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Sample records for replicative-form viral dna

  1. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

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    Lina Li

    Full Text Available 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.

  2. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    Science.gov (United States)

    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.

  3. Structure of the replicative form of bacteriophage φX174 : VI. Studies on alkali-denatured double-stranded φX DNA

    NARCIS (Netherlands)

    Pouwels, P.H.; Knijnenburg, C.M.; Rotterdam, J. van; Cohen, J.A.; Jansz, H.S.

    1968-01-01

    Double-stranded φX DNA which accumulates after infection with bacteriophage φX174 in the presence of chloramphenicol consists mainly of twisted circular double-stranded DNA with no single-strand breaks (component I) and of circular double-stranded DNA, in which single-strand breaks are present (comp

  4. DNA vaccines for viral diseases

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    Donnelly J.J.

    1999-01-01

    Full Text Available DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

  5. Viral DNA in horses infected with equine infectious anemia virus.

    OpenAIRE

    Rice, N R; Lequarré, Anne-Sophie; Casey, J W; Lahn, S; Stephens, R. M.; Edwards, J.

    1989-01-01

    The amount and distribution of viral DNA were established in a horse acutely infected with the Wyoming strain of equine infectious anemia virus (EIAV). The highest concentration of viral DNA were found in the liver, lymph nodes, bone marrow, and spleen. The kidney, choroid plexus, and peripheral blood leukocytes also contained viral DNA, but at a lower level. It is estimated that at day 16 postinoculation, almost all of the viral DNA was located in the tissues, with the liver alone containing...

  6. Advances in Non-Viral DNA Vectors for Gene Therapy

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    Cinnamon L. Hardee

    2017-02-01

    Full Text Available Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic

  7. Advances in Non-Viral DNA Vectors for Gene Therapy

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    Hardee, Cinnamon L.; Arévalo-Soliz, Lirio Milenka; Hornstein, Benjamin D.; Zechiedrich, Lynn

    2017-01-01

    Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic. PMID:28208635

  8. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage phi29

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    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2016-01-01

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine3+ causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation. PMID:24996111

  9. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29.

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    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J; Smith, Douglas E

    2014-06-20

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.

  10. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage phi29

    OpenAIRE

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2014-01-01

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine3+ causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interacti...

  11. Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

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    Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

    2002-07-01

    The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

  12. Viral discovery and sequence recovery using DNA microarrays.

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    David Wang

    2003-11-01

    Full Text Available Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

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

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    Yan Chen

    2014-10-01

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

  14. The DNA damage response in viral-induced cellular transformation.

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    Nikitin, P A; Luftig, M A

    2012-01-31

    The DNA damage response (DDR) has emerged as a critical tumour suppressor pathway responding to cellular DNA replicative stress downstream of aberrant oncogene over-expression. Recent studies have now implicated the DDR as a sensor of oncogenic virus infection. In this review, we discuss the mechanisms by which tumour viruses activate and also suppress the host DDR. The mechanism of tumour virus induction of the DDR is intrinsically linked to the need for these viruses to promote an S-phase environment to replicate their nucleic acid during infection. However, inappropriate expression of viral oncoproteins can also activate the DDR through various mechanisms including replicative stress, direct interaction with DDR components and induction of reactive oxygen species. Given the growth-suppressive consequences of activating the DDR, tumour viruses have also evolved mechanisms to attenuate these pathways. Aberrant expression of viral oncoproteins may therefore promote tumourigenesis through increased somatic mutation and aneuploidy due to DDR inactivation. This review will focus on the interplay between oncogenic viruses and the DDR with respect to cellular checkpoint control and transformation.

  15. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

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    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  16. MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

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    Qian Feng

    2012-11-01

    Full Text Available RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C, a synthetic dsRNA mimic. Here, we dissected the IFN-α/β-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/β response. IFN-α/β production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.

  17. Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini

    NARCIS (Netherlands)

    D.C. van Gent (Dik); A.A. Groeneger; R.H. Plassterk

    1993-01-01

    textabstractThe human immunodeficiency virus integrase (HIV IN) protein cleaves two nucleotides off the 3' end of viral DNA and subsequently integrates the viral DNA into target DNA. IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack by water or

  18. Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini

    NARCIS (Netherlands)

    D.C. van Gent (Dik); A.A. Groeneger; R.H. Plassterk

    1993-01-01

    textabstractThe human immunodeficiency virus integrase (HIV IN) protein cleaves two nucleotides off the 3' end of viral DNA and subsequently integrates the viral DNA into target DNA. IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack by water or

  19. DNA cleavage enzymes for treatment of persistent viral infections: Recent advances and the pathway forward

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Nicholas D., E-mail: nweber@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Aubert, Martine, E-mail: maubert@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Dang, Chung H., E-mail: cdang@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Stone, Daniel, E-mail: dstone2@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Jerome, Keith R., E-mail: kjerome@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Department of Microbiology, University of Washington, Seattle, WA 98195 (United States)

    2014-04-15

    Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.

  20. DNA-guided hepatitis B treatment: Viral load is insufficient with few exceptions

    Institute of Scientific and Technical Information of China (English)

    Pankaj Jain

    2009-01-01

    In DNA-guided hepatitis B treatment, viral load is insufficient, and requires other viral markers for treatment of hepatitis B patients as in patients with acute exacerbation of chronic hepatitis B, end-stage renal disease on dialysis, human immunodeficiency virus co-infected patients. There are exceptions to this rule:a residual level hepatitis B virus (HBV) DNA at 24 wk predicts beneficial outcome and reduced resistance at 1 year. The genotypic viral resistance to antiviral agents and occult HBV infection can be determined by HBV-DNA levels.

  1. Engineering large viral DNA genomes using the CRISPR-Cas9 system.

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    Suenaga, Tadahiro; Kohyama, Masako; Hirayasu, Kouyuki; Arase, Hisashi

    2014-09-01

    Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus-infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time-consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat-Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene-ablated HSV but also gene knock-in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein-Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.

  2. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

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    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit

  3. CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

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    Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

    2017-04-06

    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

  4. Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine.

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    Maluf, Nasib Karl; Gaussier, Hélène; Bogner, Elke; Feiss, Michael; Catalano, Carlos Enrique

    2006-12-26

    Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.

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

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    Knipe, David M., E-mail: david_knipe@hms.harvard.edu

    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.

  6. Viral reverse transcriptases show selective high affinity binding to DNA-DNA primer-templates that resemble the polypurine tract.

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    Gauri R Nair

    Full Text Available Previous results using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment-based approach that selected DNA primer-template duplexes binding with high affinity to HIV reverse transcriptase (RT showed that primers mimicking the 3' end, and in particular the six nt terminal G tract, of the RNA polypurine tract (PPT; HIV PPT: 5'-AAAAGAAAAGGGGGG-3' were preferentially selected. In this report, two viral (Moloney murine leukemia virus (MuLV and avian myeloblastosis virus (AMV and one retrotransposon (Ty3 RTs were used for selection. Like HIV RT, both viral RTs selected duplexes with primer strands mimicking the G tract at the PPT 3' end (AMV PPT: 5'-AGGGAGGGGGA-3'; MuLV PPT: 5'-AGAAAAAGGGGGG-3'. In contrast, Ty3, whose PPT lacks a G tract (5'-GAGAGAGAGGAA-3' showed no selective binding to any duplex sequences. Experiments were also conducted with DNA duplexes (termed DNA PPTs mimicking the RNA PPT-DNA duplex of each virus and a control duplex with a random DNA sequence. Retroviral RTs bound with high affinity to all viral DNA PPT constructs, with HIV and MuLV RTs showing comparable binding to the counterpart DNA PPT duplexes and reduced affinity to the AMV DNA PPT. AMV RT showed similar behavior with a modest preference for its own DNA PPT. Ty3 RT showed no preferential binding for its own or any other DNA PPT and viral RTs bound the Ty3 DNA PPT with relatively low affinity. In contrast, binding affinity of HIV RT to duplexes containing the HIV RNA PPT was less dependent on the G tract, which is known to be pivotal for efficient extension. We hypothesize that the G tract on the RNA PPT helps shift the binding orientation of RT to the 3' end of the PPT where extension can occur.

  7. DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

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    Rowles, Daniell L; Tsai, Yuan-Chin; Greco, Todd M; Lin, Aaron E; Li, Minghao; Yeh, Justin; Cristea, Ileana M

    2015-06-01

    Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.

  8. Consistent detection of Felis domesticus papillomavirus 2 DNA sequences within feline viral plaques.

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    Munday, John S; Peters-Kennedy, Jeanine

    2010-11-01

    Viral plaques are well recognized skin lesions of cats. They are thought to be caused by papillomavirus infection; however, the causative papillomavirus is uncertain. In the current study, polymerase chain reaction using 2 consensus primer sets and 1 primer set specific for Felis domesticus papillomavirus 2 (FdPV-2) was used to amplify DNA from a series of 14 feline viral plaques. The FdPV-2 sequences were detected in all 14 viral plaques by the specific primers but in only 1 of 14 feline cutaneous trichoblastomas. Papillomavirus DNA was amplified from 8 plaques using the consensus primers. Sequences from FdPV-2 were amplified using the consensus primers from 4 plaques. In addition, 3 plaques contained papillomavirus DNA sequences from Felis domesticus papillomavirus sequence MY1, and a previously unreported papillomavirus DNA sequence was amplified from 1 plaque. As FdPV-2 was consistently present within the plaques, this suggests that this papillomavirus is the likely etiologic agent. Feline viral plaques can undergo neoplastic transformation to Bowenoid in situ carcinomas (BISCs). As FdPV-2 DNA is frequently present within BISCs, this suggests that FdPV-2 induces viral plaque formation and then remains detectible after neoplastic transformation.

  9. Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR).

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    Hebia-Fellah, Imen; Léauté, Anne; Fiéni, Francis; Zientara, Stéphan; Imbert-Marcille, Berthe-Marie; Besse, Bernard; Fortier, Guillaume; Pronost, Stephane; Miszczak, Fabien; Ferry, Bénédicte; Thorin, Chantal; Pellerin, Jean-Louis; Bruyas, Jean-François

    2009-06-01

    In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility. Samples of stallion semen (n=390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test. EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (pherpes viruses in other species.

  10. Evidence for and Localization of Vegetative Viral DNA Replication by Autoradiographic Detection of RNA·DNA Hybrids in Sections of Tumors Induced by Shope Papilloma Virus

    Science.gov (United States)

    Orth, Gérard; Jeanteur, Philippe; Croissant, Odile

    1971-01-01

    The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis. Images PMID:4331563

  11. Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging.

    Science.gov (United States)

    Berndsen, Zachary T; Keller, Nicholas; Grimes, Shelley; Jardine, Paul J; Smith, Douglas E

    2014-06-10

    Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.

  12. Increment of DNA topoisomerases in chemically and virally transformed cells

    Energy Technology Data Exchange (ETDEWEB)

    Crespi, M.D.; Mladovan, A.G.; Baldi, A. (Instituto de Biologia y Medicina Experimental, Buenos Aires (Argentina))

    1988-03-01

    The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo(a)pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between {sup 32}P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo(a)pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA.

