h p.i., with the appearance of viral factories inside the nucleus. TsV assembly was exclusively nuclear. TsV-N1 genome revealed very different from previously known algae viruses (Phycodnaviridae). Putative function and/or homology could be resolved for only 9 of the 33 ORFs encoded. Among those was a surprising DNA polymerase type Delta (only found in Eukaryotes), and two genes with closest homology to genes from human parasites of the urogenital tract. These results support the idea that the diversity of microalgae viruses goes far beyond the Phycodnaviridae family and leave the door open for future studies on implications of microalgae viruses for human health.

  13. Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.

    PubMed Central

    Davis, A R; Bos, T J; Nayak, D P

    1983-01-01

    We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein. Images PMID:6306656

  14. Diversity and Abundance of Single-Stranded DNA Viruses in Human Feces▿†

    PubMed Central

    Kim, Min-Soo; Park, Eun-Jin; Roh, Seong Woon; Bae, Jin-Woo

    2011-01-01

    In this study, we investigated the abundance and diversity of single-stranded DNA (ssDNA) viruses in fecal samples from five healthy individuals through a combination of serial filtration and CsCl gradient ultracentrifugation. Virus abundance ranged from 108 to 109 per gram of feces, and virus-to-bacterium ratios were much lower (less than 0.1) than those observed in aquatic environments (5 to 10). Viral DNA was extracted and randomly amplified using phi29 polymerase and analyzed through high-throughput 454 pyrosequencing. Among 400,133 sequences, an average of 86.2% viromes were previously uncharacterized in public databases. Among previously known viruses, double-stranded DNA podophages (52 to 74%), siphophages (11 to 30%), myophages (1 to 4%), and ssDNA microphages (3 to 9%) were major constituents of human fecal viromes. A phylogenetic analysis of 24 large contigs of microphages based on conserved capsid protein sequences revealed five distinct newly discovered evolutionary microphage groups that were distantly related to previously known microphages. Moreover, putative capsid protein sequences of five contigs were closely related to prophage-like sequences in the genomes of three Bacteroides and three Prevotella strains, suggesting that Bacteroides and Prevotella are the sources of infecting microphages in their hosts. PMID:21948823

  15. RNase H and RNA-directed DNA polymerase: associated enzymatic activities of murine mammary tumor virus.

    PubMed Central

    Dion, A S; Williams, C J; Moore, D H

    1977-01-01

    The RNA-directed DNA polymerase of murine mammary tumor virus, a type B RNA tumor virus, was purified sequentially through DEAE-cellulose, phosphocellulose (step gradient), and phosphocellulose (linear salt gradient) chromatography followed by glycerol sedimentation centrifugation. During all stages of purification, coincident peaks of RNA-directed DNA polymerase activity, templated by polyribocytidylate-oligodeoxyguanidylate, and RNase H digestion of [3H]polyriboadenylate-polydeoxythymidylate were observed, and both enzymatic activities displayed a cation preference for magnesium. Under conditions that removed adventitiously associated nucleases, RNase H activity was found to co-purify with polymerase. The specificity of this nuclease was assayed with various prepared substrates, which indicated that the polymerase-associated RNase H activity was directed only against the RNA strand of an RNA-DNA hybrid. It is highly probable that RNase H (RNA-DNA hybrid: ribonucleotide-hydrolase, EC 3.1.4..34) and RNA-directed DNA polymerase of type B viruses are associated enzymatic activities analogous to those observed for avian and mammalian type C RNA tumor viruses. Images PMID:67221

  16. Infectious cDNA Clone of the Epidemic West Nile Virus from New York City

    PubMed Central

    Shi, Pei-Yong; Tilgner, Mark; Lo, Michael K.; Kent, Kim A.; Bernard, Kristen A.

    2002-01-01

    We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 × 109 to 5 × 109 PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3′-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV. PMID:12021317

  17. Repair of base damage and genome maintenance in the nucleo-cytoplasmic large DNA viruses.

    PubMed

    Redrejo-Rodríguez, Modesto; Salas, María L

    2014-01-22

    Among the DNA viruses, the so-called nucleo-cytoplasmic large DNA viruses (NCLDV) constitute a monophyletic group that currently consists of seven families of viruses infecting a very broad variety of eukaryotes, from unicellular marine protists to humans. Many recent papers have analyzed the sequence and structure of NCLDV genomes and their phylogeny, providing detailed analysis about their genomic structure and evolutionary history and proposing their inclusion in a new viral order named Megavirales that, according to some authors, should be considered as a fourth domain of life, aside from Bacteria, Archaea and Eukarya. The maintenance of genetic information protected from environmental attacks and mutations is essential not only for the survival of cellular organisms but also viruses. In cellular organisms, damaged DNA bases are removed in two major repair pathways: base excision repair (BER) and nucleotide incision repair (NIR) that constitute the major pathways responsible for repairing most endogenous base lesions and abnormal bases in the genome by precise repair procedures. Like cells, many NCLDV encode proteins that might constitute viral DNA repair pathways that would remove damages through BER/NIR pathways. However, the molecular mechanisms and, specially, the biological roles of those viral repair pathways have not been deeply addressed in the literature so far. In this paper, we review viral-encoded BER proteins and the genetic and biochemical data available about them. We propose and discuss probable viral-encoded DNA repair mechanisms and pathways, as compared with the functional and molecular features of known homologs proteins.

  18. Detection of Marek's disease virus DNA in Japanese quail susceptible to atherosclerosis

    SciTech Connect

    Pyrzak, R.; Shih, J.C.H.

    1986-03-01

    Marek's disease virus (MDV) was demonstrated as an etiological agent which causes atherosclerosis in the chicken. Since herpes viruses are ubiquitous, incidences of viral atherogenesis in humans and other animals were speculated. In this laboratory, the atherosclerosis susceptible (SUS) and resistant (RES) Japanese quail were developed as the animal model for atherosclerosis research. The susceptibility of the animal might be due to an infection of MDV or a related quail herpes virus (QHV). An initial attempt to isolate viruses from quail and an agar gel precipitin test for MDC were not positive. A DNA hybridization technique was used to determine whether the MDC-DNA existed in the quail cell. The gene library of MDV EcoRl DNA fragments was used to prepare the DNA probe, labeled with (/sup 32/P) by nick translation. Dot hybridizations were carried out by mixing the MDV-DNA probe with DNAs isolated from quail tissues. A high stringent condition was used. From this experiment it was found that the tissues from the SUS quail were hybridization positive, but most of them from RES quail were negative. When aortas were compared, the severe atherosclerotic had a strong hybridization (3-4 cop. of genome/cell) whereas the others hybridized moderately (1 cop./cell). It was concluded that genes from MDV or a QHV indeed existed in Japanese quail.

  19. Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

    PubMed Central

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-01-01

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

  20. Neisseria gonorrhoeae Bacterial DNA Load in the Pharynges and Saliva of Men Who Have Sex with Men.

    PubMed

    Chow, Eric P F; Tabrizi, Sepehr N; Phillips, Samuel; Lee, David; Bradshaw, Catriona S; Chen, Marcus Y; Fairley, Christopher K

    2016-10-01

    Neisseria gonorrhoeae can be cultured in the saliva of individuals with pharyngeal gonorrhea. The aim of this study was to quantify the gonococcal bacterial DNA loads in the pharynges and saliva among men who have sex with men (MSM) with untreated pharyngeal gonorrhea. Untreated MSM who tested positive for pharyngeal gonorrhea by culture and returned for antibiotic treatment within 14 days at the Melbourne Sexual Health Centre between October 2014 and March 2015 were eligible for this study. The gonococcal bacterial DNA load was measured using real-time quantitative PCR. The median gonococcal bacterial DNA loads in the pharynges and saliva were calculated and compared to culture positivity using the Mann-Whitney U test. A total of 33 men were included in this study. The median gonococcal bacterial DNA load did not differ between the pharynges in men who were culture positive (2.5 × 10(5) copies/swab) and culture negative (2.9 × 10(4) copies/swab) (P = 0.166) and the saliva (culture positive, 2.2 × 10(5) copies/ml; culture negative, 2.7 × 10(5) copies/ml) (P = 0.499). The bacterial DNA load in the pharynges (P = 0.695) and saliva (P = 0.969) did not differ between who men returned for treatment within 7 days and those who returned 8 to 14 days later. Substantial gonococcal bacterial DNA loads were detected in both saliva and pharynges among MSM with pharyngeal gonorrhea. These findings suggest that gonorrhea can be transmitted via sexual practices involving exposure to saliva, such as oroanal practices (rimming) and saliva use as a lubricant for anal sex. PMID:27413195

  1. RNA primer used in synthesis of anticomplementary DNA by reverse transcriptase of avian myeloblastosis virus.

    PubMed Central

    Myers, J C; Dobkin, C; Spiegelman, S

    1980-01-01

    When either the homologous RNA (avian myeloblastosis virus RNA) or a heterologous RNA (poliovirus RNA) was used as a template, the anticomplementary DNA synthesized in vitro by avian myeloblastosis virus reverse transcriptase (RNA-directed DNA nucleotidyltransferase, EC 2.7.7.7) was primed by fragments of the original RNA template that usually had adenosine at their 3' ends. When we used phage T/ RNA ligase (EC 6.5.1.3) to label the 3' end of the RNA template fragments contained in the RNA . cDNA hybrid intermediate, adenosine was found to be the principal nucleoside carrying the label. We infer from these results that the ribonuclease H (hybrid nuclease) activity of the reverse transcriptase creates fragments of the original RNA template with adenosine as the principal 3' terminus and that these fragments serve as primers for the synthesis of anticomplementary DNA. Images PMID:6154930

  2. Molecular analysis of bacterial isolates and total community DNA from kraft pulp mill effluent treatment systems.

    PubMed

    Fortin, N; Fulthorpe, R R; Allen, D G; Greer, C W

    1998-06-01

    Chloroaliphatics are major components of bleached kraft mill effluents. Gene probes and oligonucleotide primers were developed to monitor kraft pulp mill effluent treatment systems for the presence of key genes (dehalogenases) responsible for the dehalogenation of chloroaliphatic organics. The primers were used for polymerase chain reaction (PCR) analysis of genomic DNA extracted from dehalogenating bacterial isolates and from total community DNA extracted from water and sediments of mill effluent treatment system. PCR amplification with oligonucleotide primers designed from dhlB, encoding the haloacid dehalogenase from Xanthobacter autotrophicus, revealed the presence of dehalogenase genes in both aerated lagoons and stabilization basins. Similarly, positive results were obtained with mmoX primers designed from the soluble methane monooxygenase gene of Methylococcus capsulatus Bath. The haloacetate dehalogenase encoding gene (dehH2) from Moraxella sp. was typically not detected in mill effluent treatment systems unless the biomass was selectively enriched. DNA sequence analysis of several PCR fragaments revealed significant similarity to known dehalogenase amd methane monooxygenase genes. The results indicated a broad distribution of known dehalogenation genes and bacteria with chloroorganic-degrading potential in the mill effluent treatment systems. PMID:9734304

  3. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  4. DNA damage promotes Herpes Simplex Virus-1 protein expression in a neuroblastoma cell line

    PubMed Central

    Volcy, Ketna; Fraser, Nigel W.

    2013-01-01

    Although the induction of the cellular DNA damage response by Herpes simplex virus-1 (HSV-1) infection of epithelial cells in tissue culture promotes productive infection, there has been no experimental observation of the effect of the cellular DNA damage response on HSV-1 infection in vivo or in neuronal derived cell lines in tissue culture. Thus, it has been speculated that the lack of cellular DNA damage induction during infection of neurons may promote latency in these cells. This work examines the profile of HSV-1 promoter induction and protein expression, in the absence or presence of infection; using cellular DNA damage inducing topoisomerase inhibitors (Camptothecin and Etoposide) on a neuroblastoma cell line (C1300) in which HSV-1 infection fails to induce the DNA damage response. In the absence of infection, a plasmid expressing the immediate early ICP0 promoter was the most induced by the DNA damage drug treatments compared to the early (RR) and late (VP16) gene promoters. Similarly, drug treatment of C1300 cells infected with HSV-1 virus showed enhanced protein expression for ICP0, but not ICP4 and VP16 proteins. However, when the cells were infected with a HSV-1 virus defective in the immediate early gene trans-activator VP16 (in814) and treated with the DNA damaging drugs, there was enhanced expression of immediate early and late HSV-1 proteins. Although, viral infection of the neuroblastoma cell alone did not induce DNA damage, cellular DNA damage induced by drug treatments facilitated viral promoter induction and viral protein expression. This implicates a mechanism by which HSV-1 viral genes in a quiescent or latent state may become induced by cellular DNA damage in neuronal cells to facilitate productive infection. PMID:23354549

  5. Insertion of reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of a very virulent Marek's disease virus alters its pathogenicity.