  13. Viral hemorrhagic fevers of animals caused by DNA viruses

    Science.gov (United States)

    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...

  14. cis-acting sequences that control the level of viral DNA synthesis in the polyomavirus late region.

    Science.gov (United States)

    Melucci-Vigo, G; Ciotta, C; Risuleo, G

    1989-01-01

    A deletion in the polyomavirus late region results in a drastic reduction of viral replication, as shown after transfection of viral DNA into 3T6 cells. This mutation is cis acting, since cotransfection with wild-type DNA did not restore the normal phenotype. Viral DNA synthesis returned to normal levels only after reintroduction of the authentic sequences in either orientation. The data presented here suggest that these sequences are involved in the binding of a factor(s) that controls the level of viral replication. Images PMID:2552181

  15. Heterologous Protection Against Influenza by Injection of DNA Encoding a Viral Protein

    Science.gov (United States)

    Ulmer, Jeffrey B.; Donnelly, John J.; Parker, Suezanne E.; Rhodes, Gary H.; Felgner, Philip L.; Dwarki, V. J.; Gromkowski, Stanislaw H.; Deck, R. Randall; Dewitt, Corrille M.; Friedman, Arthur; Hawe, Linda A.; Leander, Karen R.; Martinez, Douglas; Perry, Helen C.; Shiver, John W.; Montgomery, Donna L.; Liu, Margaret A.

    1993-03-01

    Cytotoxic T lymphocytes (CTLs) specific for conserved viral antigens can respond to different strains of virus, in contrast to antibodies, which are generally strain-specific. The generation of such CTLs in vivo usually requires endogenous expression of the antigen, as occurs in the case of virus infection. To generate a viral antigen for presentation to the immune system without the limitations of direct peptide delivery or viral vectors, plasmid DNA encoding influenza A nucleoprotein was injected into the quadriceps of BALB/c mice. This resulted in the generation of nucleoprotein-specific CTLs and protection from a subsequent challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass loss, and increased survival.

  16. Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice.

    Science.gov (United States)

    Qi, Zhihua; Li, Gaiyun; Hu, Hao; Yang, Chunhui; Zhang, Xiaoming; Leng, Qibin; Xie, Youhua; Yu, Demin; Zhang, Xinxin; Gao, Yueqiu; Lan, Ke; Deng, Qiang

    2014-07-01

    It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. Importance: (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was

  17. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis.

    Science.gov (United States)

    Lu, Huading; Dai, Yuhu; Lv, Lulu; Zhao, Huiqing

    2014-01-01

    The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.

  18. Small terminase couples viral DNA binding to genome-packaging ATPase activity.

    Science.gov (United States)

    Roy, Ankoor; Bhardwaj, Anshul; Datta, Pinaki; Lander, Gabriel C; Cingolani, Gino

    2012-08-08

    Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here we describe the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ∼23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology.

  19. [The presence of non-integrated SV40 viral DNA in nonproductive cells transformed by this virus].

    Science.gov (United States)

    Daya-Grosjean, L; Bénichou, D; Monier, R

    1975-09-08

    The hot phenol extraction of nuclic acids reveals the presence of small amounts of nonintegrated SV 40 DNA in transformed syrian hamster or mouse cells. The extractibility of the viral DNA is influenced by its conformation; SV 40 DNA, form I is preferentially extracted by contrast with form III DNA.

  20. Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

    Science.gov (United States)

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-04-01

    Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

  1. SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products.

    Directory of Open Access Journals (Sweden)

    Gregory A Sowd

    2014-12-01

    Full Text Available Simian virus 40 (SV40 and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PK(cs kinase activity, facilitates some aspects of double strand break (DSB repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR and do not colocalize with non-homologous end joining (NHEJ factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PK(cs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5' to 3' end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication.

  2. SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products.

    Science.gov (United States)

    Sowd, Gregory A; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L; Fanning, Ellen

    2014-12-01

    Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PK(cs) kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PK(cs) and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5' to 3' end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication.

  3. Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation.

    Science.gov (United States)

    Kaufer, Benedikt B; Jarosinski, Keith W; Osterrieder, Nikolaus

    2011-03-14

    Some herpesviruses, particularly lymphotropic viruses such as Marek's disease virus (MDV) and human herpesvirus 6 (HHV-6), integrate their DNA into host chromosomes. MDV and HHV-6, among other herpesviruses, harbor telomeric repeats (TMRs) identical to host telomeres at either end of their linear genomes. Using MDV as a natural virus-host model, we show that herpesvirus TMRs facilitate viral genome integration into host telomeres and that integration is important for establishment of latency and lymphoma formation. Integration into host telomeres also aids in reactivation from the quiescent state of infection. Our results and the presence of TMRs in many herpesviruses suggest that integration mediated by viral TMRs is a conserved mechanism, which ensures faithful virus genome maintenance in host cells during cell division and allows efficient mobilization of dormant viral genomes. This finding is of particular importance as reactivation is critical for virus spread between susceptible individuals and is necessary for continued herpesvirus evolution and survival.

  4. Viral oncogene-induced DNA damage response is activated in Kaposi sarcoma tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Sonja Koopal

    2007-09-01

    Full Text Available Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV-infected tumor cells that express endothelial cell (EC markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

  5. Fusion Wheat Histone H4 Protein Increases Transfection Efficiency of Non-viral DNA Vector

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-yan; ZHANG Yu-jing

    2011-01-01

    The lack of efficient and non-toxic gene delivery, preferably with non-viral DNA vectors, is generally regarded as a major limitation for gene therapy. In this study, a wheat histone H4 gene was cloned from Triticum aestivum, sequenced, modified and expressed in E. coli. The wheat histone H4 gene and reconstructed H4TL gene encoded wheat histone H4 and a recombinant protein of 141 amino acids with an approximate molecular weight of 15500. Gel electrophoresis mobility shift assays demonstrated that the purified protein had high affinity for DNA.Most significantly, the complex of plasmid pEGFP/Cl with H4TL was transfected with increased efficiency into MCF-7, HO8910, LNCap, A549 and HeLa cells in vitro. These results demonstrate that the targeting of non-viral vectors to tumor-specific receptors provides a cheap, simple and highly efficient tool for gene delivery.

  6. Small terminase couples viral DNA-binding to genome-packaging ATPase activity

    OpenAIRE

    Roy, Ankoor; Bhardwaj, Anshul; Datta, Pinaki; Lander, Gabriel C.; Cingolani, Gino

    2012-01-01

    Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here, we describe the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ~23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essen...

  7. Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication.

    Science.gov (United States)

    Paran, Nir; De Silva, Frank S; Senkevich, Tatiana G; Moss, Bernard

    2009-12-17

    Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

  8. p53 represses human papillomavirus type 16 DNA replication via the viral E2 protein

    Directory of Open Access Journals (Sweden)

    Morgan Iain M

    2008-01-01

    Full Text Available Abstract Background Human papillomavirus (HPV DNA replication can be inhibited by the cellular tumour suppressor protein p53. However, the mechanism through which p53 inhibits viral replication and the role that this might play in the HPV life cycle are not known. The papillomavirus E2 protein is required for efficient HPV DNA replication and also regulates viral gene expression. E2 represses transcription of the HPV E6 and E7 oncogenes and can thereby modulate indirectly host cell proliferation and survival. In addition, the E2 protein from HPV 16 has been shown to bind p53 and to be capable of inducing apoptosis independently of E6 and E7. Results Here we use a panel of E2 mutants to confirm that mutations which block the induction of apoptosis via this E6/E7-independent pathway, have little or no effect on the induction of apoptosis by the E6/E7-dependent pathway. Although these mutations in E2 do not affect the ability of the protein to mediate HPV DNA replication, they do abrogate the repressive effects of p53 on the transcriptional activity of E2 and prevent the inhibition of E2-dependent HPV DNA replication by p53. Conclusion These data suggest that p53 down-regulates HPV 16 DNA replication via the E2 protein.

  9. DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding.

    Science.gov (United States)

    Li, Chun Mei; Zheng, Lin Ling; Yang, Xiao Xi; Wan, Xiao Yan; Wu, Wen Bi; Zhen, Shu Jun; Li, Yuan Fang; Luo, Ling Fei; Huang, Cheng Zhi

    2016-01-01

    Viral infections have caused numerous diseases and deaths worldwide. Due to the emergence of new viruses and frequent virus variation, conventional antiviral strategies that directly target viral or cellular proteins are limited because of the specificity, drug resistance and rapid clearance from the human body. Therefore, developing safe and potent antiviral agents with activity against viral infection at multiple points in the viral life cycle remains a major challenge. In this report, we propose a new modality to inhibit viral infection by fabricating DNA conjugated gold nanoparticle (DNA-AuNP) networks on cell membranes as a protective barrier. The DNA-AuNPs networks were found, via a plaque formation assay and viral titers, to have potent antiviral ability and protect host cells from human respiratory syncytial virus (RSV). Confocal immunofluorescence image analysis showed 80 ± 3.8% of viral attachment, 91.1 ± 0.9% of viral entry and 87.9 ± 2.8% of viral budding were inhibited by the DNA-AuNP networks, which were further confirmed by real-time fluorescence imaging of the RSV infection process. The antiviral activity of the networks may be attributed to steric effects, the disruption of membrane glycoproteins and limited fusion of cell membrane bilayers, all of which play important roles in viral infection. Therefore, our results suggest that the DNA-AuNP networks have not only prophylactic effects to inhibit virus attachment and entry, but also therapeutic effects to inhibit viral budding and cell-to-cell spread. More importantly, this proof-of-principle study provides a pathway for the development of a universal, broad-spectrum antiviral therapy.

  10. Temporal order of evolution of DNA replication systems inferred by comparison of cellular and viral DNA polymerases

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2006-12-01

    Full Text Available Abstract Background The core enzymes of the DNA replication systems show striking diversity among cellular life forms and more so among viruses. In particular, and counter-intuitively, given the central role of DNA in all cells and the mechanistic uniformity of replication, the core enzymes of the replication systems of bacteria and archaea (as well as eukaryotes are unrelated or extremely distantly related. Viruses and plasmids, in addition, possess at least two unique DNA replication systems, namely, the protein-primed and rolling circle modalities of replication. This unexpected diversity makes the origin and evolution of DNA replication systems a particularly challenging and intriguing problem in evolutionary biology. Results I propose a specific succession for the emergence of different DNA replication systems, drawing argument from the differences in their representation among viruses and other selfish replicating elements. In a striking pattern, the DNA replication systems of viruses infecting bacteria and eukaryotes are dominated by the archaeal-type B-family DNA polymerase (PolB whereas the bacterial replicative DNA polymerase (PolC is present only in a handful of bacteriophage genomes. There is no apparent mechanistic impediment to the involvement of the bacterial-type replication machinery in viral DNA replication. Therefore, I hypothesize that the observed, markedly unequal distribution of the replicative DNA polymerases among the known cellular and viral replication systems has a historical explanation. I propose that, among the two types of DNA replication machineries that are found in extant life forms, the archaeal-type, PolB-based system evolved first and had already given rise to a variety of diverse viruses and other selfish elements before the advent of the bacterial, PolC-based machinery. Conceivably, at that stage of evolution, the niches for DNA-viral reproduction have been already filled with viruses replicating with the

  11. Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging.

    Science.gov (United States)

    Dhar, Alok; Feiss, Michael

    2005-03-18

    DNA packaging by large DNA viruses such as the tailed bacteriophages and the herpesviruses involves DNA translocation into a preformed protein shell, called the prohead. Translocation is driven by an ATP hydrolysis-powered DNA packaging motor. The bacteriophages encode a heterodimeric viral DNA packaging protein, called terminase. The terminases have an ATPase center located in the N terminus of the large subunit implicated in DNA translocation. In previous work with phage lambda, lethal mutations that changed ATP-reactive residues 46 and 84 of gpA, the large terminase subunit, were studied. These mutant enzymes retained the terminase endonuclease and helicase activities, but had severe defects in virion assembly, and lacked the terminase high-affinity ATPase activity. Surprisingly, in the work described here, we found that enzymes with the conservative gpA changes Y46F and Y46A had only mild packaging defects. These mild defects contrast with their profound virion assembly defects. Thus, these mutant enzymes have, in addition to the mild DNA packaging defects, a severe post-DNA packaging defect. In contrast, the gpA K84A enzyme had similar virion assembly and DNA packaging defects. The DNA packaging energy budget, i.e. DNA packaged/ATP hydrolyzed, was unchanged for the mutant enzymes, indicating that DNA translocation is tightly coupled to ATP hydrolysis. A model is proposed in which gpA residues 46 and 84 are important for terminase's high-affinity ATPase activity. Assembly of the translocation complex remodels this ATPase so that residues 46 and 84 are not crucial for the activated translocation ATPase. Changing gpA residues 46 and 84 primarily affects assembly, rather than the activity, of the translocation complex.

  12. An Epstein-Barr virus mutant produces immunogenic defective particles devoid of viral DNA.

    Science.gov (United States)

    Pavlova, Sophia; Feederle, Regina; Gärtner, Kathrin; Fuchs, Walter; Granzow, Harald; Delecluse, Henri-Jacques

    2013-02-01

    Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists of producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4(+) T-cell response that was indistinguishable from the one obtained with wild-type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.

  13. Impact of two different commercial DNA extraction methods on BK virus viral load

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    Massimiliano Bergallo

    2016-03-01

    Full Text Available Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France and manual QIAGEN extraction (QIAGEN Hilden, Germany. BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit, for the extraction of BK virus from serum and urine specimens.

  14. Optical tweezers studies of viral DNA packaging: Motor function and DNA confinement in Bacteriophages phi29, lambda, and T4

    Science.gov (United States)

    Smith, Douglas

    2007-03-01

    In the assembly of many viruses a powerful molecular motor translocates the genome into a pre-assembled capsid. We use optical tweezers to directly measure translocation of a single DNA molecule into the viral capsid. Improved techniques allow us to measure initiation and early stages of packaging. With phi29 the DNA terminal protein was found to cause large variations in the starting point of packaging. Removal of this protein results in terminal initiation, permitting more accurate assessment of motor function and DNA confinement forces. We investigated the role of electrostatic repulsion by varying ionic screening of the DNA. The observed trends are in accord with those theoretically expected considering counter-ion competition; however the forces are larger than expected in comparison with recent theories and DNA ejection measurements. We have recently succeeded in extending our methods to study two other phages: lambda and T4. These systems have unique structural and functional features, presenting an opportunity for comparative studies in this family of molecular motors. Initial measurements show that lambda and T4 translocate DNA several times faster than the phi29 motor, but are more sensitive to applied load.

  15. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    Directory of Open Access Journals (Sweden)

    Huzhang Mao

    2016-03-01

    Full Text Available Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

  16. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    Science.gov (United States)

    Reyes-Aldrete, Emilio; Sherman, Michael B.; Woodson, Michael; Atz, Rockney; Grimes, Shelley; Jardine, Paul J.; Morais, Marc C.

    2016-01-01

    SUMMARY Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring, and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate. PMID:26904950

  17. Retroviral DNA integration: viral and cellular determinants of target-site selection.

    Directory of Open Access Journals (Sweden)

    Mary K Lewinski

    2006-06-01

    Full Text Available Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV integrates preferentially within active transcription units, whereas murine leukemia virus (MLV integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN coding region into HIV (to make HIVmIN caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I-hypersensitive sites (i.e., +/- 1 kb, and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.

  18. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

    Directory of Open Access Journals (Sweden)

    Maja Kiselinova

    2016-03-01

    Full Text Available The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6 between time point 1 and 2; and median of 31 days (IQR: 28-36 between time point 2 and 3. Patients were median of 6 years (IQR: 3-12 on ART, and plasma viral load (<50 copies/ml was suppressed for median of 4 years (IQR: 2-8. Total HIV-1 DNA, unspliced (us and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85, us HIV-1 RNA (p = 0.029, R² = 0.40, and VOA (p = 0.041, R2 = 0.44. Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54. The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1. Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the

  19. Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

    Institute of Scientific and Technical Information of China (English)

    Yun-Qing Yao; Wei-Ping Zhou; Hong Ren; Qi Liu; Shu-Hua Guo; Ding-Feng Zhang; Ni Tang; Ai-Long Huang; Xiao-Yi Zou; Jiang-Feng Xiao; Yun Luo; Da-Zhi Zhang; Bo Wang

    2005-01-01

    AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs).METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System.Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group.RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group.CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species.HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

  20. DNA-guided hepatitis B treatment, viral load is essential,but not sufficient

    Institute of Scientific and Technical Information of China (English)

    Rafael Bárcena Marugán; Silvia García Garzón

    2009-01-01

    Hepatitis B virus (HBV) infection is a global public health problem that concerns 350 million people worldwide. Individuals with chronic hepatitis B (CHB) are at increased risk of developing liver cirrhosis,hepat i c de- compensation and hepatocellular carcinoma. To maintain undetectable viral load reduces chronic infection complications. There is no treatment that eradicates HBV infection. Current drugs are expensive, are associated with adverse events, and are of limited efficacy. Current guidelines try to standardize the clinical practice. Nevertheless, controversy remains about management of asymptomatic patients with CHB who are hepatitis B e antigen (HBeAg)-positive with normal alanine aminotransferase, and what is the cut-off value of viral load to distinguish HBeAgnegative CHB patients and inactive carriers. We discuss in detail why DNA level alone is not sufficient to begin treatment of CHB.

  1. Role of biofilm in protection of the replicative form of Legionella pneumophila.

    Science.gov (United States)

    Andreozzi, Elisa; Di Cesare, Andrea; Sabatini, Luigia; Chessa, Elisa; Sisti, Davide; Rocchi, Marco; Citterio, Barbara

    2014-12-01

    The dual nature of Legionella pneumophila enables its survival in free and intracellular environments and underpins its infection and spread mechanisms. Experiments using bacterial cultures and improved RTqPCR protocols were devised to gain fresh insights into the role of biofilm in protecting the replicative form of L. pneumophila. mip gene expression was used as a marker of virulence in sessile (biofilm-bound) and planktonic (free-floating) cells of L. pneumophila serotype 1 ATCC 33152. The ratio of mip gene expression to transcriptionally active Legionella cells increased both in sessile and free-floating cells demonstrating an up-regulation of mip gene under nutrient depletion. However, a different trend was observed between the two forms, in planktonic cells the mip gene expression/transcriptionally active Legionella cells increased until the end of the experiment, while in the biofilm such increase was observed at the end of the experiment. These findings suggest a possible association between the switch to the transmissive phase of Legionella and a mip up-regulation and a role for biofilm in preserving Legionella cells in replicative form. Moreover, it has been shown that improved RTqPCR protocols are valuable tools to explore bacterial virulence.

  2. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities

    DEFF Research Database (Denmark)

    Guo, Yang; Kragelund, Birthe Brandt; White, Malcolm F.

    2015-01-01

    The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), bot...... for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.......The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both...... encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U...

  3. Viral eukaryogenesis: was the ancestor of the nucleus a complex DNA virus?

    Science.gov (United States)

    Bell, P J

    2001-09-01

    In the theory of viral eukaryogenesis I propose here, the eukaryotic nucleus evolved from a complex DNA virus. It is proposed that the virus established a persistent presence in the cytoplasm of a methanogenic mycoplasma and evolved into the eukaryotic nucleus by acquiring a set of essential genes from the host genome and eventually usurping its role. It is proposed that several characteristic features of the eukaryotic nucleus derive from its viral ancestry. These include mRNA capping, linear chromosomes, and separation of transcription from translation. In the model, phagocytosis and other membrane fusion-based processes are derived from viral membrane fusion processes and evolved in concert with the nucleus. The coevolution of phagocytosis and the nucleus rendered much of the host archaeal genome redundant since the protoeukaryote could obtain raw materials and energy by engulfing bacterial syntrophs/prey. This redundancy allowed loss of the archaeal chromosome, generating an organism with eukaryotic features. The evolution of phagocytosis allowed the eukaryotes to be the first organisms to occupy the niche of predator.

  4. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

    Science.gov (United States)

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanpin; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

  5. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication

    Indian Academy of Sciences (India)

    Guili Wang; Gaowei Ren; Xin Cui; Yanpin Ma; Ying Qi; Yujing Huang; Zhongyang Liu; Zhengrong Sun; Qiang Ruan

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns. Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer in HCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

  6. A novel DNA vaccine technology conveying protection against a lethal herpes simplex viral challenge in mice.

    Directory of Open Access Journals (Sweden)

    Julie L Dutton

    Full Text Available While there are a number of licensed veterinary DNA vaccines, to date, none have been licensed for use in humans. Here, we demonstrate that a novel technology designed to enhance the immunogenicity of DNA vaccines protects against lethal herpes simplex virus 2 (HSV-2 challenge in a murine model. Polynucleotides were modified by use of a codon optimization algorithm designed to enhance immune responses, and the addition of an ubiquitin-encoding sequence to target the antigen to the proteasome for processing and to enhance cytotoxic T cell responses. We show that a mixture of these codon-optimized ubiquitinated and non-ubiquitinated constructs encoding the same viral envelope protein, glycoprotein D, induced both B and T cell responses, and could protect against lethal viral challenge and reduce ganglionic latency. The optimized vaccines, subcloned into a vector suitable for use in humans, also provided a high level of protection against the establishment of ganglionic latency, an important correlate of HSV reactivation and candidate endpoint for vaccines to proceed to clinical trials.

  7. Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.

    Directory of Open Access Journals (Sweden)

    Michael J Moser

    Full Text Available Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR. Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth and two-enzyme (MMLV RT/Taq RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.

  8. Epigenetic control of viral life-cycle by a DNA-methylation dependent transcription factor.

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    Kirsty Flower

    Full Text Available Epstein-Barr virus (EBV encoded transcription factor Zta (BZLF1, ZEBRA, EB1 is the prototype of a class of transcription factor (including C/EBPalpha that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.