    PubMed

    Mays, Jody K; Silva, Robert F; Kim, Taejoong; Fadly, Aly

    2012-01-01

    Co-cultivation of the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in the generation of a recombinant MDV containing the REV long terminal repeat (LTR) named the RM1 strain of MDV, a strain that was highly attenuated for oncogenicity but induced severe bursal and thymic atrophy. We hypothesize that the phenotypic changes were solely due to the LTR insertion. Furthermore, we hypothesize that insertion of REV LTR into an analogous location in a different MDV would result in a similar phenotypic change. To test these hypotheses, we inserted the REV LTR into a bacterial artificial chromosome (BAC) clone of a very virulent strain of MDV, Md5, and designated the virus rMd5-RM1-LTR. The rMd5-RM1-LTR virus and the rMd5 virus were passaged in duck embryo fibroblast cells for up to 40 passages before pathogenicity studies. Susceptible chickens were inoculated intra-abdominally at hatch with the viruses rMd5-RM1-LTR, rMd5 BAC parental virus, wild-type strain Md5, or strain RM1 of MDV. The rMd5-RM1-LTR virus was attenuated at cell culture passage 40, whereas the rMd5 BAC without RM1 LTR retained its pathogenicity at cell culture passage 40. Using polymerase chain analysis, the RM1 LTR insert was detected in MDV isolated from buffy coat cells collected from chickens inoculated with rMd5-RM1-LTR, but only at 1 week post inoculation. The data suggest that the presence of the RM1 LTR insert within MDV genome for 1 week post inoculation with virus at hatch is sufficient to cause a reduction in pathogenicity of strain Md5 of MDV.

  6. GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases

    PubMed Central

    Roberts, Jane L.; Tavallai, Mehrad; Nourbakhsh, Aida; Fidanza, Abigail; Cruz‐Luna, Tanya; Smith, Elizabeth; Siembida, Paul; Plamondon, Pascale; Cycon, Kelly A.; Doern, Christopher D.; Booth, Laurence

    2015-01-01

    Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi‐kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug‐treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged ‘rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre‐treatment with the ‘Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. ‘Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of ‘Rafenib drugs/pazopanib restored antibiotic sensitivity in pan‐antibiotic resistant bacteria including multiple strains of bla kpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. J. Cell. Physiol. 230: 2552–2578, 2015. © 2015 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc. PMID:25858032

  7. GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases.

    PubMed

    Roberts, Jane L; Tavallai, Mehrad; Nourbakhsh, Aida; Fidanza, Abigail; Cruz-Luna, Tanya; Smith, Elizabeth; Siembida, Paul; Plamondon, Pascale; Cycon, Kelly A; Doern, Christopher D; Booth, Laurence; Dent, Paul

    2015-10-01

    Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged 'rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the 'Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. 'Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of 'Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. PMID:25858032

  8. GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases

    PubMed Central

    ROBERTS, JANE L.; TAVALLAI, MEHRAD; NOURBAKHSH, AIDA; FIDANZA, ABIGAIL; CRUZ-LUNA, TANYA; SMITH, ELIZABETH; SIEMBIDA, PAUL; PLAMONDON, PASCALE; CYCON, KELLY A.; DOERN, CHRISTOPHER D.; BOOTH, LAURENCE; DENT, PAUL

    2016-01-01

    Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged ‘rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the ‘Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. ‘Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of ‘Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. PMID:25858032

  9. GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases.

    PubMed

    Roberts, Jane L; Tavallai, Mehrad; Nourbakhsh, Aida; Fidanza, Abigail; Cruz-Luna, Tanya; Smith, Elizabeth; Siembida, Paul; Plamondon, Pascale; Cycon, Kelly A; Doern, Christopher D; Booth, Laurence; Dent, Paul

    2015-10-01

    Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged 'rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the 'Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. 'Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of 'Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections.

  10. Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

    PubMed

    Pandya, P R; Singh, K M; Parnerkar, S; Tripathi, A K; Mehta, H H; Rank, D N; Kothari, R K; Joshi, C G

    2010-01-01

    Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85-89% similar to 16S rDNA database sequences. For the remaining 3.14%; the similarity was lower than 85% Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of the Cytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good's coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen. PMID:20720314

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

    SciTech Connect

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

    2014-10-15

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

  12. Infectious hepatitis B virus from cloned DNA of known nucleotide sequence.

    PubMed Central

    Will, H; Cattaneo, R; Darai, G; Deinhardt, F; Schellekens, H; Schaller, H

    1985-01-01

    The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA. Images PMID:2983320

  13. Nutrition status and small intestinal bacterial overgrowth in patients with virus-related cirrhosis.

    PubMed

    Yao, Jia; Chang, Le; Yuan, Lili; Duan, Zhongping

    2016-01-01

    Malnutrition and small intestinal bacterial overgrowth (SIBO) is frequently present in patients with liver cirrhosis (LC). However, the direct relationship between SIBO and nutrition status in the LC patients has not been elucidated. The aim of this study was to investigate whether there was an association between nutrition status, evaluated by the subjective global assessment (SGA) and SIBO in patients with Hepatitis B virus (HBV) or hepatitis C virus (HCV) related cirrhosis. A total of 120 patients with HBV or HCV-related cirrhosis and 30 healthy controls were included. Nutritional status was determined according to SGA and anthropometry. All patients and healthy controls underwent a glucose hydrogen breath test for SIBO. The prevalence of malnutrition for the patients with HBV or HCV related cirrhosis ranged 19.4%-60% in China. The highest prevalence of malnutrition was detected by SGA, the lowest by triceps skinfold thickness. The frequency of SIBO was significantly higher in the malnourished (SGA-B/C) than in the well-nourished (SGA-A) patients with HBV or HCV related cirrhosis [41/72 (56.9%) vs 12/48 (25.0%) (p=0.001)]. Univariate analysis showed that SIBO, ascites, and Child-Turcotte-Pugh (CTP) class were associated with malnutrition. Multivariate analysis demonstrated that SIBO [odds ratio (OR) 8.10; p=0.002] and ascites (OR 4.56; p=0.022) were independently associated with the occurrence of malnutrition (SGA-B/C) in the same subjects. SIBO is independently related to the occurrence of malnutrition (SGA-B/C) in patients with HBV or HCV cirrhosis. We deduce that SIBO may play an important role in nutrition status in patients with HBV or HCV cirrhosis.

  14. Nutrition status and small intestinal bacterial overgrowth in patients with virus-related cirrhosis.

    PubMed

    Yao, Jia; Chang, Le; Yuan, Lili; Duan, Zhongping

    2016-01-01

    Malnutrition and small intestinal bacterial overgrowth (SIBO) is frequently present in patients with liver cirrhosis (LC). However, the direct relationship between SIBO and nutrition status in the LC patients has not been elucidated. The aim of this study was to investigate whether there was an association between nutrition status, evaluated by the subjective global assessment (SGA) and SIBO in patients with Hepatitis B virus (HBV) or hepatitis C virus (HCV) related cirrhosis. A total of 120 patients with HBV or HCV-related cirrhosis and 30 healthy controls were included. Nutritional status was determined according to SGA and anthropometry. All patients and healthy controls underwent a glucose hydrogen breath test for SIBO. The prevalence of malnutrition for the patients with HBV or HCV related cirrhosis ranged 19.4%-60% in China. The highest prevalence of malnutrition was detected by SGA, the lowest by triceps skinfold thickness. The frequency of SIBO was significantly higher in the malnourished (SGA-B/C) than in the well-nourished (SGA-A) patients with HBV or HCV related cirrhosis [41/72 (56.9%) vs 12/48 (25.0%) (p=0.001)]. Univariate analysis showed that SIBO, ascites, and Child-Turcotte-Pugh (CTP) class were associated with malnutrition. Multivariate analysis demonstrated that SIBO [odds ratio (OR) 8.10; p=0.002] and ascites (OR 4.56; p=0.022) were independently associated with the occurrence of malnutrition (SGA-B/C) in the same subjects. SIBO is independently related to the occurrence of malnutrition (SGA-B/C) in patients with HBV or HCV cirrhosis. We deduce that SIBO may play an important role in nutrition status in patients with HBV or HCV cirrhosis. PMID:27222411

  15. Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase.

    PubMed Central

    Saito, K; Miura, N; Yamazaki, M; Hirano, H; Murakoshi, I

    1992-01-01

    Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach. Images PMID:1518833

  16. NKLP27: a teleost NK-lysin peptide that modulates immune response, induces degradation of bacterial DNA, and inhibits bacterial and viral infection.

    PubMed

    Zhang, Min; Li, Mo-fei; Sun, Li

    2014-01-01

    NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

  17. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    PubMed

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  18. Search for varicella zoster virus DNA in saliva of healthy individuals aged 20-59 years.

    PubMed

    Birlea, Marius; Cohrs, Randall J; Bos, Nathan; Mehta, Satish K; Pierson, Duane L; Gilden, Don

    2014-02-01

    All neurological and ocular complications of varicella zoster virus (VZV) reactivation can occur without rash. Virological verification requires detection of VZV DNA or anti-VZV IgG antibody in cerebrospinal fluid (CSF), or anti-VZV IgM antibody in serum or CSF. If VZV were readily detected in other tissue in patients with neurological disease without rash and found to correlate with tests listed above, more invasive tests such as lumbar puncture might be obviated. Saliva is a potential source of VZV DNA. To study the potential diagnostic value of detecting VZV DNA in saliva from patients with neurological disease, saliva of healthy adults was searched for VZV DNA. A single saliva sample obtained by passive drool was centrifuged at 16,000g for 20 min. DNA was extracted from the supernatant and cell pellet and examined in triplicate for VZV DNA by real time PCR. A single random saliva sample from 80 healthy men and women aged 20-59 years revealed no VZV DNA (Table ), but was uniformly positive for cell (GAPdH) DNA. Because VZV DNA was not found in a random saliva sample from 80 individuals 20-59-year-old, a VZV-positive sample during neurologic disease may have potential significance. Further studies will determine whether VZV DNA in saliva correlates with VZV DNA or anti-VZV antibody in CSF in patients with neurological disease.

  19. Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE.

    PubMed

    Mew, A J; Ionas, G; Clarke, J K; Robinson, A J; Marshall, R B

    1985-12-01

    Sixteen isolates of Mycoplasma ovipneumoniae recovered from the nasal tract or lungs of sheep from different flocks in New Zealand were examined by bacterial restriction endonuclease DNA analysis (BRENDA) using EcoR1 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). All isolates gave BRENDA patterns which differed entirely from one another. Following 20 serial passages (corresponding to approximately 67 generations) of an isolate, no change was detected in the BRENDA pattern. When eight isolates were examined by SDS-PAGE most bands were common but, nevertheless, each isolate was unique in the sense that they differed from one another in one or more bands. The marked heterogeneity of patterns observed when strains of M. ovipneumoniae are compared by BRENDA, together with the stability of such patterns over many generations, will enable this approach to be used to study the epidemiology of individual strains of M. ovipneumoniae within a flock.

  20. Dynamics of the expanding DNA nucleoid released from a bacterial cell

    NASA Astrophysics Data System (ADS)

    Odijk, T.

    2000-03-01

    A semiquantitative theoretical discussion is given of a DNA nucleoid escaping from a bacterial cell after lysis. There is a strong excluded-volume effect causing the nucleoid to expand, though it may be held together globally by various constraints. It is argued that the friction is dominated by the fairly local motion of superhelical segments. In an intermediate regime, hydrodynamic screening is strong. In a diffusion model a self-similar solution is presented of the non-linear diffusion equation. An affinely deforming model for the expanding nucleoid is also given. The predicted rate of expansion seems to be in qualitative accord with recent, preliminary experiments on lysed Escherichia coli viewed under the microscope.