  9. Development of Viral Capsid DNA Aptamer Conjugates as Cell-Targeted Delivery Vehicles

    Science.gov (United States)

    Tong, Gary Jen-Wei

    The ability to generate semi-synthetic DNA-protein conjugates has become increasingly important in the fields of chemical biology and nanobiotechnology. As applications in these fields become more complex, there is also an increased need for methods of attaching synthetic DNA to protein substrates in a well-defined manner. This work outlines the development of new methods for site-specific DNA-protein bioconjugation, as well as the development of novel viral capsid DNA aptamer conjugates for cell-targeting purposes. In order to generate DNA-protein conjugates in a site-specific manner, chemistries orthogonal to native functional groups present on DNA and proteins were exploited. In one method, the attachment of DNA to proteins was achieved via oxime formation. This strategy involved the in situ deprotection of an allyloxycarbonyl-protected alkoxyamine-bearing DNA in the presence of a protein containing a single ketone group. The utility of this approach was demonstrated in the synthesis of a DNA-GFP conjugate. In addition to the oxime formation route, two oxidative coupling methods were also developed for DNA-protein bioconjugation. The first reaction coupled phenylenediamine-containing DNA to anilines, which had been site-specifically incorporated into proteins, in the presence of NaIO4. These reaction conditions were demonstrated on the proteins bacteriophage MS2 and GFP, and were mild enough for the components to retain both protein structure and DNA base-pairing capabilities. The second oxidative coupling reaction conjugated aniline-containing proteins to DNA bearing an o-aminophenol moiety. This reaction occurred under similarly mild conditions; however, higher coupling yields were achieved on MS2 at shorter reaction times by using this strategy. In all three of these methods, the generation of a singly-modified product was achieved. Using one of our oxidative coupling strategies, MS2-DNA aptamer conjugates were synthesized for the development of multivalent

  10. Improving the safety of viral DNA vaccines: development of vectors containing both 5' and 3' homologous regulatory sequences from non-viral origin.

    Science.gov (United States)

    Martinez-Lopez, A; Encinas, P; García-Valtanen, P; Gomez-Casado, E; Coll, J M; Estepa, A

    2013-04-01

    Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5' and 3' regulatory sequences of β-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies.

  11. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    Science.gov (United States)

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  12. Uracil DNA glycosylase counteracts APOBEC3G-induced hypermutation of hepatitis B viral genomes: excision repair of covalently closed circular DNA.

    Directory of Open Access Journals (Sweden)

    Kouichi Kitamura

    Full Text Available The covalently closed circular DNA (cccDNA of the hepatitis B virus (HBV plays an essential role in chronic hepatitis. The cellular repair system is proposed to convert cytoplasmic nucleocapsid (NC DNA (partially double-stranded DNA into cccDNA in the nucleus. Recently, antiviral cytidine deaminases, AID/APOBEC proteins, were shown to generate uracil residues in the NC-DNA through deamination, resulting in cytidine-to-uracil (C-to-U hypermutation of the viral genome. We investigated whether uracil residues in hepadnavirus DNA were excised by uracil-DNA glycosylase (UNG, a host factor for base excision repair (BER. When UNG activity was inhibited by the expression of the UNG inhibitory protein (UGI, hypermutation of NC-DNA induced by either APOBEC3G or interferon treatment was enhanced in a human hepatocyte cell line. To assess the effect of UNG on the cccDNA viral intermediate, we used the duck HBV (DHBV replication model. Sequence analyses of DHBV DNAs showed that cccDNA accumulated G-to-A or C-to-T mutations in APOBEC3G-expressing cells, and this was extensively enhanced by UNG inhibition. The cccDNA hypermutation generated many premature stop codons in the P gene. UNG inhibition also enhanced the APOBEC3G-mediated suppression of viral replication, including reduction of NC-DNA, pre-C mRNA, and secreted viral particle-associated DNA in prolonged culture. Enhancement of APOBEC3G-mediated suppression by UNG inhibition was not observed when the catalytic site of APOBEC3G was mutated. Transfection experiments of recloned cccDNAs revealed that the combination of UNG inhibition and APOBEC3G expression reduced the replication ability of cccDNA. Taken together, these data indicate that UNG excises uracil residues from the viral genome during or after cccDNA formation in the nucleus and imply that BER pathway activities decrease the antiviral effect of APOBEC3-mediated hypermutation.

  13. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    Science.gov (United States)

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

  14. Rapid, sensitive and cost effective method for isolation of viral DNA from feacal samples of dogs

    Directory of Open Access Journals (Sweden)

    Savi.

    2010-06-01

    Full Text Available A simple method for viral DNA extraction using chelex resin was developed. The method used was eco-friendly and cost effective compared to other methods such as phenol chloroform method which use health hazardous organic reagents. Further, a polymerase chain reaction (PCR based detection of canine parvovirus (CPV using primers from conserved region of VP2 gene was developed. To increase the sensitivity and specificity of reaction, nested PCR was designed. PCR reaction was optimized to amplify 747bp product of VP2 gene. The assay can be completed in few hours and doesn’t need hazardous chemicals. Thus, the sample preparation using chelating resin along with nested PCR seems to be a sensitive, specific and practical method for the detection of CPV in diarrhoeal feacal samples. [Vet. World 2010; 3(3.000: 105-106

  15. Leeches as a source of mammalian viral DNA and RNA - a study in medicinal leeches

    DEFF Research Database (Denmark)

    Kampmann, Marie-Louise; Schnell, Ida Bærholm; Jensen, Randi Holm

    2017-01-01

    leeches suggest that host viruses may also be detectable. To systematically test this hypothesis, we performed a proof of concept study using quantitative PCR (qPCR) to detect DNA viruses (bovine herpesvirus [BHV], human adenovirus [HAdV]) and RNA viruses (influenza A [InfA] and measles morbillivirus [Me......V]) from nucleic acids extracted from medicinal leeches fed with blood spiked with each virus. All viruses except BHV showed a gradual decline in concentration from day 1 to 50, and all except BHV were detectable in at least half of the samples even after 50 days. BHV exhibited a rapid decline at day 27...... and was undetectable at day 50. Our findings in medicinal leeches indicate that leeches collected in the wild might be an untapped resource for detecting vertebrate viruses and could provide new opportunities to study wildlife viral diseases of rare species in challenging environments, where capturing and handling...

  16. KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

    Science.gov (United States)

    Gupta, Namrata; Thakker, Suhani; Verma, Subhash C.

    2016-09-01

    The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA.

  17. Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA.

    Science.gov (United States)

    Yang, Teng-Chieh; Maluf, Nasib Karl

    2012-02-21

    Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

  18. Occurrence of viral DNA in paired samples of corneal rim and cornea preservation fluid.

    Science.gov (United States)

    Broniek, G; Langwińska-Wośko, E; Sybilska, M; Szaflik, J P; Przybylski, M; Wróblewska, M

    2017-04-01

    Corneal transplants have one of the highest success rates among all transplantological procedures. Corneas intended for transplantation are stored in a preservation fluid, which is then tested for bacterial and fungal infections. Among all analyses of infectious complications following corneal transplants, infections caused by bacteria or fungi are the most prominent. Surprisingly, however, apart from a few publications, there is a lack of data regarding the occurrence of viruses in donor corneas and the risk of transmitting these to their recipients. The intention of this research was therefore to determine the frequency with which human herpesvirus 1 (HHV-1), human herpesvirus 2 (HHV-2), and human adenovirus (HAdV) occur in transplanted corneal tissue, as well as in samples of preservation fluid. The study comprised 57 paired samples, with each pair consisting of a fragment of the corneal tissue remaining after its trepanation for transplantation surgery and a sample of corneal preservation fluid. Sample pairs were all tested for the presence of the DNA of three viruses (HHV-1, HHV-2, and HAdV) using real time PCR technique. Viral DNA was found in three of the tested corneas-HHV-1 DNA in one paired sample (1.8%) and adenovirus DNA in two single samples (3.5%). We postulate that virological testing of corneas for transplantation should be considered, particularly in the case of donors with increased risk factors for herpesvirus and adenovirus reactivation. J. Med. Virol. 89:732-736, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Immunity to viral haemorrhagic septicaemia (VHS) following DNA vaccination of rainbow trout at an early life-stage

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja

    2001-01-01

    Rainbow trout fry of average weight 0.5 g were vaccinated against viral haemorrhagic septicaemia (VHS) by intramuscular injection of 1 mug of plasmid DNA encoding the VHS virus glycoprotein gene. Challenge with a lethal dose of virus at two different time points, 9 and 71 days post...

  20. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities.

    Science.gov (United States)

    Guo, Yang; Kragelund, Birthe B; White, Malcolm F; Peng, Xu

    2015-06-19

    The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5' → 3' ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.

  1. Substrate recognition and motion mode analyses of PFV integrase in complex with viral DNA via coarse-grained models.

    Directory of Open Access Journals (Sweden)

    Jianping Hu

    Full Text Available HIV-1 integrase (IN is an important target in the development of drugs against the AIDS virus. Drug design based on the structure of IN was markedly hampered due to the lack of three-dimensional structure information of HIV-1 IN-viral DNA complex. The prototype foamy virus (PFV IN has a highly functional and structural homology with HIV-1 IN. Recently, the X-ray crystal complex structure of PFV IN with its cognate viral DNA has been obtained. In this study, both Gaussian network model (GNM and anisotropy network model (ANM have been applied to comparatively investigate the motion modes of PFV DNA-free and DNA-bound IN. The results show that the motion mode of PFV IN has only a slight change after binding with DNA. The motion of this enzyme is in favor of association with DNA, and the binding ability is determined by its intrinsic structural topology. Molecular docking experiments were performed to gain the binding modes of a series of diketo acid (DKA inhibitors with PFV IN obtained from ANM, from which the dependability of PFV IN-DNA used in the drug screen for strand transfer (ST inhibitors was confirmed. It is also found that the functional groups of keto-enol, bis-diketo, tetrazole and azido play a key role in aiding the recognition of viral DNA, and thus finally increase the inhibition capability for the corresponding DKA inhibitor. Our study provides some theoretical information and helps to design anti-AIDS drug based on the structure of IN.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    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

  3. Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant

    Directory of Open Access Journals (Sweden)

    Victoria M. Pearson

    2016-10-01

    Full Text Available Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (Circoviridae and Geminiviridae, and model organisms for genetics and evolution studies (Microviridae. Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such as Circoviridae, Geminiviridae, and Microviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.

  4. Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.

    Science.gov (United States)

    Glushakova, Lyudmyla G; Sharma, Nidhi; Hoshika, Shuichi; Bradley, Andrea C; Bradley, Kevin M; Yang, Zunyi; Benner, Steven A

    2015-11-15

    Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-μl sample. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Utke, Katrin; Kock, Holger; Schuetze, Heike

    2008-01-01

    To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger...... injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system....... of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells...

  6. Concatemeric intermediates of equine herpesvirus type 1 DNA replication contain frequent inversions of adjacent long segments of the viral genome.

    Science.gov (United States)

    Slobedman, B; Simmons, A

    1997-03-17

    In common with other alpha-herpesviruses, the genome of equine herpesvirus type-1 (EHV-1) comprises covalently linked long and short unique sequences of DNA, each flanked by inverted repeats. Equimolar amounts of two genomic isomers, generated by free inversion of the short segment, relative to the long segment, are packaged into EHV-1 virions. In contrast with herpes simplex virus (HSV), inversion of genomic long segments has not been described. In the current work, the structures of high molecular weight intermediates of EHV-1 DNA replication were studied by field inversion gel electrophoresis. It is shown that adjacent long segments of the viral genome are frequently inverted in concatemeric intermediates of EHV-1 DNA replication. Further, like HSV concatemers, high molecular weight intermediates of EHV-1 replication are flanked exclusively by the long segment of the viral genome. Hence, despite the fact that only two, rather than four, isomers of EHV-1 DNA are packaged into virions, the intermediates of EHV-1 DNA replication closely resemble those of herpes simplex virus type 1 in structure. These data have implications relating to the mechanisms involved in packaging of alpha-herpesvirus DNA.

  7. Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: evidence for their involvement in regulation of the viral DNA replication.

    Science.gov (United States)

    Saribas, A Sami; Mun, Sarah; Johnson, Jaslyn; El-Hajmoussa, Mohammad; White, Martyn K; Safak, Mahmut

    2014-01-20

    JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation.

  8. Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

    Directory of Open Access Journals (Sweden)

    Fowke Keith R

    2005-10-01

    Full Text Available Abstract Background In addition to mediating the integration process, HIV-1 integrase (IN has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s and/or motif(s within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection. Results Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV and sequence Q (211KELQKQITK in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA exhibited more severe defect of induction of β-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-β-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C

  9. Is passive transmission of non-viral vectors through artificial insemination of sperm-DNA mixtures sufficient for chicken transgenesis?

    OpenAIRE

    CHAPARIAN, Shahram; ABDULAHNEJAD, Ahad; RASHIDI, Farzad; Toghyani, Majid; Gheisari, Abbasali; Eghbalsaied, Shahin

    2016-01-01

    DNA uptake in the post-acrosomal region of the spermatozoa takes place exclusively in immotile spermatozoa that are naturally unable to fertilize eggs. The present study aimed to assess whether passive transmission of non-viral vectors to the surrounding areas of chicken embryos could be an alternate mechanism in chicken sperm-mediated gene transfer. First, the presence of nucleases in rooster seminal plasma was evaluated. Semen ejaculates from five roosters were centrifuged and the supernata...

  10. Bio-replicated forming of the biomimetic drag-reducing surfaces in large area based on shark skin

    Institute of Scientific and Technical Information of China (English)

    HAN Xin; ZHANG DeYuan; LI Xiang; LI YuanYue

    2008-01-01

    On the investigation of biomimetic drag-reducing surface, direct replication of the firm scarfskins on low-resistance creatures to form biomimetic drag-reducing surfaces with relatively vivid morphology relative to the living prototype is a new attempt of the bio-replicated forming technology. Taking shark skin as the bio-replication template, the hot embossing method was applied to the micro-replication of its outward morphology. Furthermore, the skins were jointed together to form the drag-reducing sur-face in large area. The results of the resistance measurements in a water tunnel according to the flat-plate sample pieces have shown that the biomimetic shark-skin coating fabricated by the bio-replicated forming method has significant drag reduction effect, and that the drag reduction effi-ciency reached 8.25% in the test conditions.

  11. Investigating Signs of Recent Evolution in the Pool of Pro-viral DNA during Years of Successful HAART

    DEFF Research Database (Denmark)

    Mens, H.; Pedersen, Anders Gorm; Jørgensen, L. B.;

    2007-01-01

    In order to shed light on the nature of the persistent reservoir of human immunodeficiency virus type 1 (HIV-1), we investigated signs of recent evolution in the pool of proviral DNA in patients on successful HAART. Pro-viral DNA, corresponding to the C2-V3-C3 region of the HIV-1 env gene......, and then determine the support for models that imply evolution between time points. Model fit and model-selection uncertainty was assessed using the Akaike information criterion (AIC) and Akaike weights. The consensus sequence data was also analyzed using a range of phylogenetic techniques to determine whether...

  12. Investigating Signs of Recent Evolution in the Pool of Pro-viral DNA during Years of Successful HAART

    DEFF Research Database (Denmark)

    Mens, H.; Pedersen, Anders Gorm; Jørgensen, L. B.

    2007-01-01

    In order to shed light on the nature of the persistent reservoir of human immunodeficiency virus type 1 (HIV-1), we investigated signs of recent evolution in the pool of proviral DNA in patients on successful HAART. Pro-viral DNA, corresponding to the C2-V3-C3 region of the HIV-1 env gene......, was collected from PBMCs isolated from 57 patients. Both "consensus" (57 patients) and clonal (7 patients) sequences were obtained from five time points spanning a 24-month period. The main computational strategy was to use maximum likelihood to fit a set of alternative phylogenetic models to the clonal data...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. The Role of DNA Twist in the Packaging of Viral Genomes☆

    OpenAIRE

    Rollins, Geoffrey C.; Petrov, Anton S.; Harvey, Stephen C.

    2008-01-01

    We performed molecular dynamics simulations of the genome packaging of bacteriophage P4 using two coarse-grained models of DNA. The first model, 1DNA6 (one pseudo-atom per six DNA basepairs), represents DNA as a string of beads, for which DNA torsions are undefined. The second model, 3DNA6 (three pseudo-atoms per six DNA basepairs), represents DNA as a series of base planes with torsions defined by the angles between successive planes. Bacteriophage P4 was packaged with 1DNA6, 3DNA6 in a tors...

  15. Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: a new concept for improvement of non-viral therapeutic gene delivery.

    Science.gov (United States)

    Tolmachov, Oleg E

    2012-05-01

    The cell-specific and long-term expression of therapeutic transgenes often requires a full array of native gene control elements including distal enhancers, regulatory introns and chromatin organisation sequences. The delivery of such extended gene expression modules to human cells can be accomplished with non-viral high-molecular-weight DNA vectors, in particular with several classes of linear DNA vectors. All high-molecular-weight DNA vectors are susceptible to damage by shear stress, and while for some of the vectors the harmful impact of shear stress can be minimised through the transformation of the vectors to compact topological configurations by supercoiling and/or knotting, linear DNA vectors with terminal loops or covalently attached terminal proteins cannot be self-compacted in this way. In this case, the only available self-compacting option is self-entangling, which can be defined as the folding of single DNA molecules into a configuration with mutual restriction of molecular motion by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the entangling by bringing DNA segments into proximity and by interfering with the DNA slipping away from the entanglement. I propose that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, inter-segment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. It is expected that the non-B-DNA based entanglement of the linear DNA vectors would consist of the initial transient and co-operative non-B-DNA mediated binding events followed by tight self-ensnarement of the vector DNA. Once in the nucleoplasm of the target human cells, the DNA can be disentangled by type II

  16. Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-viral Gene Therapy for Leber's Congenital Amaurosis.

    Science.gov (United States)

    Sun, Da; Sahu, Bhubanananda; Gao, Songqi; Schur, Rebecca M; Vaidya, Amita M; Maeda, Akiko; Palczewski, Krzysztof; Lu, Zheng-Rong

    2017-06-16

    Development of a gene delivery system with high efficiency and a good safety profile is essential for successful gene therapy. Here we developed a targeted non-viral delivery system using a multifunctional lipid ECO for treating Leber's congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65(-/-) mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Epstein-Barr Virus Immediate-Early Protein Zta Co-Opts Mitochondrial Single-Stranded DNA Binding Protein To Promote Viral and Inhibit Mitochondrial DNA Replication▿

    Science.gov (United States)

    Wiedmer, Andreas; Wang, Pu; Zhou, Jing; Rennekamp, Andrew J.; Tiranti, Valeria; Zeviani, Massimo; Lieberman, Paul M.

    2008-01-01

    Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. To better understand these functions and identify cellular collaborators of Zta, we purified an epitope-tagged version of Zta in cells capable of supporting lytic replication. FLAG-tagged Zta was purified from a nuclear fraction using FLAG antibody immunopurification and peptide elution. Zta-associated proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. The Zta-associated proteins included members of the HSP70 family and various single-stranded DNA and RNA binding proteins. The nuclear replication protein A subunits (RPA70 and RPA32) and the human mitochondrial single-stranded DNA binding protein (mtSSB) were confirmed by Western blotting to be specifically enriched in the FLAG-Zta immunopurified complex. mtSSB coimmunoprecipitated with endogenous Zta during reactivation of EBV-positive Burkitt lymphoma and lymphoblastoid cell lines. Small interfering RNA depletion of mtSSB reduced Zta-induced lytic replication of EBV but had only a modest effect on transcription activation function. A point mutation in the Zta DNA binding domain (C189S), which is known to reduce lytic cycle replication, eliminated mtSSB association with Zta. The predominantly mitochondrial localization of mtSSB was shifted to partly nuclear localization in cells expressing Zta. Mitochondrial DNA synthesis and genome copy number were reduced by Zta-induced EBV lytic replication. We conclude that Zta interaction with mtSSB serves the dual function of facilitating viral and blocking mitochondrial DNA replication. PMID:18305033

  18. Epstein-Barr virus immediate-early protein Zta co-opts mitochondrial single-stranded DNA binding protein to promote viral and inhibit mitochondrial DNA replication.

    Science.gov (United States)

    Wiedmer, Andreas; Wang, Pu; Zhou, Jing; Rennekamp, Andrew J; Tiranti, Valeria; Zeviani, Massimo; Lieberman, Paul M

    2008-05-01

    Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. To better understand these functions and identify cellular collaborators of Zta, we purified an epitope-tagged version of Zta in cells capable of supporting lytic replication. FLAG-tagged Zta was purified from a nuclear fraction using FLAG antibody immunopurification and peptide elution. Zta-associated proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. The Zta-associated proteins included members of the HSP70 family and various single-stranded DNA and RNA binding proteins. The nuclear replication protein A subunits (RPA70 and RPA32) and the human mitochondrial single-stranded DNA binding protein (mtSSB) were confirmed by Western blotting to be specifically enriched in the FLAG-Zta immunopurified complex. mtSSB coimmunoprecipitated with endogenous Zta during reactivation of EBV-positive Burkitt lymphoma and lymphoblastoid cell lines. Small interfering RNA depletion of mtSSB reduced Zta-induced lytic replication of EBV but had only a modest effect on transcription activation function. A point mutation in the Zta DNA binding domain (C189S), which is known to reduce lytic cycle replication, eliminated mtSSB association with Zta. The predominantly mitochondrial localization of mtSSB was shifted to partly nuclear localization in cells expressing Zta. Mitochondrial DNA synthesis and genome copy number were reduced by Zta-induced EBV lytic replication. We conclude that Zta interaction with mtSSB serves the dual function of facilitating viral and blocking mitochondrial DNA replication.

  19. Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation.

    Science.gov (United States)

    Dixit, Aparna Banerjee; Ray, Krishanu; Black, Lindsay W

    2012-12-11

    Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.