  1. New Type of Papillomavirus and Novel Circular Single Stranded DNA Virus Discovered in Urban Rattus norvegicus Using Circular DNA Enrichment and Metagenomics.

    PubMed

    Hansen, Thomas Arn; Fridholm, Helena; Frøslev, Tobias Guldberg; Kjartansdóttir, Kristín Rós; Willerslev, Eske; Nielsen, Lars Peter; Hansen, Anders Johannes

    2015-01-01

    Rattus norvegicus (R. norvegicus) are ubiquitous and their presence has several effects on the human populations in our urban areas on a global scale. Both historically and presently, this close interaction has facilitated the dissemination of many pathogens to humans, making screening for potentially zoonotic and emerging viruses in rats highly relevant. We have investigated faecal samples from R. norvegicus collected from urban areas using a protocol based on metagenomic enrichment of circular DNA genomes and subsequent sequencing. We found a new type of papillomavirus, with a L1 region 82% identical to that of the known R. norvegicus Papillomavirus 2. Additionally, we found 20 different circular replication associated protein (Rep)-encoding single stranded DNA (CRESS-DNA) virus-like genomes, one of which has homology to the replication-associated gene of Beak and feather disease virus. Papillomaviruses are a group of viruses known for their carcinogenic potential, and although they are known to infect several different vertebrates, they are mainly studied and characterised in humans. CRESS-DNA viruses are found in many different environments and tissue types. Both papillomaviruses and CRESS-DNA viruses are known to have pathogenic potential and screening for novel and known viruses in R. norvegicus could help identify viruses with pathogenic potential. PMID:26559957

  2. Detection of virus-specific RNA in simian sarcoma-leukemia virus-infected cells in in situ hybridization to viral complementary DNA.

    PubMed Central

    Kaufman, S L; Gallo, R C; Miller, N R

    1979-01-01

    An in situ molecular hybridization system which will detect retrovirus RNA in the cytoplasm of individual virus-infected cells has been developed. The technique was applied to cells infected with simian sarcoma-leukemia virus, where the virus-specific RNA was detected by hybridization to simian sarcoma-leukemia virus 3H-labeled complementary DNA. The system is useful for detecting viral RNA-containing cells in the presence of an excess of virus-negative cells and for determining which type of cell in a heterogenous population is expressing viral RNA. Images PMID:224220

  3. De novo reconstruction of plant RNA and DNA virus genomes from viral siRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In antiviral defense, plants produce massive quantities of 21-24 nucleotide siRNAs. Here we demonstrate that the complete genomes of DNA and RNA viruses and viroids can be reconstructed by deep sequencing and de novo assembly of viral/viroid siRNAs from experimentally- and naturally-infected plants....

  4. Assessment of [3H]Thymidine Incorporation into DNA as a Method To Determine Bacterial Productivity in Stream Bed Sediments

    PubMed Central

    Kaplan, Louis A.; Bott, Thomas L.; Bielicki, John K.

    1992-01-01

    We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25°C, but hydrolysis at 120°C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 μM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats. PMID:16348806

  5. Molecular cloning of covalently closed circular DNA of bovine leukemia virus.

    PubMed Central

    Kashmiri, S V; Mehdi, R; Ferrer, J F

    1984-01-01

    The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs. Images PMID:6319758

  6. Novel DNA virus isolated from samples showing endothelial cell necrosis in the Japanese eel, Anguilla japonica.

    PubMed

    Mizutani, Tetsuya; Sayama, Yusuke; Nakanishi, Akira; Ochiai, Hideharu; Sakai, Kouji; Wakabayashi, Kouji; Tanaka, Nozomi; Miura, Emi; Oba, Mami; Kurane, Ichiro; Saijo, Masayuki; Morikawa, Shigeru; Ono, Shin-ichi

    2011-03-30

    Economic loss due to viral endothelial cell necrosis of eel (VECNE) of Anguilla japonica is a serious problem for the cultured Japanese eel market. However, the viral genome responsible for VECNE is unknown. We recently developed a rapid determination system for viral nucleic acid sequences (RDV) to determine viral genome sequences. In this study, viral DNA fragments were obtained using RDV, and approximately 15-kbp circular full genome sequences were determined using a next-generation sequencing system, overlapping PCR, and Southern blot analysis. One open reading frame (ORF) was homologous to the large T-antigen of polyomavirus; other ORFs have no homology with any nucleic or amino acid sequences of polyomavirus. Therefore, as this DNA virus might comprise a novel virus family, we provisionally named it Japanese eel endothelial cells-infecting virus (JEECV). JEECV was detected in both naturally and experimentally infected eels, suggesting that JEECV potentially causes VECNE. PMID:21277610

  7. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology

    PubMed Central

    Legendre, Matthieu; Bartoli, Julia; Shmakova, Lyubov; Jeudy, Sandra; Labadie, Karine; Adrait, Annie; Lescot, Magali; Poirot, Olivier; Bertaux, Lionel; Bruley, Christophe; Couté, Yohann; Rivkina, Elizaveta; Abergel, Chantal; Claverie, Jean-Michel

    2014-01-01

    The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 μm in diameter and adenine–thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 μm in length and guanine–cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 μm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health. PMID:24591590

  8. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.

    PubMed

    Legendre, Matthieu; Bartoli, Julia; Shmakova, Lyubov; Jeudy, Sandra; Labadie, Karine; Adrait, Annie; Lescot, Magali; Poirot, Olivier; Bertaux, Lionel; Bruley, Christophe; Couté, Yohann; Rivkina, Elizaveta; Abergel, Chantal; Claverie, Jean-Michel

    2014-03-18

    The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 μm in diameter and adenine-thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 μm in length and guanine-cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 μm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health.

  9. An active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells.

    PubMed

    Bagh, Sangram; Mandal, Mahuya; Ang, Jordan; McMillen, David R

    2011-03-01

    Synthetic biology includes an effort to logically control cellular behavior. One long-term goal is to implement medical interventions inside living cells, creating intracellular "disease fighters"; one may imagine a system that detects viral infection and responds to halt the spread of the virus. Here, we explore a system designed to display some of the qualitative features that such disease prevention systems should have, while not claiming that the system itself has any medical application. An intracellular disease prevention mechanism should: lie dormant in the absence of the disease state; detect the onset of a lethal disease pathway; respond to halt or mitigate the disease's effects; and be subject to external deactivation when required. We have created a device that displays these properties, in the highly simplified case of a bacterial viral disease. Our system detects the onset of the lytic phase of bacteriophage lambda in Escherichia coli, responds by preventing this lethal pathway from being followed, and is deactivated by a temperature shift. We have formulated a mathematical model of the engineered system, using parameters obtained from the literature and by local experimental measurement, and shown that the model captures the essential experimental behavior of the system in most parameter regimes.

  10. Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes.

    PubMed

    Minkah, Nana; Hwang, Young; Perry, Kay; Van Duyne, Gregory D; Hendrickson, Robert; Lefkowitz, Elliot J; Hannenhalli, Sridhar; Bushman, Frederic D

    2007-08-15

    Topoisomerase enzymes regulate superhelical tension in DNA resulting from transcription, replication, repair, and other molecular transactions. Poxviruses encode an unusual type IB topoisomerase that acts only at conserved DNA sequences containing the core pentanucleotide 5'-(T/C)CCTT-3'. In X-ray structures of the variola virus topoisomerase bound to DNA, protein-DNA contacts were found to extend beyond the core pentanucleotide, indicating that the full recognition site has not yet been fully defined in functional studies. Here we report quantitation of DNA cleavage rates for an optimized 13 bp site and for all possible single base substitutions (40 total sites), with the goals of understanding the molecular mechanism of recognition and mapping topoisomerase sites in poxvirus genome sequences. The data allow a precise definition of enzyme-DNA interactions and the energetic contributions of each. We then used the resulting "action matrix" to show that favorable topoisomerase sites are distributed all along the length of poxvirus DNA sequences, consistent with a requirement for local release of superhelical tension in constrained topological domains. In orthopox genomes, an additional central cluster of sites was also evident. A negative correlation of predicted topoisomerase sites was seen relative to early terminators, but no correlation was seen with early or late promoters. These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo.

  11. DNA vaccine protects ornamental koi (Cyprinus carpio koi) against North American spring viremia of carp virus

    USGS Publications Warehouse

    Emmenegger, E.J.; Kurath, G.

    2008-01-01

    The emergence of spring viremia of carp virus (SVCV) in the United States constitutes a potentially serious alien pathogen threat to susceptible fish stocks in North America. A DNA vaccine with an SVCV glycoprotein (G) gene from a North American isolate was constructed. In order to test the vaccine a challenge model utilizing a specific pathogen-free domestic koi stock and a cold water stress treatment was also developed. We have conducted four trial studies demonstrating that the pSGnc DNA vaccine provided protection in vaccinated fish against challenge at low, moderate, and high virus doses of the homologous virus. The protection was significant (p < 0.05) as compared to fish receiving a mock vaccine construct containing a luciferase reporter gene and to non-vaccinated controls in fish ranging in age from 3 to 14 months. In all trials, the SVCV-G DNA immunized fish were challenged 28-days post-vaccination (546 degree-days) and experienced low mortalities varying from 10 to 50% with relative percent survivals ranging from 50 to 88%. The non-vaccinated controls and mock construct vaccinated fish encountered high cumulative percent mortalities ranging from 70 to 100%. This is the first report of a SVCV DNA vaccine being tested successfully in koi. These experiments prove that the SVCV DNA (pSGnc) vaccine can elicit specific reproducible protection and validates its potential use as a prophylactic vaccine in koi and other vulnerable North American fish stocks.

  12. Histone-Like Proteins of the Dinoflagellate Crypthecodinium cohnii Have Homologies to Bacterial DNA-Binding Proteins

    PubMed Central

    Wong, J. T. Y.; New, D. C.; Wong, J. C. W.; Hung, V. K. L.

    2003-01-01

    The dinoflagellates have very large genomes encoded in permanently condensed and histoneless chromosomes. Sequence alignment identified significant similarity between the dinoflagellate chromosomal histone-like proteins of Crypthecodinium cohnii (HCCs) and the bacterial DNA-binding and the eukaryotic histone H1 proteins. Phylogenetic analysis also supports the origin of the HCCs from histone-like proteins of bacteria. PMID:12796310

  13. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in th...

  14. Recurrent DNA virus domestication leading to different parasite virulence strategies

    PubMed Central

    Pichon, Apolline; Bézier, Annie; Urbach, Serge; Aury, Jean-Marc; Jouan, Véronique; Ravallec, Marc; Guy, Julie; Cousserans, François; Thézé, Julien; Gauthier, Jérémy; Demettre, Edith; Schmieder, Sandra; Wurmser, François; Sibut, Vonick; Poirié, Marylène; Colinet, Dominique; da Silva, Corinne; Couloux, Arnaud; Barbe, Valérie; Drezen, Jean-Michel; Volkoff, Anne-Nathalie

    2015-01-01

    Relics of ancient infections are abundant in eukaryote genomes, but little is known about how they evolve when they confer a functional benefit on their host. We show here, for the first time, that the virus-like particles shown to protect Venturia canescens eggs against host immunity are derived from a nudivirus genome incorporated by the parasitic wasp into its own genetic material. Nudivirus hijacking was also at the origin of protective particles from braconid wasps. However, we show here that the viral genes produce “liposomes” that wrap and deliver V. canescens virulence proteins, whereas the particles are used as gene transfer agents in braconid wasps. Our findings indicate that virus domestication has occurred repeatedly during parasitic wasp evolution but with different evolutionary trajectories after endogenization, resulting in different virulence molecule delivery strategies. PMID:26702449

  15. A Catalytic DNA Activated by a Specific Strain of Bacterial Pathogen

    PubMed Central

    Shen, Zhifa; Wu, Zaisheng; Chang, Dingran; Zhang, Wenqing; Tram, Kha; Lee, Christine; Kim, Peter; Salena, Bruno J.