  20. Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals.

    Directory of Open Access Journals (Sweden)

    Dana Willner

    Full Text Available The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota.

  1. Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma by using viral metagenomics.

    Science.gov (United States)

    Ng, Terry Fei Fan; Manire, Charles; Borrowman, Kelly; Langer, Tammy; Ehrhart, Llewellyn; Breitbart, Mya

    2009-03-01

    Viral metagenomics, consisting of viral particle purification and shotgun sequencing, is a powerful technique for discovering viruses associated with diseases with no definitive etiology, viruses that share limited homology with known viruses, or viruses that are not culturable. Here we used viral metagenomics to examine viruses associated with sea turtle fibropapillomatosis (FP), a debilitating neoplastic disease affecting sea turtles worldwide. By means of purifying and shotgun sequencing the viral community directly from the fibropapilloma of a Florida green sea turtle, a novel single-stranded DNA virus, sea turtle tornovirus 1 (STTV1), was discovered. The single-stranded, circular genome of STTV1 was approximately 1,800 nucleotides in length. STTV1 has only weak amino acid level identities (25%) to chicken anemia virus in short regions of its genome; hence, STTV1 may represent the first member of a novel virus family. A total of 35 healthy turtles and 27 turtles with FP were tested for STTV1 using PCR, and only 2 turtles severely afflicted with FP were positive. The affected turtles were systemically infected with STTV1, since STTV1 was found in blood and all major organs. STTV1 exists as a quasispecies, with several genome variants identified in the fibropapilloma of each positive turtle, suggesting rapid evolution of this virus. The STTV1 variants were identical over the majority of their genomes but contained a hypervariable region with extensive divergence. This study demonstrates the potential of viral metagenomics for discovering novel viruses directly from animal tissue, which can enhance our understanding of viral evolution and diversity.

  2. Two doses of bovine viral diarrhea virus DNA vaccine delivered by electroporation induce long-term protective immune responses.

    Science.gov (United States)

    van Drunen Littel-van den Hurk, Sylvia; Lawman, Zoe; Snider, Marlene; Wilson, Don; van den Hurk, Jan V; Ellefsen, Barry; Hannaman, Drew

    2013-02-01

    Bovine viral diarrhea virus (BVDV) is a pathogen of major importance in cattle, so there is a need for new effective vaccines. DNA vaccines induce balanced immune responses and are relatively inexpensive and thus promising for both human and veterinary applications. In this study, newborn calves with maternal antibodies were vaccinated intramuscularly (i.m.) with a BVDV E2 DNA vaccine with the TriGrid Delivery System for i.m. delivery (TDS-IM). Two doses of this vaccine spaced 6 or 12 weeks apart were sufficient to induce significant virus-neutralizing antibody titers, numbers of activated T cells, and reduction in viral shedding and clinical presentations after BVDV-2 challenge. In contrast to the placebo-treated animals, the vaccinated calves did not lose any weight, which is an excellent indicator of the well-being of an animal and has a significant economic impact. Furthermore, the interval between the two vaccinations did not influence the magnitude of the immune responses or degree of clinical protection, and a third immunization was not necessary or beneficial. Since electroporation may enhance not only the magnitude but also the duration of immunity after DNA immunization, the interval between vaccination and challenge was extended in a second trial, which showed that two doses of this E2 DNA vaccine again significantly reduced clinical disease against BVDV for several months. These results are promising and support this technology for use against infectious diseases in cattle and large species, including humans, in general.

  3. Continuous allosteric regulation of a viral packaging motor by a sensor that detects the density and conformation of packaged DNA.

    Science.gov (United States)

    Berndsen, Zachary T; Keller, Nicholas; Smith, Douglas E

    2015-01-20

    We report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage ϕ29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral capsid, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside. This signal continuously regulates the motor speed and pausing in response to changes in either density or conformation of the packaged DNA, and slows the motor before the buildup of large forces resisting DNA confinement. Analysis of motor slipping reveals that the force resisting packaging remains low (<1 pN) until ∼ 70% and then rises sharply to ∼ 23 pN at high filling, which is a several-fold lower value than was previously estimated under the assumption that force alone slows the motor. These findings are consistent with recent studies of the stepping kinetics of the motor. The allosteric regulatory mechanism we report allows double-stranded DNA viruses to achieve rapid, high-density packing of their genomes by limiting the buildup of nonequilibrium load forces on the motor.

  4. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    Science.gov (United States)

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

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

    Science.gov (United States)

    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

  6. Precore/basal core promoter mutants and hepatitis B viral DNA levels as predictors for liver deaths and hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Myron J Tong; Lawrence M Blatt; Jia-Horng Kao; Jason Tzuying Cheng; William G Corey

    2006-01-01

    AIM: To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma.METHODS: The mean follow-up time was 83.6 ± 39.6mo. Alpha-fetoprotein test and abdominal ultrasound were used for cancer surveillance. Hepatitis B basal core promoter mutants, precore mutants, genotypes,hepatitis B viral DNA (HBV DNA) level and hepatitis B e antigen (HBeAg) were measured. Univariate analysis and logistic regression were used to assess odds ratios for viral factors related to liver deaths and hepatocellular carcinoma development.RESULTS: During follow-up, 38 patients had liver deaths not related to hepatocellular carcinoma. On multivariate analysis, older age [odds ratio: 95.74 (12.13-891.31);P < 0.0001], male sex [odds ratio: 7.61 (2.20-47.95);P = 0.006], and higher log10 HBV DNA [odds ratio:4.69 (1.16-20.43); P < 0.0001] were independently predictive for these liver related deaths. Also, 31 patients developed hepatocellular carcinoma. Multivariate analysis showed that older age [odds ratio: 26.51 (2.36-381.47);P = 0.007], presence of precore mutants [odds ratio:4.23 (1.53-19.58); P = 0.02] and presence of basal core promoter mutants [odds ratio: 2.93 (1.24-7.57); P =0.02] were independent predictors for progression to hepatocellular carcinoma.CONCLUSION: Our results show that high levels of baseline serum HBV DNA are associated with nonhepatocellular carcinoma-related deaths of liver failure,while genetic mutations in the basal core promoter and precore regions are predictive for development of hepatocellular carcinoma.

  7. The role of DNA twist in the packaging of viral genomes.

    Science.gov (United States)

    Rollins, Geoffrey C; Petrov, Anton S; Harvey, Stephen C

    2008-03-01

    We performed molecular dynamics simulations of the genome packaging of bacteriophage P4 using two coarse-grained models of DNA. The first model, 1DNA6 (one pseudo-atom per six DNA basepairs), represents DNA as a string of beads, for which DNA torsions are undefined. The second model, 3DNA6 (three pseudo-atoms per six DNA basepairs), represents DNA as a series of base planes with torsions defined by the angles between successive planes. Bacteriophage P4 was packaged with 1DNA6, 3DNA6 in a torsionally relaxed state, and 3DNA6 in a torsionally strained state. We observed good agreement between the packed conformation of 1DNA6 and the packed conformations of 3DNA6. The free energies of packaging were in agreement, as well. Our results suggest that DNA torsions can be omitted from coarse-grained bacteriophage packaging simulations without significantly altering the DNA conformations or free energies of packaging that the simulations predict.

  8. Insights into specific DNA recognition during the assembly of a viral genome packaging machine.

    Science.gov (United States)

    de Beer, Tonny; Fang, Jenny; Ortega, Marcos; Yang, Qin; Maes, Levi; Duffy, Carol; Berton, Nancy; Sippy, Jean; Overduin, Michael; Feiss, Michael; Catalano, Carlos Enrique

    2002-05-01

    Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses. In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy. Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold. Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif. Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence. The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.

  9. Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication.

    Science.gov (United States)

    Kim, Young-Eui; Park, Mi Young; Kang, Kyeong Jin; Han, Tae Hee; Lee, Chan Hee; Ahn, Jin-Hyun

    2015-08-01

    The UL112-113 region of the human cytomegalovirus (HCMV) genome encodes four phosphoproteins of 34, 43, 50, and 84 kDa that promote viral DNA replication. Co-transfection assays have demonstrated that self-interaction of these proteins via the shared N-termini is necessary for their intranuclear distribution as foci and for the efficient relocation of a viral DNA polymerase processivity factor (UL44) to the viral replication sites. However, the requirement of UL112-113 N-terminal residues for viral growth and DNA replication has not been fully elucidated. Here, we investigated the effect of deletion of the N-terminal regions of UL112-113 proteins on viral growth and oriLyt-dependent DNA replication. A deletion of the entire UL112 region or the region encoding the 25 N-terminal amino-acid residues from the HCMV (Towne strain) bacmid impaired viral growth in bacmid-transfected human fibroblast cells, indicating their requirement for viral growth. In co-immunoprecipitation assays using the genomic gene expressing the four UL112-113 proteins together, the 25 N-terminal amino-acid residues were found to be necessary for stable expression of UL112-113 proteins and their self-interaction. These residues were also required for efficient binding to and relocation of UL44, but not for interaction with IE2, an origin-binding transcription factor. In co-transfection/replication assays, replication of the oriLyt-containing plasmid was promoted by expression of intact UL112-113 proteins, but not by the expression of 25-amino-acid residue-deleted proteins. Our results demonstrate that the 25 N-terminal amino-acid residues of UL112-113 proteins that mediate self-interaction contribute to viral growth by promoting their binding to UL44 and the initiation of oriLyt-dependent DNA replication.

  10. Influence of temperature on the efficacy of homologous and heterologous DNA vaccines against viral hemorrhagic septicemia in Pacific Herring

    Science.gov (United States)

    Hart, Lucas; Lorenzen, Niels; Einer-Jensen, Katja; Purcell, Maureen; Hershberger, Paul

    2017-01-01

    Homologous and heterologous (genogroup Ia) DNA vaccines against viral hemorrhagic septicemia virus (genogroup IVa) conferred partial protection in Pacific Herring Clupea pallasii. Early protection at 2 weeks postvaccination (PV) was low and occurred only at an elevated temperature (12.6°C, 189 degree days), where the relative percent survival following viral exposure was similar for the two vaccines (IVa and Ia) and higher than that of negative controls at the same temperature. Late protection at 10 weeks PV was induced by both vaccines but was higher with the homologous vaccine at both 9.0°C and 12.6°C. Virus neutralization titers were detected among 55% of all vaccinated fish at 10 weeks PV. The results suggest that the immune response profile triggered by DNA vaccination of herring was similar to that reported for Rainbow Trout Oncorhynchus mykiss by Lorenzen and LaPatra in 2005, who found interferon responses in the early days PV and the transition to adaptive response later. However, the protective effect was far less prominent in herring, possibly reflecting different physiologies or adaptations of the two fish species.

  11. Packaging Double-Helical DNA into Viral Capsids: Structures, Forces, and Energetics

    OpenAIRE

    Petrov, Anton S.; Harvey, Stephen C.