    2015-01-01

    Abstract Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD‐CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD‐CD1 was derived by an in vitro selection approach where a random‐sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross‐reactivities to unintended C. difficile strains and other bacteria species. RFD‐CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that in vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain‐specific recognition of bacterial pathogens. PMID:26676768

  16. Bacterial evolution by genomic island transfer occurs via DNA transformation in planta.

    PubMed

    Lovell, Helen C; Mansfield, John W; Godfrey, Scott A C; Jackson, Robert W; Hancock, John T; Arnold, Dawn L

    2009-09-29

    Our understanding of the evolution of microbial pathogens has been advanced by the discovery of "islands" of DNA that differ from core genomes and contain determinants of virulence. The acquisition of genomic islands (GIs) by horizontal gene transfer (HGT) is thought to have played a major role in microbial evolution. There are, however, few practical demonstrations of the acquisition of genes that control virulence, and, significantly, all have been achieved outside the animal or plant host. Loss of a GI from the bean pathogen Pseudomonas syringae pv. phaseolicola (Pph) is driven by exposure to the stress imposed by the plant's resistance response. Here, we show that the complete episomal island, which carries pathogenicity genes including the effector avrPphB, transfers between strains of Pph by transformation in planta and inserts at a specific att site in the genome of the recipient. Our results show that the evolution of bacterial pathogens by HGT may be achieved via transformation, the simplest mechanism of DNA exchange. This process is activated by exposure to plant defenses, when the pathogen is in greatest need of acquiring new genetic traits to alleviate the antimicrobial stress imposed by plant innate immunity.

  17. Stochastic Self-Assembly of ParB Proteins Builds the Bacterial DNA Segregation Apparatus.

    PubMed

    Sanchez, Aurore; Cattoni, Diego I; Walter, Jean-Charles; Rech, Jérôme; Parmeggiani, Andrea; Nollmann, Marcelo; Bouet, Jean-Yves

    2015-08-26

    Many canonical processes in molecular biology rely on the dynamic assembly of higher-order nucleoprotein complexes. In bacteria, the assembly mechanism of ParABS, the nucleoprotein super-complex that actively segregates the bacterial chromosome and many plasmids, remains elusive. We combined super-resolution microscopy, quantitative genome-wide surveys, biochemistry, and mathematical modeling to investigate the assembly of ParB at the centromere-like sequences parS. We found that nearly all ParB molecules are actively confined around parS by a network of synergistic protein-protein and protein-DNA interactions. Interrogation of the empirically determined, high-resolution ParB genomic distribution with modeling suggests that instead of binding only to specific sequences and subsequently spreading, ParB binds stochastically around parS over long distances. We propose a new model for the formation of the ParABS partition complex based on nucleation and caging: ParB forms a dynamic lattice with the DNA around parS. This assembly model and approach to characterizing large-scale, dynamic interactions between macromolecules may be generalizable to many unrelated machineries that self-assemble in superstructures. PMID:27135801

  18. Identification of novel bacterial DNA gyrase inhibitors: An in silico study.

    PubMed

    Rahimi, Hamzeh; Najafi, Ali; Eslami, Habib; Negahdari, Babak; Moghaddam, Mehrdad Moosazadeh

    2016-01-01

    Owing to essential role in bacterial survival, DNA gyrase has been exploited as a validated drug target. However, rapidly emerging resistance to gyrase-targeted drugs such as widely utilized fluoroquinolones reveals the necessity to develop novel compounds with new mechanism of actions against this enzyme. Here, an attempt has been made to identify new drug-like molecules for Shigella flexneri DNA gyrase inhibition through in silico approaches. The structural similarity search was carried out using the natural product simocyclinone D8, a unique gyrase inhibitor, to virtually screen ZINC database. A total of 11830 retrieved hits were further screened for selection of high-affinity compounds by implementing molecular docking followed by investigation of druggability according to Lipinski's rule, biological activity and physiochemical properties. Among the hits initially identified, three molecules were then confirmed to have reasonable gyrase-binding affinity and to follow Lipinski's rule. Based on these in silico findings, three compounds with different chemical structures from previously identified gyrase inhibitors were proposed as potential candidates for the treatment of fluoroquinolone-resistant strains and deserve further investigations. PMID:27499795

  19. Identification of novel bacterial DNA gyrase inhibitors: An in silico study

    PubMed Central

    Rahimi, Hamzeh; Najafi, Ali; Eslami, Habib; Negahdari, Babak; Moghaddam, Mehrdad Moosazadeh

    2016-01-01

    Owing to essential role in bacterial survival, DNA gyrase has been exploited as a validated drug target. However, rapidly emerging resistance to gyrase-targeted drugs such as widely utilized fluoroquinolones reveals the necessity to develop novel compounds with new mechanism of actions against this enzyme. Here, an attempt has been made to identify new drug-like molecules for Shigella flexneri DNA gyrase inhibition through in silico approaches. The structural similarity search was carried out using the natural product simocyclinone D8, a unique gyrase inhibitor, to virtually screen ZINC database. A total of 11830 retrieved hits were further screened for selection of high-affinity compounds by implementing molecular docking followed by investigation of druggability according to Lipinski’s rule, biological activity and physiochemical properties. Among the hits initially identified, three molecules were then confirmed to have reasonable gyrase-binding affinity and to follow Lipinski’s rule. Based on these in silico findings, three compounds with different chemical structures from previously identified gyrase inhibitors were proposed as potential candidates for the treatment of fluoroquinolone-resistant strains and deserve further investigations. PMID:27499795

  20. Dynamic vs static bending rigidities for DNA and M13 virus

    NASA Astrophysics Data System (ADS)

    Schurr, J. M.; Song, Lu; Kim, Ug-Sung

    1991-05-01

    A recently developed theory for weakly bending rods provides a means to estimate the dynamic bending rigidities of filamentous macromolecules from transient electro-optic and dynamic light scattering experiments. This theory is applied to analyze transient electric dichroisrn data from Prschke''s laboratory and transient electric birefringence data from Eden''s laboratory for DNA restriction fragments with 95 to 250 bp and also to analyze dynamic light scattering data for M13 virus. Surprisingly the dynamic bending rigidity of DNA is about three times the consensus static value inferred from its equilibrium persistence length. This implies that the bending potential of DNA is non-simple and/or biased. In contrast the dynamic and static bending rigidities of M13 virus are nearly identical so its bending potential is simple and unbiased. 1.

  1. Immunogenicity of West Nile virus infectious DNA and its noninfectious derivatives

    SciTech Connect

    Seregin, Alexey; Nistler, Ryan; Borisevich, Victoria; Yamshchikov, Galina; Chaporgina, Elena; Kwok, Chun Wai; Yamshchikov, Vladimir . E-mail: yaximik@ku.edu

    2006-12-20

    The exceptionally high virulence of the West Nile NY99 strain makes its suitability in the development of a live WN vaccine uncertain. The aim of this study is to investigate the immunogenicity of noninfectious virus derivatives carrying pseudolethal mutations, which preclude virion formation without affecting preceding steps of the viral infectious cycle. When administered using DNA immunization, such constructs initiate an infectious cycle but cannot lead to a viremia. While the magnitude of the immune response to a noninfectious replication-competent construct was lower than that of virus or infectious DNA, its overall quality and the protective effect were similar. In contrast, a nonreplicating construct of similar length induced only a marginally detectable immune response in the dose range used. Thus, replication-competent noninfectious constructs derived from infectious DNA may offer an advantageous combination of the safety of noninfectious formulations with the quality of the immune response characteristic of infectious vaccines.

  2. Physical mapping of BK virus DNA with SacI, MboII, and AluI restriction endonucleases.

    PubMed Central

    Yang, R C; Wu, R

    1978-01-01

    A new restriction endonuclease, SacI from Streptomyces achromogenes cleaves BK virus (strain MM) DNA into 3 fragments, whereas MboII from Moraxella bovis and AluI from Arthrobacter luteus give 22 and 30 fragments, respectively. All these specific DNA fragments were ordered and mapped on the viral genome by two methods first, by the reciprocal digestion method using uniformly 32P-labeled DNA; and second, by the partial digestion technique using the single-end 32P-labeled DNA. This study, together with those reported earlier, defined the location of 90 cleavage sites on the BK virus DNA. Images PMID:215783

  3. Expression of the 1918 Influenza A Virus PB1-F2 Enhances the Pathogenesis of Viral and Secondary Bacterial Pneumonia

    PubMed Central

    McAuley, Julie L.; Hornung, Felicita; Boyd, Kelli L.; Smith, Amber M.; McKeon, Raelene; Bennink, Jack; Yewdell, Jonathan W.; McCullers, Jonathan A.

    2007-01-01

    Secondary bacterial pneumonia frequently claimed the lives of victims during the devastating 1918 influenza A virus pandemic. Little is known about the viral factors contributing to the lethality of the 1918 pandemic. Here we show that expression of the viral accessory protein PB1-F2 enhances inflammation during primary viral infection of mice and increases both the frequency and severity of secondary bacterial pneumonia. The priming effect of PB1-F2 on bacterial pneumonia could be recapitulated in mice by intranasal delivery of a synthetic peptide derived from the C-terminal portion of the PB1-F2. Relative to its isogenic parent, an influenza virus engineered to express a PB1-F2 with coding changes matching the 1918 pandemic strain was more virulent in mice, induced more pulmonary immunopathology, and led to more severe secondary bacterial pneumonia. These findings help explain both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic. PMID:18005742

  4. Immunization with non-replicating E. coli minicells delivering both protein antigen and DNA protects mice from lethal challenge with lymphocytic choriomeningitis virus

    PubMed Central

    Giacalone, Matthew J.; Zapata, Juan C.; Berkley, Neil L.; Sabbadini, Roger A.; Chu, Yen-Lin; Salvato, Maria S.; McGuire, Kathleen L.

    2008-01-01

    In the midst of new investigations into the mechanisms of both delivery and protection of new vaccines and vaccine carriers, it has become clear that immunization with delivery mechanisms that do not involve living, replicating organisms are vastly preferred. In this report, non-replicating bacterial minicells simultaneously co-delivering the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) and the corresponding DNA vaccine were tested for the ability to generate protective cellular immune responses in mice. It was found that good protection (89%) was achieved after intramuscular administration, moderate protection (31%) was achieved after intranasal administration, and less protection (7%) was achieved following gastric immunization. These results provide a solid foundation on which to pursue the use of bacterial minicells as a non-replicating vaccine delivery platform. PMID:17258845

  5. The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

    PubMed

    Mitsudome, Yuya; Takahama, Mamiko; Hirose, Jun; Yoshida, Naoto

    2014-01-01

    Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for

  6. Structural mimics of viruses through peptide/DNA co-assembly.

    PubMed

    Ni, Rong; Chau, Ying

    2014-12-31

    A synthetic mimic of viral structure has been constructed by the synergistic co-assembly of a 16-amino acid peptide and plasmid DNA. The rational design of this short peptide, including segments for binding DNA and forming β-sheet, is inspired by viral capsid protein. The resulting nanostructures, which we term nanococoons, appear as ellipsoids of virus-like dimension (65 × 47 nm) and display repeating stripes of ∼4 nm wide. We propose that the co-assembly process involves DNA as a template to assist the organization of peptide strands by electrostatic interaction, while the bilayer β-sheets and their lateral association stabilize the peptide "capsid" and organize the DNA within. The hierarchy affords an extremely stable structure, protecting peptide and DNA against enzymatic digestion. It opens a new and facile avenue to fabricate viral alternatives with diverse functions.

  7. Nuclear Sensing of Viral DNA, Epigenetic Regulation of Herpes Simplex Virus Infection, and Innate Immunity

    PubMed Central

    Knipe, David M.