    2008-01-01

    Small, icosahedral double-stranded DNA bacteriophage pack their genomes tightly into preformed protein capsids using an ATP-driven motor. Coarse-grain molecular-mechanics models provide a detailed picture of DNA packaging in bacteriophage, revealing how conformation depends on capsid size and shape, and the presence or absence of a protein core. The forces that oppose packaging have large contributions from both electrostatic repulsions and the entropic penalty of confining the DNA into the c...

  12. Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions.

    Science.gov (United States)

    Wolfe, Annie; Phipps, Kara; Weitao, Tao

    2014-01-01

    DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.

  13. Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space

    Science.gov (United States)

    Graziano, Vito; McGrath, William J.; Suomalainen, Maarit; Greber, Urs F.; Freimuth, Paul; Blainey, Paul C.; Luo, Guobin; Xie, X. Sunney; Mangel, Walter F.

    2013-01-01

    The precursor to adenovirus protein VI, pVI, is a multifunctional protein with different roles early and late in virus infection. Here, we focus on two roles late in infection, binding of pVI to DNA and to the major capsid protein hexon. pVI bound to DNA as a monomer independent of DNA sequence with an apparent equilibrium dissociation constant, Kd(app), of 46 nm. Bound to double-stranded DNA, one molecule of pVI occluded 8 bp. Upon the binding of pVI to DNA, three sodium ions were displaced from the DNA. A ΔG00 of −4.54 kcal/mol for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy resulted from nonspecific interactions between pVI and DNA. The proteolytically processed, mature form of pVI, protein VI, also bound to DNA; its Kd(app) was much higher, 307 nm. The binding assays were performed in 1 mm MgCl2 because in the absence of magnesium, the binding to pVI or protein VI to DNA was too tight to determine a Kd(app). Three molecules of pVI bound to one molecule of the hexon trimer with an equilibrium dissociation constant Kd(app) of 1.1 nm. PMID:23043136

  14. The Q motif of a viral packaging motor governs its force generation and communicates ATP recognition to DNA interaction.

    Science.gov (United States)

    Tsay, James M; Sippy, Jean; Feiss, Michael; Smith, Douglas E

    2009-08-25

    A key step in the assembly of many viruses is the packaging of DNA into preformed procapsids by an ATP-powered molecular motor. To shed light on the motor mechanism we used single-molecule optical tweezers measurements to study the effect of mutations in the large terminase subunit in bacteriophage lambda on packaging motor dynamics. A mutation, K84A, in the putative ATPase domain driving DNA translocation was found to decrease motor velocity by approximately 40% but did not change the force dependence or decrease processivity substantially. These findings support the hypothesis that a deviant "Walker A-like" phosphate-binding motif lies adjacent to residue 84. Another mutation, Y46F, was also found to decrease motor velocity by approximately 40% but also increase slipping during DNA translocation by >10-fold. These findings support the hypothesis that viral DNA packaging motors contain an adenine-binding motif that regulates ATP hydrolysis and substrate affinity analogous to the "Q motif" recently identified in DEAD-box RNA helicases. We also find impaired force generation for the Y46F mutant, which shows that the Q motif plays an important role in determining the power and efficiency of the packaging motor.

  15. Single DNA molecule jamming and history-dependent dynamics during motor-driven viral packaging

    Science.gov (United States)

    Keller, Nicholas; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2016-08-01

    In many viruses, molecular motors forcibly pack single DNA molecules to near-crystalline density into ~50-100 nm prohead shells. Unexpectedly, we found that packaging frequently stalls in conditions that induce net attractive DNA-DNA interactions. Here, we present findings suggesting that this stalling occurs because the DNA undergoes a nonequilibrium jamming transition analogous to that observed in many soft-matter systems, such as colloidal and granular systems. Experiments in which conditions are changed during packaging to switch DNA-DNA interactions between purely repulsive and net attractive reveal strongly history-dependent dynamics. An abrupt deceleration is usually observed before stalling, indicating that a transition in DNA conformation causes an abrupt increase in resistance. Our findings suggest that the concept of jamming can be extended to a single polymer molecule. However, compared with macroscopic samples of colloidal particles we find that single DNA molecules jam over a much larger range of densities. We attribute this difference to the nanoscale system size, consistent with theoretical predictions for jamming of attractive athermal particles.

  16. VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells.

    Science.gov (United States)

    Liu, Xin; Dong, Ying; Wang, Jingquan; Li, Long; Zhong, Zhenmin; Li, Yun-Pan; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

    2017-06-28

    Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Mut(s)) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

  17. Identification of viral DNA (Anelloviridae) in a 200-year-old dental pulp sample (Napoleon's Great Army, Kaliningrad, 1812).

    Science.gov (United States)

    Bédarida, Sandra; Dutour, Olivier; Buzhilova, Alexandra P; de Micco, Philippe; Biagini, Philippe

    2011-03-01

    Ancient human remains are potential sources of biological information including traces of past infections, since previous studies have demonstrated the effective detection of several bacterial agents or host-integrated viruses in old biological remnants like tissues or teeth. Studies of skeletal dental pulp samples are of particular interest since this location is potentially exposed to bloodborne agents during life through its vascularization, and could be considered as well preserved from environment after death of the host. DNA viruses belonging to the family Anelloviridae are highly present in human populations where they harbor an extreme genetic diversity but a yet undefined implication in hosts' health. We hypothesized that anelloviruses would be detected in ancient remains and that they may also serve as tracer viruses for the study of other viral agents. We analyzed 200-year-old dental pulp samples from remains of soldiers of Napoleon's Great Army during the Russian Retreat. Successful detection of Anelloviridae DNA by PCR was obtained for 1/21 ancient samples tested. The sequence identified showed 23% nucleotide divergence with the closest group of modern isolates (genus Gammatorquevirus), and was confirmed as phylogenetically distinct from those identified in saliva samples from the two investigators in charge of the study (genera Alphatorquevirus and Betatorquevirus). PCR directed toward the human beta globin gene was also performed. Negative controls were negative. Our results demonstrate that an ubiquitary, non-integrated, DNA virus is detectable from ancient biological material, with potential developments in terms of evolution studies or subsequent molecular investigations involving further viral agents.

  18. Barrier to auto integration factor becomes dephosphorylated during HSV-1 Infection and Can Act as a host defense by impairing viral DNA replication and gene expression.

    Science.gov (United States)

    Jamin, Augusta; Thunuguntla, Prasanth; Wicklund, April; Jones, Clinton; Wiebe, Matthew S

    2014-01-01

    BAF (Barrier to Autointegration Factor) is a highly conserved DNA binding protein that senses poxviral DNA in the cytoplasm and tightly binds to the viral genome to interfere with DNA replication and transcription. To counteract BAF, a poxviral-encoded protein kinase phosphorylates BAF, which renders BAF unable to bind DNA and allows efficient viral replication to occur. Herein, we examined how BAF phosphorylation is affected by herpes simplex virus type 1 (HSV-1) infection and tested the ability of BAF to interfere with HSV-1 productive infection. Interestingly, we found that BAF phosphorylation decreases markedly following HSV-1 infection. To determine whether dephosphorylated BAF impacts HSV-1 productive infection, we employed cell lines stably expressing a constitutively unphosphorylated form of BAF (BAF-MAAAQ) and cells overexpressing wild type (wt) BAF for comparison. Although HSV-1 production in cells overexpressing wtBAF was similar to that in cells expressing no additional BAF, viral growth was reduced approximately 80% in the presence of BAF-MAAAQ. Experiments were also performed to determine the mechanism of the antiviral activity of BAF with the following results. BAF-MAAAQ was localized to the nucleus, whereas wtBAF was dispersed throughout cells prior to infection. Following infection, wtBAF becomes dephosphorylated and relocalized to the nucleus. Additionally, BAF was associated with the HSV-1 genome during infection, with BAF-MAAAQ associated to a greater extent than wtBAF. Importantly, unphosphorylated BAF inhibited both viral DNA replication and gene expression. For example, expression of two regulatory proteins, ICP0 and VP16, were substantially reduced in cells expressing BAF-MAAAQ. However, other viral genes were not dramatically affected suggesting that expression of certain viral genes can be differentially regulated by unphosphorylated BAF. Collectively, these results suggest that BAF can act in a phosphorylation-regulated manner to impair

  19. Proficient Replication of the Yeast Genome by a Viral DNA Polymerase.

    Science.gov (United States)

    Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M

    2016-05-27

    DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication.

  20. DNA-transporting nanoparticles : design and in vitro evaluation of DNA and formulation for non-viral gene delivery

    NARCIS (Netherlands)

    van Gaal, E.V.B.|info:eu-repo/dai/nl/30483629X

    2010-01-01

    The aim of gene therapy is to treat, cure or prevent a disease by replacing defective genes, introducing new genes or changing the expression of a person’s genes. Success of gene therapy is dependent on successful delivery of DNA from the site of administration into cell nuclei. Naturally occurring

  1. Clinical application of real time-polymerase chain reaction in determining cytomegalovirus viral DNA load in renal transplant recipients

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chuan-bao; LAI Hui-ying; XU Hong-tao; WANG Da-guang; XIAO Fei

    2012-01-01

    Background Cytomegalovirus (CMV) remains a significant clinical problem among immunosuppressed renal transplant patients.Quantitative PCR assays have become the most common methods in the determination of CMV infections in transplant patients.This study was to determine the relationship between CMV infection and the acute rejection of the transplanted kidney.Methods Plasma samples from 77 renal transplant patients that were pre-transplant negative for CMV infection were tested using real-time quantitative PCR and CMV gene-specific primers.The detected viral loads were retrospectively compared with the acute rejection rate and the chronic or mild rejection rates of the renal transplant.Results CMV-DNA was detected in 29 of 77 recipients,yielding a positive rate of detection of 37.7% for this procedure.Twelve of the 21 recipients (57.1%) who suffered acute rejection had positive CMV-DNA.Among the 56 recipients suffered from chronic or mild rejection,17 (30.4%) had positive CMV-DNA plasma.Moreover,of the 29 recipients who had detectable CMV-DNA after transplant,12 (41.4%) suffered from acute rejection; of the 48 recipients with undetectable CMV-DNA,only nine (18.8%) developed acute rejection.Post-transplant patients with acute rejection had a higher rate (57.1% vs.30.4%,P=0.03) of post-transplant CMV infection than those with chronic or mild rejection.Conclusion CMV infection is a risk factor of acute renal transplant rejection and CMV infection should be prevented and treated in renal transplant recipients.Chin Med J 2012; 125(19):3575-3577

  2. An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

    Directory of Open Access Journals (Sweden)

    Hayate Merad

    Full Text Available BACKGROUND: Integrase (IN of the type 1 human immunodeficiency virus (HIV-1 catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149-186 in the crystal structure of the catalytic core (CC of the IN-Phe185Lys variant that consists of the alpha(4 and alpha(5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. PRINCIPAL FINDINGS: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the alpha(4 and alpha(5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat ends of virus DNA with a K(d (dissociation constant in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix, it is the N terminal helix (alpha(4 which has the role of DNA recognition helix, while the C terminal helix (alpha(5 would rather contribute to the motif stabilization by interactions with the alpha(4 helix. CONCLUSION: The motif, termed HTHi (i, for inverted emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.