    2015-01-01

    Herpes simplex virus (HSV) undergoes a lytic infection in epithelial cells and a latent infection in neuronal cells, and epigenetic mechanisms play a major role in the differential gene expression under the two conditions. Herpes viron DNA is not associated with histones but is rapidly loaded with heterochromatin upon entry into the cell. Viral proteins promote reversal of the epigenetic silencing in epithelial cells while the viral latency-associated transcript promotes additional heterochromatin in neuronal cells. The cellular sensors that initiate the chromatinization of foreign DNA have not been fully defined. IFI16 and cGAS are both essential for innate sensing of HSV DNA, and new evidence shows how they work together to initiate innate signaling. IFI16 also plays a role in the heterochromatinization of HSV DNA, and this review will examine how IFI16 integrates epigenetic regulation and innate sensing of foreign viral DNA to show how these two responses are related. PMID:25742715

  8. Symbiotic Virus at the Evolutionary Intersection of Three Types of Large DNA Viruses; Iridoviruses, Ascoviruses, and Ichnoviruses

    PubMed Central

    Bigot, Yves; Renault, Sylvaine; Nicolas, Jacques; Moundras, Corinne; Demattei, Marie-Véronique; Samain, Sylvie; Bideshi, Dennis K.; Federici, Brian A.

    2009-01-01

    Background The ascovirus, DpAV4a (family Ascoviridae), is a symbiotic virus that markedly increases the fitness of its vector, the parasitic ichneumonid wasp, Diadromus puchellus, by increasing survival of wasp eggs and larvae in their lepidopteran host, Acrolepiopsis assectella. Previous phylogenetic studies have indicated that DpAV4a is related to the pathogenic ascoviruses, such as the Spodoptera frugiperda ascovirus 1a (SfAV1a) and the lepidopteran iridovirus (family Iridoviridae), Chilo iridescent virus (CIV), and is also likely related to the ancestral source of certain ichnoviruses (family Polydnaviridae). Methodology/Principal Findings To clarify the evolutionary relationships of these large double-stranded DNA viruses, we sequenced the genome of DpAV4a and undertook phylogenetic analyses of the above viruses and others, including iridoviruses pathogenic to vertebrates. The DpAV4a genome consisted of 119,343 bp and contained at least 119 open reading frames (ORFs), the analysis of which confirmed the relatedness of this virus to iridoviruses and other ascoviruses. Conclusions Analyses of core DpAV4a genes confirmed that ascoviruses and iridoviruses are evolutionary related. Nevertheless, our results suggested that the symbiotic DpAV4a had a separate origin in the iridoviruses from the pathogenic ascoviruses, and that these two types shared parallel evolutionary paths, which converged with respect to virion structure (icosahedral to bacilliform), genome configuration (linear to circular), and cytopathology (plasmalemma blebbing to virion-containing vesicles). Our analyses also revealed that DpAV4a shared more core genes with CIV than with other ascoviruses and iridoviruses, providing additional evidence that DpAV4a represents a separate lineage. Given the differences in the biology of the various iridoviruses and ascoviruses studied, these results provide an interesting model for how viruses of different families evolved from one another. PMID:19636425

  9. Characterization of a complete genome of a circular single-stranded DNA virus from porcine stools in Korea.

    PubMed

    Kim, A Reum; Chung, Hee Chun; Kim, Hye Kwon; Kim, Eun Ok; Nguyen, Van Giap; Choi, Min Gyung; Yang, Hye Jung; Kim, Jung Ah; Park, Bong Kyun

    2014-02-01

    Porcine circular single-stranded DNA viruses have been just identified from swine feces in Korea. This virus was mentioned as bovine stool-associated circular DNA virus (BoSCV)-like virus discovered from porcine stools. However, the thorough characteristics of the virus were not identified. Therefore, this research focuses on finding a full genome sequence and analyzing the genetic features of the virus. The virus, now called porcine stool-associated circular DNA virus in Korea (PoSCV Kor), consists of 2,589 bases forming circular structure. It has two major ORFs inversely encoding replicase and capsid protein, with each stem-loop structure between 5' ends and 3' ends of the two putative ORFs. This characteristics is the same as PoSCV in New Zealand, but different from chimpanzee stool-associated circular virus (ChiSCVs) and BoSCV, which have one stem-loop structure. Therefore, it would be sure that PoSCV Kor is very similar to PoSCV in respect to the genetic aspect; the same number of nucleotide bases and the amino acid identity of replicase and capsid protein (96 and 93 %, respectively). This fact could be certified through the finding that PoSCV Kor and PoSCV are in the same cluster by phylogenetic analysis based on the comparison with full-sequences of other circular ssDNA viruses.

  10. Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site

    SciTech Connect

    Patel, Asmita; Yakovleva, Lyudmila; Shuman, Stewart; Mondragón, Alfonso

    2010-10-22

    Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a {approx}21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

  11. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

    PubMed Central

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J.; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-01-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton–virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  12. DNA Microarray Platform for Detection and Surveillance of Viruses Transmitted by Small Mammals and Arthropods

    PubMed Central

    Khan, Mohd Jaseem; Trabuco, Amanda Cristina; Alfonso, Helda Liz; Figueiredo, Mario Luis; Batista, Weber Cheli; Badra, Soraya Jabur; Figueiredo, Luiz Tadeu; Lavrador, Marco Aurélio

    2016-01-01

    Viruses transmitted by small mammals and arthropods serve as global threats to humans. Most emergent and re-emergent viral agents are transmitted by these groups; therefore, the development of high-throughput screening methods for the detection and surveillance of such viruses is of great interest. In this study, we describe a DNA microarray platform that can be used for screening all viruses transmitted by small mammals and arthropods (SMAvirusChip) with nucleotide sequences that have been deposited in the GenBank. SMAvirusChip was designed with more than 15,000 oligonucleotide probes (60-mers), including viral and control probes. Two SMAvirusChip versions were designed: SMAvirusChip v1 contains 4209 viral probes for the detection of 409 viruses, while SMAvirusChip v2 contains 4943 probes for the detection of 416 viruses. SMAvirusChip was evaluated with 20 laboratory reference-strain viruses. These viruses could be specifically detected when alone in a sample or when artificially mixed within a single sample. The sensitivity of SMAvirusChip was evaluated using 10-fold serial dilutions of dengue virus (DENV). The results showed a detection limit as low as 2.6E3 RNA copies/mL. Additionally, the sensitivity was one log10 lower (2.6E2 RNA copies/mL) than quantitative real-time RT-PCR and sufficient to detect viral genomes in clinical samples. The detection of DENV in serum samples of DENV-infected patients (n = 6) and in a whole blood sample spiked with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples. In addition, in a pool of mosquito samples spiked with DENV, the virus was also detectable. SMAvirusChip was able to specifically detect viruses in cell cultures, serum samples, total blood samples and a pool of mosquitoes, confirming that cellular RNA/DNA did not interfere with the assay. Therefore, SMAvirusChip may represent an innovative surveillance method for the rapid identification of viruses transmitted by small

  13. Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling

    PubMed Central

    Zhen, Huajun; Han, Taewon; Fennell, Donna E.

    2013-01-01

    We report here that stress experienced by bacteria due to aerosolization and air sampling can result in severe membrane impairment, leading to the release of DNA as free molecules. Escherichia coli and Bacillus atrophaeus bacteria were aerosolized and then either collected directly into liquid or collected using other collection media and then transferred into liquid. The amount of DNA released was quantified as the cell membrane damage index (ID), i.e., the number of 16S rRNA gene copies in the supernatant liquid relative to the total number in the bioaerosol sample. During aerosolization by a Collison nebulizer, the ID of E. coli and B. atrophaeus in the nebulizer suspension gradually increased during 60 min of continuous aerosolization. We found that the ID of bacteria during aerosolization was statistically significantly affected by the material of the Collison jar (glass > polycarbonate; P < 0.001) and by the bacterial species (E. coli > B. atrophaeus; P < 0.001). When E. coli was collected for 5 min by filtration, impaction, and impingement, its ID values were within the following ranges: 0.051 to 0.085, 0.16 to 0.37, and 0.068 to 0.23, respectively; when it was collected by electrostatic precipitation, the ID values (0.011 to 0.034) were significantly lower (P < 0.05) than those with other sampling methods. Air samples collected inside an equine facility for 2 h by filtration and impingement exhibited ID values in the range of 0.30 to 0.54. The data indicate that the amount of cell damage during bioaerosol sampling and the resulting release of DNA can be substantial and that this should be taken into account when analyzing bioaerosol samples. PMID:24096426

  14. Analysis of Aleutian disease virus infection in vitro and in vivo: demonstration of Aleutian disease virus DNA in tissues of infected mink.

    PubMed Central

    Bloom, M E; Race, R E; Aasted, B; Wolfinbarger, J B

    1985-01-01

    Aleutian disease virus (ADV) infection was analyzed in vivo and in vitro to compare virus replication in cell culture and in mink. Initial experiments compared cultures of Crandell feline kidney (CRFK) cells infected with the avirulent ADV-G strain or the highly virulent Utah I ADV. The number of ADV-infected cells was estimated by calculating the percentage of cells displaying ADV antigen by immunofluorescence (IFA), and several parameters of infection were determined. Infected cells contained large quantities of viral DNA (more than 10(5) genomes per infected cell) as estimated by dot-blot DNA-DNA hybridization, and much of the viral DNA, when analyzed by Southern blot hybridization, was found to be of a 4.8-kilobase-pair duplex monomeric replicative form (DM DNA). Furthermore, the cultures contained 7 to 67 fluorescence-forming units (FFU) per infected cell, and the ADV genome per FFU ratio ranged between 2 X 10(3) and 164 X 10(3). Finally, the pattern of viral antigen detected by IFA was characteristically nuclear, although cytoplasmic fluorescence was often found in the same cells. Because no difference was noted between the two virus strains when cultures containing similar numbers of infected cells were compared, it seemed that both viruses behaved similarly in infected cell culture. These data were used as a basis for the analysis of infection of mink by virulent Utah I ADV. Ten days after infection, the highest levels of viral DNA were detected in spleen (373 genomes per cell), mesenteric lymph node (MLN; 750 genomes per cell), and liver (373 genomes per cell). In marked contrast to infected CRFK cells, the predominant species of ADV DNA in all tissues was single-stranded virion DNA; however, 4.8-kilobase-pair DM DNA was found in MLN and spleen. This observation suggested that MLN and spleen were sites of virus replication, but that the DNA found in liver reflected sequestration of virus produced elsewhere. A final set of experiments examined MLN taken

  15. Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity.

    PubMed

    Erttmann, Saskia F; Härtlova, Anetta; Sloniecka, Marta; Raffi, Faizal A M; Hosseinzadeh, Ava; Edgren, Tomas; Rofougaran, Reza; Resch, Ulrike; Fällman, Maria; Ek, Torben; Gekara, Nelson O

    2016-07-19

    The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1β (IL-1β) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection. PMID:27421701

  16. A temperature-induced narrow DNA curvature range sustains the maximum activity of a bacterial promoter in vitro.

    PubMed

    Prosseda, Gianni; Mazzola, Alessia; Di Martino, Maria Letizia; Tielker, Denis; Micheli, Gioacchino; Colonna, Bianca

    2010-04-01

    Among the molecular strategies bacteria have set up to quickly match their transcriptional program to new environments, changes in sequence-mediated DNA curvature play a crucial role. Bacterial promoters, especially those of mesophilic bacteria, are in general preceded by a curved region. The marked thermosensitivity of curved DNA stretches allows bacteria to rapidly sense outer temperature variations and affects transcription by favoring the binding of activators or repressors. Curved DNA is also able to influence the transcriptional activity of a bacterial promoter directly, without the involvement of trans-acting regulators. This study attempts to quantitatively analyze the role of DNA curvature in thermoregulated gene expression using a real-time in vitro transcription model system based on a specific fluorescence molecular beacon. By analyzing the temperature-dependent expression of a reporter gene in a construct carrying a progressively decreasing bent sequence upstream from the promoter, we show that with a decrease in temperature a narrow curvature range accounts for a significant enhancement of promoter activity. This strengthens the view that DNA curvature-mediated regulation of gene expression is likely a strategy offering fine-tuning control possibilities and that, considering the widespread presence of curved sequences upstream from bacterial promoters, it may represent one of the most primitive forms of gene regulation.

  17. Detection of Bacterial Indicators and Human and Bovine Enteric Viruses in Surface Water and Groundwater Sources Potentially Impacted by Animal and Human Wastes in Lower Yakima Valley, Washington▿

    PubMed Central

    Gibson, Kristen E.; Schwab, Kellogg J.

    2011-01-01

    Tangential flow ultrafiltration (UF) was used to concentrate and recover bacterial indicators and enteric viruses from 100 liters of groundwater (GW; n = 10) and surface water (SW; n = 11) samples collected in Lower Yakima Valley, WA. Human and bovine enteric viruses were analyzed in SW and GW concentrates by real-time PCR by using integrated inhibition detection. PMID:21075875

  18. Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy

    NASA Technical Reports Server (NTRS)

    Ling, Paul D.; Vilchez, Regis A.; Keitel, Wendy A.; Poston, David G.; Peng, Rong Sheng; White, Zoe S.; Visnegarwala, Fehmida; Lewis, Dorothy E.; Butel, Janet S.

    2003-01-01

    Patients with human immunodeficiency virus type 1 (HIV-1) infection are at high risk of developing Epstein-Barr virus (EBV)-associated lymphoma. However, little is known of the EBV DNA loads in patients receiving highly active antiretroviral therapy (HAART). Using a real-time quantitative polymerase chain reaction assay, we demonstrated that significantly more HIV-1-infected patients receiving HAART than HIV-1-uninfected volunteers had detectable EBV DNA in blood (57 [81%] of 70 vs. 11 [16%] of 68 patients; P=.001) and saliva (55 [79%] of 68 vs. 37 [54%] of 68 patients; P=.002). The mean EBV loads in blood and saliva samples were also higher in HIV-1-infected patients than in HIV-1-uninfected volunteers (P=.001). The frequency of EBV detection in blood was associated with lower CD4+ cell counts (P=.03) among HIV-1-infected individuals, although no differences were observed in the EBV DNA loads in blood or saliva samples in the HIV-1-infected group. Additional studies are needed to determine whether EBV-specific CD4+ and CD8+ cells play a role in the pathogenesis of EBV in HIV-1-infected patients receiving HAART.

  19. QDs-DNA nanosensor for the detection of hepatitis B virus DNA and the single-base mutants.

    PubMed

    Wang, Xiang; Lou, Xinhui; Wang, Yi; Guo, Qingchuan; Fang, Zheng; Zhong, Xinhua; Mao, Hongju; Jin, Qinghui; Wu, Lei; Zhao, Hui; Zhao, Jianlong

    2010-04-15

    We report here a quantum dots-DNA (QDs-DNA) nanosensor based on fluorescence resonance energy transfer (FRET) for the detection of the target DNA and single mismatch in hepatitis B virus (HBV) gene. The proposed one-pot DNA detection method is simple, rapid and efficient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide (TOPO) on the surface of QDs with 3-mercaptopropionic acid (MPA). Subsequently, oligonucleotides were attached to the QDs surface to form functional QDs-DNA conjugates. Along with the addition of DNA targets and Cy5-modified signal DNAs into the QDs-DNA conjugates, sandwiched hybrids were formed. The resulting assembly brings the Cy5 fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to fluorescence emission from the acceptor by means of FRET on illumination of the donor. In order to efficiently detect single-base mutants in HBV gene, oligonucleotide ligation assay was employed. If there existed a single-base mismatch, which could be recognized by the ligase, the detection probe was not ligated and no Cy5 emission was produced due to the lack of FRET. The feasibility of the proposed method was also demonstrated in the detection of synthetic 30-mer oliginucleotide targets derived from the HBV with a sensitivity of 4.0nM by using a multilabel counter. The method enables a simple and efficient detection that could be potentially used for high throughput and multiplex detections of target DNA and the mutants.

  20. Comparison of Herpes simplex virus plaque development after viral treatment with anti-DNA or antilipid agents

    SciTech Connect

    Coohill, T.P.; Babich, M.; Taylor, W.D.; Snipes, W.

    1980-06-01

    The plaque development of Herpes simplex virus type 1 (HSV) is slower for viruses treated with two anti-DNA agents: ultraviolet radiation (uv) or n-acetoxy-2-acetyl-aminofluorene. For HSV treated with three antimembrane agents - butylated hydroxytoluene, acridine plus near uv radiation, or ether - the plaque development time is the same as for untreated viruses. These differences hold even for viruses that survived treatment that lowered viability below the 1% level. Gamma ray inactivation of HSV produces no change in plaque development even though this agent is believed to preferentially affect viral DNA.

  1. Artificially 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.

    PubMed

    Kim, Taejoong; Mays, Jody; Fadly, Aly; Silva, Robert F

    2011-06-01

    Researchers reported that co-cultivating the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in an REV long terminal repeat (LTR) being inserted into the internal repeat short (IRS) region of JM/102W. When the resulting recombinant virus was serially passed in cell culture, the initial LTR was duplicated and a second LTR spontaneously appeared in the terminal repeat short (TRS) region of the MDV genome. The virus, designated RM1, was significantly attenuated but still induced severe bursal and thymic atrophy (Isfort et al. PNAS 89:991-995). To determine whether the altered phenotype was due solely to the LTR, we cloned the LTR from the RM1 IRS region and inserted it into the IRS region of a very virulent bacterial artificial clone (BAC) of the Md5 strain of MDV, which we designated rMd5-RM1-LTR. During blind passage in duck embryo fibroblast cultures, the initial LTR in the rMd5-RM1-LTR was also duplicated, with LTRs appearing in both IRS and TRS regions of the MDV genome. The inserted LTR sequences and transcripts associated with the MDV open reading frames MDV085, MDV086, SORF2, US1, and US10 were molecularly characterized. The parental Md5 BAC contains a family of transcripts of 3, 2, and 1 kb that all terminate at the end of the US10 gene. The rMd5-RM1-LTR and RM1 viruses both express an additional 4 kb transcript that originates in the LTR and also terminates after US10. Collectively, the data suggest that our engineered rMd5-RM1-LTR virus very closely resembles the RM1 virus in its structure and transcription patterns.

  2. Epstein–Barr virus DNA level as a novel prognostic factor in nasopharyngeal carcinoma

    PubMed Central

    Zhang, Jing; Shu, Chi; Song, Yanlin; Li, Qingfang; Huang, Jingwen; Ma, Xuelei

    2016-01-01

    Abstract Background: The plasma Epstein–Barr virus (EBV) DNA level in patients with nasopharyngeal carcinoma (NPC) performs as an appealing prognostic factor, but conclusions of its prognostic values from previous studies are inconsistent. In this study, we performed a comprehensive meta-analysis to evaluate the prognostic value of EBV DNA level in patients with NPC. Methods: Published studies were searched in PubMed. The baseline characteristics of patients, overall survival (OS), and other survival outcomes were extracted. Pooled hazard ratio (HR), 95% confidence interval (CI), and P value were calculated to estimate the prognostic value of EBV DNA level. Each cut-off value mentioned in the studies was obtained. Kaplan–Meier curves were used to extract data, and graphical survival plots were extracted for calculating HR when the study did not describe the information directly. Results: This meta-analysis pooled 23 eligible studies including 10,732 patients with NPC. The pooled HR (95% CI) of pretreatment plasma EBV DNA level (pre-DNA) for OS was 2.78 (2.19, 3.55), and the HR (95% CI) of posttreatment plasma EBV DNA level (post-DNA) for OS was 5.43 (2.72, 10.82), suggesting that EBV DNA level was significantly correlated to the outcomes of patients with NPC. Conclusion: High expression levels of EBV DNA predicts poor prognosis in NPC. PMID:27749596

  3. Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Nichols, Krista M.; Winton, James R.; Kurath, Gael; Thorgaard, Gary H.; Wheeler, Paul; Hansen, John D.; Herwig, Russell P.; Park, Linda K.

    2006-01-01

    The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

  4. DNA buckling in bacteriophage cavities as a mechanism to aid virus assembly.

    PubMed

    Hirsh, Andrew D; Perkins, N C

    2015-03-01

    While relatively simple biologically, bacteriophages are sophisticated biochemical machines that execute a precise sequence of events during virus assembly, DNA packaging, and ejection. These stages of the viral life cycle require intricate coordination of viral components whose structures are being revealed by single molecule experiments and high resolution (cryo-electron microscopy) reconstructions. For example, during packaging, bacteriophages employ some of the strongest known molecular motors to package DNA against increasing pressure within the viral capsid shell. Located upstream of the motor is an elaborate portal system through which DNA is threaded. A high resolution reconstruction of the portal system for bacteriophage ϕ29 reveals that DNA buckles inside a small cavity under large compressive forces. In this study, we demonstrate that DNA can also buckle in other bacteriophages including T7 and P22. Using a computational rod model for DNA, we demonstrate that a DNA buckle can initiate and grow within the small confines of a cavity under biologically-attainable force levels. The forces of DNA-cavity contact and DNA-DNA electrostatic repulsion ultimately limit cavity filling. Despite conforming to very different cavity geometries, the buckled DNA within T7 and P22 exhibits near equal volumetric energy density (∼1kT/nm(3)) and energetic cost of packaging (∼22kT). We hypothesize that a DNA buckle creates large forces on the cavity interior to signal the conformational changes to end packaging. In addition, a DNA buckle may help retain the genome prior to tail assembly through significantly increased contact area with the portal. PMID:25613203

  5. Comparative Genomics of Amphibian-like Ranaviruses, Nucleocytoplasmic Large DNA Viruses of Poikilotherms

    PubMed Central

    Price, Stephen J.

    2015-01-01

    Recent research on genome evolution of large DNA viruses has highlighted a number of incredibly dynamic processes that can facilitate rapid adaptation. The genomes of amphibian-like ranaviruses – double-stranded DNA viruses infecting amphibians, reptiles, and fish (family Iridoviridae) – were examined to assess variation in genome content and evolutionary processes. The viruses studied were closely related, but their genome content varied considerably, with 29 genes identified that were not present in all of the major clades. Twenty-one genes had evidence of recombination, while a virus isolated from a captive reptile appeared to be a mosaic of two divergent parents. Positive selection was also found to be acting on more than a quarter of Ranavirus genes and was found most frequently in the Spanish common midwife toad virus, which has had a severe impact on amphibian host communities. Efforts to resolve the root of this group by inclusion of an outgroup were inconclusive, but a set of core genes were identified, which recovered a well-supported species tree. PMID:27812275

  6. The complete sequence of soybean chlorotic mottle virus DNA and the identification of a novel promoter.

    PubMed

    Hasegawa, A; Verver, J; Shimada, A; Saito, M; Goldbach, R; Van Kammen, A; Miki, K; Kameya-Iwaki, M; Hibi, T

    1989-12-11

    The complete nucleotide sequence of an infectious clone of soybean chlorotic mottle virus (SoyCMV) DNA was determined and compared with those of three other caulimoviruses, cauliflower mosaic virus (CaMV), carnation etched ring virus and figwort mosaic virus. The double-stranded DNA genome of SoyCMV (8,175 bp) contained nine open reading frames (ORFs) and one large intergenic region. The primer binding sites, gene organization and size of ORFs were similar to those of the other caulimoviruses, except for ORF I, which was split into ORF Ia and Ib. The amino acid sequences deduced from each ORF showed only short, highly homologous regions in several of the corresponding ORFs of the three other caulimoviruses. A promoter fragment of 378 bp in SoyCMV ORF III showed a strong expression activity, comparable to that of the CaMV 35S promoter, in tobacco mesophyll protoplasts as determined by a beta-glucuronidase assay using electrotransfection. The fragment contained CAAT and TATA boxes but no transcriptional enhancer signal as reported for the CaMV 35S promoter. Instead, it had sequences homologous to a part of the translational enhancer signal reported for the 5'-leader sequence of tobacco mosaic virus RNA.

  7. Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication

    SciTech Connect

    Evans, E.V.A.

    1989-01-01

    The poxvirus, vaccinia, is large DNA virus which replicates in the cytoplasma of the host cell. The virus is believed to encode most or all of the functions required for the temporally regulated transcription and replication of its 186 kilobase genome. Physical and genetic autonomy from the host make vaccinia a useful eukaryotic organism in which to study replication genes and proteins, using a combination of biochemical and genetic techniques. Essential viral functions for replication are identified by conditional lethal mutants that fail to synthesize DNA at the non-permissive temperatures. One such group contains the non-complementing alleles ts17, ts24, ts69 (WR strain). Studies were undertaken to define the phenotype of ts mutants, and to identify and characterize the affected gene and protein. Mutant infection was essentially normal at 32{degree}C, but at 39{degree}C the mutants did not incorporate {sup 3}H-thymidine into nascent viral DNA or synthesize late viral proteins. If mutant cultures were shifted to non-permissive conditions at the height of replication, DNA synthesis was halted rapidly, implying that the mutants are defective in DNA elongation. The gene affected in the WR mutants and in ts6389, a DNA-minus mutant of the IHD strain, was mapped by marker rescue and corresponds to open reading frame 5 (orfD5) of the viral HindIII D fragment.

  8. Virion-associated HIV-1 Vpr: variable amount in virus particles derived from cells upon virus infection or proviral DNA transfection.

    PubMed

    Singh, S P; Tungaturthi, P; Cartas, M; Tomkowicz, B; Rizvi, T A; Khan, S A; Kalyanaraman, V S; Srinivasan, A

    2001-04-25

    Human immunodeficiency virus type-1 (HIV-1) Vpr is a virion-associated protein implicated to have a role in AIDS pathogenesis. In regard to the amount of Vpr incorporated into virus particles, the published data vary widely. To address this, we quantitated Vpr in virus particles derived from diverse sources that are used to evaluate the biological effect of Vpr. Virus particles from infected cells showed only a small amount of Vpr. Interestingly, virus particles from cells cotransfected with HIV-1 proviral DNA lacking Vpr coding sequences (NLDeltaVpr) and a Vpr expression plasmid showed a drastic increase (29.4-fold) in the incorporation of Vpr. Furthermore, cotransfection involving NLDeltaVpr and different concentrations of Vpr expression plasmid resulted in virus particles containing Vpr in proportion to the Vpr expression plasmid used. The differences in virus particles with respect to Vpr as revealed by these studies should be taken into account in assessing the effect of Vpr.

  9. Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

    SciTech Connect

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S.; Pushko, Peter

    2014-11-15

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

  10. Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication

    SciTech Connect

    Traut, W.; Fanning, E.

    1988-02-01

    The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity, but its functional role was not defined. In the present report, the authors used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlate with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.

  11. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  12. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    NASA Astrophysics Data System (ADS)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  13. Chimeric cDNA studies of Theiler's murine encephalomyelitis virus neurovirulence.

    PubMed Central

    Zhang, L; Senkowski, A; Shim, B; Roos, R P

    1993-01-01

    Strain GDVII and other members of the GDVII subgroup of Theiler's murine encephalomyelitis virus are highly neurovirulent and rapidly fatal, while strain DA and other members of the TO subgroup produce a chronic, demyelinating disease. GDVII/DA chimeric cDNA studies suggest that a major neurovirulence determinant is within the GDVII 1B through 1D capsid protein coding region, although the additional presence of upstream GDVII sequences, including the 5' untranslated region, contributes to full neurovirulence. Our studies indicate that there are limitations in precisely delineating neurovirulence determinants with chimeric cDNAs between evolutionarily diverged viruses, such as GDVII and DA. PMID:8510228

  14. Chimeric cDNA studies of Theiler's murine encephalomyelitis virus neurovirulence.

    PubMed

    Zhang, L; Senkowski, A; Shim, B; Roos, R P

    1993-07-01

    Strain GDVII and other members of the GDVII subgroup of Theiler's murine encephalomyelitis virus are highly neurovirulent and rapidly fatal, while strain DA and other members of the TO subgroup produce a chronic, demyelinating disease. GDVII/DA chimeric cDNA studies suggest that a major neurovirulence determinant is within the GDVII 1B through 1D capsid protein coding region, although the additional presence of upstream GDVII sequences, including the 5' untranslated region, contributes to full neurovirulence. Our studies indicate that there are limitations in precisely delineating neurovirulence determinants with chimeric cDNAs between evolutionarily diverged viruses, such as GDVII and DA.

  15. Direct Detection and Quantification of Bacterial Genes Associated with Inflammation in DNA Isolated from Stool

    PubMed Central

    Gómez-Moreno, Ramón; Robledo, Iraida E.; Baerga-Ortiz, Abel

    2014-01-01

    Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer. PMID:25635239

  16. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    PubMed

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

  17. Association of a DNA virus with grapevines affected by red blotch disease in California.

    PubMed

    Al Rwahnih, Maher; Dave, Ashita; Anderson, Michael M; Rowhani, Adib; Uyemoto, Jerry K; Sudarshana, Mysore R

    2013-10-01

    In the Napa Valley of California, vineyards of 'Cabernet Franc' (CF) clone 214, 'Cabernet Sauvignon' clone 337, and 'Zinfandel' clone 1A (Z1A) with grapevines exhibiting foliar symptoms of red blotches, marginal reddening, and red veins that were accompanied by reduced sugar accumulation in fruit at harvest were initially suspected to be infected with leafroll-associated viruses. However, reverse-transcription polymerase chain reaction (PCR) tests were negative for all known leafroll-associated viruses, with the exception of Grapevine leafroll-associated virus 2 in Z1A. Metagenomic analysis of cDNA libraries obtained from double-stranded RNA enriched nucleic acid (NA) preparations from bark scrapings of dormant canes on an Illumina platform revealed sequences having a distant relationship with members of the family Geminiviridae. Sequencing of products obtained by PCR assays using overlapping primers and rolling circle amplification (RCA) confirmed the presence of a single circular genome of 3,206 nucleotides which was nearly identical to the genome of a recently reported Grapevine cabernet franc-associated virus found in declining grapevines in New York. We propose to call this virus "Grapevine red blotch-associated virus" (GRBaV) to describe its association with grapevine red blotch disease. Primers specific to GRBaV amplified a product of expected size (557 bp) from NA preparations obtained from petioles of several diseased source vines. Chip bud inoculations successfully transmitted GRBaV to test plants of CF, as confirmed by PCR analysis. This is the first report of a DNA virus associated with red blotch disease of grapevines in California. PMID:23656312

  18. A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

    PubMed Central

    Stuart, D T; Upton, C; Higman, M A; Niles, E G; McFadden, G

    1993-01-01

    Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability. Images PMID:8474156

  19. Integrated State of Oncornavirus DNA in Normal Chicken Cells and in Cells Transformed by Avian Myeloblastosis Virus

    PubMed Central

    Markham, P. D.; Baluda, M. A.

    1973-01-01

    The covalent linkage of oncornavirus-specific DNA to chicken DNA was investigated in normal chicken embryo fibroblasts (CEF) and in virus-producing leukemic cells transformed by avian myeloblastosis virus (AMV). The virus-specific sequences present in cellular DNA fractionated by different methods were detected by DNA-RNA hybridization by using 70S AMV RNA as a probe. In CEF and in leukemic cells, the viral DNA appeared to be present only in the nucleus. After cesium chloride-ethidium bromide density equilibrium sedimentation, the viral DNA was present as linear, double-stranded molecules not separable from linear chicken DNA. After extraction by the Hirt procedure, the viral DNA precipitated with the high-molecular-weight DNA. After alkaline sucrose velocity sedimentation, the viral DNA cosedimented with the high-molecular-weight cellular DNA. The results indicate that in both types of cells studied, the oncornavirus-specific DNA sequences were linked by alkali stable bonds to nuclear cellular DNA of high molecular weight and did not appear to be present in free form of any size. PMID:4359950

  20. An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8.

    PubMed Central

    Lee, C K; Knipe, D M

    1985-01-01

    An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences. Images PMID:2987527

  1. The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

    SciTech Connect

    Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.; DeBeer, Madeleine A.P.; Lopper, Matthew E.

    2010-05-25

    Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 {angstrom} resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.

  2. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    PubMed

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-01-01

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

  3. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    PubMed

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-12-10

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

  4. Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques.

    PubMed

    Raviprakash, K; Porter, K R; Kochel, T J; Ewing, D; Simmons, M; Phillips, I; Murphy, G S; Weiss, W R; Hayes, C G

    2000-07-01

    A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d. -inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.

  5. Bacterial Diversity Associated with Wild Caught Anopheles Mosquitoes from Dak Nong Province, Vietnam Using Culture and DNA Fingerprint

    PubMed Central

    Ngo, Chung Thuy; Aujoulat, Fabien; Veas, Francisco; Jumas-Bilak, Estelle; Manguin, Sylvie

    2015-01-01

    Background Microbiota of Anopheles midgut can modulate vector immunity and block Plasmodium development. Investigation on the bacterial biodiversity in Anopheles, and specifically on the identification of bacteria that might be used in malaria transmission blocking approaches, has been mainly conducted on malaria vectors of Africa. Vietnam is an endemic country for both malaria and Bancroftian filariasis whose parasitic agents can be transmitted by the same Anopheles species. No information on the microbiota of Anopheles mosquitoes in Vietnam was available previous to this study. Method The culture dependent approach, using different mediums, and culture independent (16S rRNA PCR – TTGE) method were used to investigate the bacterial biodiversity in the abdomen of 5 Anopheles species collected from Dak Nong Province, central-south Vietnam. Molecular methods, sequencing and phylogenetic analysis were used to characterize the microbiota. Results and Discussion The microbiota in wild-caught Anopheles was diverse with the presence of 47 bacterial OTUs belonging to 30 genera, including bacterial genera impacting Plasmodium development. The bacteria were affiliated with 4 phyla, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, the latter being the dominant phylum. Four bacterial genera are newly described in Anopheles mosquitoes including Coxiella, Yersinia, Xanthomonas, and Knoellia. The bacterial diversity per specimen was low ranging from 1 to 4. The results show the importance of pairing culture and fingerprint methods to better screen the bacterial community in Anopheles mosquitoes. Conclusion Sampled Anopheles species from central-south Vietnam contained a diverse bacterial microbiota that needs to be investigated further in order to develop new malaria control approaches. The combination of both culture and DNA fingerprint methods allowed a thorough and complementary screening of the bacterial community in Anopheles mosquitoes. PMID:25747513

  6. Norwalk-like virus and bacterial pathogens associated with cases of gastroenteritis onboard a US Navy ship.

    PubMed

    Oyofo, B A; Soderquist, R; Lesmana, M; Subekti, D; Tjaniadi, P; Fryauff, D J; Corwin, A L; Richie, E; Lebron, C

    1999-12-01

    Acute gastroenteritis is a potential cause of substantial morbidity in U.S. military personnel during deployment. This study investigated the microbial causes of diarrhea in U.S. troops on exercises in Southeast Asia aboard the U.S.S. Germantown from March through May 1996. A total of 49 (7%) patients with diarrhea reported to sick call during a 3-month deployment involving 721 personnel. Diarrheal samples from 49 patients were subjected to bacterial and parasitologic examination, but sufficient samples from only 47 of 49 were available for analysis of the presence of Norwalk-like virus (NLV). Of the 49 diarrhea cases, 10 (20.4%) appeared to be due to bacterial etiology alone, 10 (20.4%) due to bacteria and the prototype Taunton agent (TNA), 11 (22.4%) due to TNA only, and 4 (8.0%) due to parasites. Norwalk-like virus RNA was present in 21 (45%) of 47 stool samples from the diarrhea cases, 10 with bacterial etiologies and 11 without bacterial or parasitic etiologies. No pathogen was detected in 14 (29%) of the cases. Four of the controls showed the presence of parasitic organisms. Of the 11 cases in which enterotoxigenic Escherichia coli was isolated, 8 were positive for colonization factor antigen (CFA/IV), and 3 were CFA-negative. The bacterial pathogens tested were all susceptible to gentamicin, and furadantin, but were resistant to ceftriaxone and norfloxacin, including 75% of the Campylobacter spp. These data support the view that the major cause of diarrhea for troops deployed in this geographic area is most likely NLVs.

  7. Occurrence of Deformed wing virus, Chronic bee paralysis virus and mtDNA variants in haplotype K of Varroa destructor mites in Syrian apiaries.

    PubMed

    Elbeaino, Toufic; Daher-Hjaij, Nouraldin; Ismaeil, Faiz; Mando, Jamal; Khaled, Bassem Solaiman; Kubaa, Raied Abou

    2016-05-01

    A small-scale survey was conducted on 64 beehives located in four governorates of Syria in order to assess for the first time the presence of honeybee-infecting viruses and of Varroa destructor mites in the country. RT-PCR assays conducted on 192 honeybees (Apis mellifera L.) using virus-specific primers showed that Deformed wing virus (DWV) was present in 49 (25.5%) of the tested samples and Chronic bee paralysis virus (CBPV) in 2 (1.04%), whereas Acute bee paralysis virus, Sacbrood virus, Black queen cell virus and Kashmir bee virus were absent. Nucleotide sequences of PCR amplicons obtained from DWV and CBPV genomes shared 95-97 and 100% identity with isolates reported in the GenBank, respectively. The phylogenetic tree grouped the Syrian DWV isolates in one cluster, distinct from all those of different origins reported in the database. Furthermore, 19 adult V. destructor females were genetically analyzed by amplifying and sequencing four fragments in cytochrome oxidase subunit 1 (cox1), ATP synthase 6 (atp6), cox3 and cytochrome b (cytb) mitochondrial DNA (mtDNA) genes. Sequences of concatenated V. destructor mtDNA genes (2696 bp) from Syria were similar to the Korean (K) haplotype and were found recurrently in all governorates. In addition, two genetic lineages of haplotype K with slight variations (0.2-0.3%) were present only in Tartous and Al-Qunaitra governorates. PMID:26914360

  8. Occurrence of Deformed wing virus, Chronic bee paralysis virus and mtDNA variants in haplotype K of Varroa destructor mites in Syrian apiaries.

    PubMed

    Elbeaino, Toufic; Daher-Hjaij, Nouraldin; Ismaeil, Faiz; Mando, Jamal; Khaled, Bassem Solaiman; Kubaa, Raied Abou

    2016-05-01

    A small-scale survey was conducted on 64 beehives located in four governorates of Syria in order to assess for the first time the presence of honeybee-infecting viruses and of Varroa destructor mites in the country. RT-PCR assays conducted on 192 honeybees (Apis mellifera L.) using virus-specific primers showed that Deformed wing virus (DWV) was present in 49 (25.5%) of the tested samples and Chronic bee paralysis virus (CBPV) in 2 (1.04%), whereas Acute bee paralysis virus, Sacbrood virus, Black queen cell virus and Kashmir bee virus were absent. Nucleotide sequences of PCR amplicons obtained from DWV and CBPV genomes shared 95-97 and 100% identity with isolates reported in the GenBank, respectively. The phylogenetic tree grouped the Syrian DWV isolates in one cluster, distinct from all those of different origins reported in the database. Furthermore, 19 adult V. destructor females were genetically analyzed by amplifying and sequencing four fragments in cytochrome oxidase subunit 1 (cox1), ATP synthase 6 (atp6), cox3 and cytochrome b (cytb) mitochondrial DNA (mtDNA) genes. Sequences of concatenated V. destructor mtDNA genes (2696 bp) from Syria were similar to the Korean (K) haplotype and were found recurrently in all governorates. In addition, two genetic lineages of haplotype K with slight variations (0.2-0.3%) were present only in Tartous and Al-Qunaitra governorates.

  9. A nanovirus-like DNA component associated with yellow vein disease of Ageratum conyzoides: evidence for interfamilial recombination between plant DNA viruses.

    PubMed

    Saunders, K; Stanley, J

    1999-11-10

    Yellow vein disease of Ageratum conyzoides, a weed species that is widely distributed throughout Asia, has been attributed to infection by the geminivirus Ageratum yellow vein virus (AYVV). In addition to a single AYVV genomic component (DNA A), we have previously demonstrated that infected plants contain chimeric defective viral components, comprising DNA A and nongeminiviral sequences, that act as defective interfering DNAs. A database search has revealed that the nongeminiviral sequences of one such defective component (def19) show significant homology with sequences of nanovirus components that encode replication-associated proteins (Reps). Primers designed to hybridise to the nongeminiviral DNA were used to PCR-amplify a full-length nanovirus-like component, referred to as DNA 1, from an extract of infected A. conyzoides. DNA 1 is unrelated to AYVV DNA A but resembles nanovirus components that encode Reps and is most closely related (73% identity) to a nanovirus-like DNA recently isolated from geminivirus-infected cotton. DNA 1 is dependent on AYVV DNA A for systemic infection of A. conyzoides and Nicotiana benthamiana and can systemically infect N. benthamiana in the presence of the bipartite geminivirus African cassava mosaic virus. A. conyzoides plants coinfected with AYVV DNA A and DNA 1 remain asymptomatic, indicating that additional factors are required to elicit yellow vein disease. Our results provide direct evidence for recombination between distinct families of plant single-stranded DNA viruses and suggest that coinfection by geminivirus and nanovirus-like pathogens may be a widespread phenomenon. The ability of plant DNA viruses to recombine in this way may greatly increase their scope for diversification.

  10. Adeno-associated Virus as a Mammalian DNA Vector

    PubMed Central

    SALGANIK, MAX; HIRSCH, MATTHEW L.; SAMULSKI, RICHARD JUDE

    2015-01-01

    In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells. PMID:26350320

  11. Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

    PubMed

    Lo, Y M; Lo, E S; Mehal, W Z; Sampietro, M; Fiorelli, G; Ronchi, G; Tse, C H; Fleming, K A

    1993-04-01

    AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV.

  12. Nuclear sensing of viral DNA, epigenetic regulation of herpes simplex virus infection, and innate immunity

    SciTech Connect

    Knipe, David M.

    2015-05-15

    Herpes simplex virus (HSV) undergoes a lytic infection in epithelial cells and a latent infection in neuronal cells, and epigenetic mechanisms play a major role in the differential gene expression under the two conditions. HSV viron DNA is not associated with histones but is rapidly loaded with heterochromatin upon entry into the cell. Viral proteins promote reversal of the epigenetic silencing in epithelial cells while the viral latency-associated transcript promotes additional heterochromatin in neuronal cells. The cellular sensors that initiate the chromatinization of foreign DNA have not been fully defined. IFI16 and cGAS are both essential for innate sensing of HSV DNA, and new evidence shows how they work together to initiate innate signaling. IFI16 also plays a role in the heterochromatinization of HSV DNA, and this review will examine how IFI16 integrates epigenetic regulation and innate sensing of foreign viral DNA to show how these two responses are related. - Highlights: • HSV lytic and latent gene expression is regulated differentially by epigenetic processes. • The sensors of foreign DNA have not been defined fully. • IFI16 and cGAS cooperate to sense viral DNA in HSV-infected cells. • IFI16 plays a role in both innate sensing of HSV DNA and in restricting its expression.

  13. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    SciTech Connect

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  14. An upconversion fluorescent resonant energy transfer biosensor for hepatitis B virus (HBV) DNA hybridization detection.

    PubMed

    Zhu, Hao; Lu, Feng; Wu, Xing-Cai; Zhu, Jun-Jie

    2015-11-21

    A novel fluorescent resonant energy transfer (FRET) biosensor was fabricated for the detection of hepatitis B virus (HBV) DNA using poly(ethylenimine) (PEI) modified upconversion nanoparticles (NH2-UCNPs) as energy donor and gold nanoparticles (Au NPs) as acceptor. The PEI modified upconversion nanoparticles were prepared directly with a simple one-pot hydrothermal method, which provides high quality amino-group functionalized UCNPs with uniform morphology and strong upconversion luminescence. Two single-stranded DNA strands, which were partially complementary to each other, were then conjugated with NH2-UCNPs and Au NPs. When DNA conjugated NH2-UCNPs and Au NPs are mixed together, the hybridization between complementary DNA sequences on UCNPs and Au NPs will lead to the quenching of the upconversion luminescence due to the FRET process. Meanwhile, upon the addition of target DNA, Au NPs will leave the surface of the UCNPs and the upconversion luminescence can be restored because of the formation of the more stable double-stranded DNA on the UCNPs. The sensor we fabricated here for target DNA detection shows good sensitivity and high selectivity, which has the potential for clinical applications in the analysis of HBV and other DNA sequences. PMID:26421323

  15. Detection of hepatitis B virus DNA sequences in infected hepatocytes by in situ cytohybridisation

    SciTech Connect

    Gowans, E.J.; Burrell, C.J.; Jilbert, A.R.; Marmion, B.P.

    1981-01-01

    Plasmid pHBV 114 DNA, which contains 73% of the genome of hepatitis B virus (HBV), was radiolabelled with tritium to 1-2 X 10(8) dpm/microgram by nick translation and used as a radioactive probe to detect HBV DNA present in sections of infected liver tissue by in situ hybridisation followed by autoradiography. Factors affecting the sensitivity of the reaction were examined, including different methods of fixation, hybridisation time, temperature, and buffers. The specificity of the reaction for detecting viral DNA was carefully established by the use of unrelated DNA probes, pretreatment of sections with DNAase, and comparing the stability of the binding of DNA probe at different temperatures, with the melting curve of double-stranded DNA in solution. In the one liver studied in detail, cells containing large amounts of viral DNA were distributed in foci corresponding to areas containing morphologically damaged hepatocytes. This observation suggested a relationship between active viral replication and cell damage. Viral DNA was found mainly in the cytoplasm, although a minority of nuclei in these foci were also positive.

  16. Electrochemical DNA biosensor based on gold nanorods for detecting hepatitis B virus.

    PubMed

    Shakoori, Zahra; Salimian, Samaneh; Kharrazi, Sharmin; Adabi, Mahdi; Saber, Reza

    2015-01-01

    The purpose of this work was to fabricate an electrochemical DNA biosensor for detecting hepatitis B virus. Gold nanorods (GNRs), which are known for their conductivity, were used to increase surface area and consequently increase the immobilization of single-stranded DNA (ss-DNA) on the modified gold electrode. The GNRs were characterized via transmission electron microscopy. The morphology of the gold electrode before and after modification with GNRs was characterized by scanning electron microscopy. Atomic-force microscopy was used to evaluate the morphology of the GNR electrode surface before and after interaction with ss-DNA. Cyclic voltammetry was used to monitor DNA immobilization and hybridization, using [Co(phen)3](3+) as an electrochemical indicator. The target DNA sequences were quantified at a linear range from 1.0 × 10(-12) to 10.0 × 10(-6) mol L(-1), with a detection limit of 2.0 × 10(-12) mol L(-1) by 3σ. The biosensor had good specificity for distinguishing complementary DNA in the presence of non-complementary and mismatched DNA sequences. PMID:25399076

  17. Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

    PubMed

    Lo, Y M; Lo, E S; Mehal, W Z; Sampietro, M; Fiorelli, G; Ronchi, G; Tse, C H; Fleming, K A

    1993-04-01

    AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV. PMID:8496385

  18. Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

    PubMed Central

    Lo, Y M; Lo, E S; Mehal, W Z; Sampietro, M; Fiorelli, G; Ronchi, G; Tse, C H; Fleming, K A

    1993-01-01

    AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV. Images PMID:8496385

  19. Varicella-zoster virus and virus DNA in the blood and oropharynx of people with latent or active varicella-zoster virus infections.

    PubMed

    Levin, Myron J

    2014-12-01

    Varicella-zoster virus (VZV) can be detected in the blood from approximately 5 days before to 4 days after varicella. VZV DNA, primarily in T-lymphocytes, is detected as early as 8-10 days prior to rash and can persist for a week. The duration and magnitude of VZV DNAemia correlates with immune status and the efficacy of antiviral therapy. VZV DNA is also readily detected in the oropharynx just prior to rash and for 1-2 weeks thereafter. Detection of VZV DNA in blood and saliva has been useful for diagnosis and prognosis in atypical cases of varicella. Herpes zoster (HZ) is also characterized by VZV DNAemia at onset and for many weeks thereafter, and VZV DNA is present in the oropharynx shortly after HZ onset. Detection of VZV DNA in blood and saliva facilitates the diagnosis of zoster sine herpete and other atypical manifestations of VZV reactivation, such as neurologic syndromes when cerebrospinal fluid is not available, Bell's palsy, and atypical pain syndromes. VZV DNA is sometimes present in the blood and saliva of asymptomatic individuals. In total these observations extend understanding of the pathophysiology and epidemiology of VZV, and increasingly contribute to the clinical management of VZV infections.

  20. Virus-derived DNA drives mosquito vector tolerance to arboviral infection

    Pu