  3. Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

    Science.gov (United States)

    Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

    2014-05-01

    Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios.

  4. Conserved balance of hepatocyte nuclear DNA content in mononuclear and binuclear hepatocyte populations during the course of chronic viral hepatitis

    Institute of Scientific and Technical Information of China (English)

    Hidenori Toyoda; Takashi Kumada; Olivier Bregerie; Christian Brechot; Chantal Desdouets

    2006-01-01

    AIM: To analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid (4n) and octoploid (8n) nuclei, and then compared mononuclear and binuclear hepatocyte populations:METHODS: The percentages of mononuclear diploid(2n), 4n, and 8n hepatocytes and those of binuclear 2× 2n, 2 × 4n, and 2 × 8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2 ×4n and 2 × 8n hepatocytes in the binuclear hepatocyte population.RESULTS: The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2 × 4n and 2 × 8n hepatocytes in binuclear hepatocytes were similar,regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus.CONCLUSION: The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.

  5. Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication - inducible cell line

    Institute of Scientific and Technical Information of China (English)

    Wei He; Li-Xia Li; Qing-Jiao Liao; Chun-Lan Liu; Xu-Lin Chen

    2011-01-01

    AIM: To analyze the antiviral mechanism of Epigallocatechin gallate (EGCG) against hepatitis B virus (HBV) replication. METHODS: In this research, the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG. Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hepatitis B virus e antigen (HBeAg) and hepatitis B virus surface antigen (HBsAg) in the supernatant were detected by enzyme-linked immunosorbent assay. Precore mRNA and pregenomic RNA (pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR) assay. The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay. HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay. RESULTS: When HepG2.117 cells were grown in the presence of EGCG, the expression of HBeAg was suppressed, however, the expression of HBsAg was not affected. HBV precore mRNA level was also downregulated by EGCG, while the transcription of precore mRNA was not impaired. The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent, however, HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment, indicating that EGCG targets only replicative intermediates of DNA synthesis. CONCLUSION: In HepG2.117 cells, EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.

  6. Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages φ29, λ, and T4

    Science.gov (United States)

    Smith, Douglas E.; Fuller, Derek N.; Raymer, Dorian M.; Rickgauer, Peter; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Catalano, Carlos E.; Kottadiel, Vishal; Rao, Venigalla B.

    2007-09-01

    A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

  7. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

    Directory of Open Access Journals (Sweden)

    Janet M. Doolittle-Hall

    2015-11-01

    Full Text Available Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV, hepatitis B virus (HBV or Merkel cell polyomavirus (MCPyV. These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

  8. DeF-GPU: Efficient and effective deletions finding in hepatitis B viral genomic DNA using a GPU architecture.

    Science.gov (United States)

    Cheng, Chun-Pei; Lan, Kuo-Lun; Liu, Wen-Chun; Chang, Ting-Tsung; Tseng, Vincent S

    2016-12-01

    Hepatitis B viral (HBV) infection is strongly associated with an increased risk of liver diseases like cirrhosis or hepatocellular carcinoma (HCC). Many lines of evidence suggest that deletions occurring in HBV genomic DNA are highly associated with the activity of HBV via the interplay between aberrant viral proteins release and human immune system. Deletions finding on the HBV whole genome sequences is thus a very important issue though there exist underlying the challenges in mining such big and complex biological data. Although some next generation sequencing (NGS) tools are recently designed for identifying structural variations such as insertions or deletions, their validity is generally committed to human sequences study. This design may not be suitable for viruses due to different species. We propose a graphics processing unit (GPU)-based data mining method called DeF-GPU to efficiently and precisely identify HBV deletions from large NGS data, which generally contain millions of reads. To fit the single instruction multiple data instructions, sequencing reads are referred to as multiple data and the deletion finding procedure is referred to as a single instruction. We use Compute Unified Device Architecture (CUDA) to parallelize the procedures, and further validate DeF-GPU on 5 synthetic and 1 real datasets. Our results suggest that DeF-GPU outperforms the existing commonly-used method Pindel and is able to exactly identify the deletions of our ground truth in few seconds. The source code and other related materials are available at https://sourceforge.net/projects/defgpu/.

  9. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

    Science.gov (United States)

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-01-01

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  10. Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

    Directory of Open Access Journals (Sweden)

    Monica Poggianella

    Full Text Available Dengue virus (DENV infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

  11. Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

    Science.gov (United States)

    Poggianella, Monica; Slon Campos, José L; Chan, Kuan Rong; Tan, Hwee Cheng; Bestagno, Marco; Ooi, Eng Eong; Burrone, Oscar R

    2015-01-01

    Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

  12. Is passive transmission of non-viral vectors through artificial insemination of sperm-DNA mixtures sufficient for chicken transgenesis?

    Science.gov (United States)

    Chaparian, Shahram; Abdulahnejad, Ahad; Rashidi, Farzad; Toghyani, Majid; Gheisari, Abbasali; Eghbalsaied, Shahin

    2016-06-17

    DNA uptake in the post-acrosomal region of the spermatozoa takes place exclusively in immotile spermatozoa that are naturally unable to fertilize eggs. The present study aimed to assess whether passive transmission of non-viral vectors to the surrounding areas of chicken embryos could be an alternate mechanism in chicken sperm-mediated gene transfer. First, the presence of nucleases in rooster seminal plasma was evaluated. Semen ejaculates from five roosters were centrifuged and the supernatant was incubated with pBL2 for 1 h. A robust nuclease cocktail was detected in the rooster semen. To overcome these nucleases, plasmid-TransIT combinations were incubated with semen for 1 h. Incubation of exogenous DNA in the lipoplex structure could considerably bypass the semen nuclease effect. Then, intravaginal insemination of 1 × 10(9) sperm mixed with lipoplexes (40 µg pBL2:40 µl TransIT) was carried out in 15 virgin hens. Neither the epithelial tissue from the inseminated female reproductive tracts nor the produced embryos following artificial insemination showed the transgene. To remove any bias in the transgene transmission possibility, the plasmid-TransIT admixture was directly injected in close vicinity of the embryos in newly laid eggs. Nonetheless, none of the produced fetuses or chicks carried the transgene. In conclusion, the results of the present study revealed a nuclease admixture in rooster seminal plasma, and passive/active transmission of the non-viral vector into close vicinity of the chicken embryo was inefficient for producing transgenic chicks.

  13. Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination.

    Science.gov (United States)

    Minke, J M; Fischer, L; Baudu, Ph; Guigal, P M; Sindle, T; Mumford, J A; Audonnet, J C

    2006-05-15

    In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. Two to 3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system.

  14. Entecavir to Telbivudine Switch Therapy in Entecavir-Treated Patients with Undetectable Hepatitis B Viral DNA.

    Science.gov (United States)

    Kim, Dong Hyun; Choi, Jong Won; Seo, Jeong Hun; Cho, Yong Suk; Won, Sun Young; Park, Byung Kyu; Jeon, Han Ho; Shin, Sang Yun; Lee, Chun Kyon

    2017-05-01

    This study examined 2-year outcome of consecutive therapy using entecavir (ETV) followed by telbivudine (LdT) in subjects with undetectable hepatitis B virus (HBV) DNA level and normal alanine aminotransferase level after the initial 6 months of ETV treatment. Sixty subjects were randomized to continue with ETV or switch to LdT. Significant difference in baseline characteristics was not found between the two groups. Persistent HBV DNA level of 20-60 IU/mL in three consecutive samples collected three months apart or singly measured HBV DNA level of >60 IU/mL was defined as virological rebound. During 96 weeks of follow-up, all subjects of the ETV-only group (n=30) resulted in undetectable HBV DNA level. On the other hand, 83.3% (n=25) of the LdT-switched group showed treatment success. Virological rebound time varied from week 24 to 84 after switching to LdT. HBV DNA level was 180 to 2940 IU/mL at rebound time. All subjects with virological rebound (n=5) showed drug-resistant mutation: three had mutation rtM204I, and two had mutation rtM204V. Consecutive treatment using ETV followed by LdT showed virological rebound in 16.7% of subjects during 96 weeks of follow-up. HBV DNA negativity during initial ETV therapy could not be achieved in patients who switched to LdT. Consecutive treatment using ETV followed by lamivudine was ineffective for treating chronic hepatitis B. LdT was found as a more potent antiviral agent than lamivudine. However, this conclusion requires larger-scale, long-term prospective reviews of the treatment effects of ETV-LdT switch therapy.

  15. Walker-A Motif Acts to Coordinate ATP Hydrolysis with Motor Output in Viral DNA Packaging.

    Science.gov (United States)

    delToro, Damian; Ortiz, David; Ordyan, Mariam; Sippy, Jean; Oh, Choon-Seok; Keller, Nicholas; Feiss, Michael; Catalano, Carlos E; Smith, Douglas E

    2016-07-03

    During the assembly of many viruses, a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A "P-loop" motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectable levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectable yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type (WT) but exhibit little ATPase or DNA packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A and K76R, while G81A and S83A exhibit strong impairments, consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest that these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling.

  16. Measurement of HIV-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of DNA and RNA.

    Science.gov (United States)

    Parkin, Neil T

    2014-01-01

    World Health Organization-recommended surveys of acquired HIV-1 drug resistance include assessment of HIV-1 viral load suppression to levels below 1,000 copies/ml and drug resistance-associated mutation patterns in subjects on antiretroviral therapy. Surveys are being conducted in regions of the world that cannot support the collection, storage, and shipping of frozen plasma. Therefore, dried blood spots are often the specimen type of choice for both genotyping and viral load measurement. Furthermore, viral load testing for individual patient management in these regions is being scaled-up in accordance with WHO 2013 Guidelines for Antiretroviral Treatment. Technical issues related to the adaptation of viral load assays to dried blood spots, especially with respect to sensitivity (limit of detection), specificity (cell-free RNA vs. cell-associated DNA or RNA), and assay method, affect the interpretation of a viral load result from dried blood spots. Amongst published studies of commercial assay performance with dried blood spots, the bioMérieux EasyQ® and Abbott RealTime assays tended to show high (> 90%) specificity and sensitivity; the Biocentric Generic or Roche TaqMan® assays tended to show high sensitivity but lower specificity, using a 1,000 copies/ml threshold. The relative contribution of cell-associated DNA or RNA to a viral load measurement is likely to vary between patients, depending on clinical parameters and treatment status. A model was developed that predicts that in patients on antiretroviral therapy with low plasma viral load, cellular DNA is the predominant source of non-plasma virus-derived nucleic acid in dried blood spots. The extent of viral load overestimation from dried blood spots becomes less important when plasma viral load is over about 5,000 copies/ml. To avoid misclassifying subjects with plasma viral load suppression, the World Health Organization-recommended threshold of 1,000 copies/ml can be applied only when an assay that can

  17. Efficacy of a glycoprotein DNA vaccine against viral haemorrhagic septicaemia (VHS) in Pacific herring, Clupea pallasii Valenciennes

    Science.gov (United States)

    Hart, L.M.; Lorenzen, Niels; LaPatra, S.E.; Grady, C.A.; Roon, S.E.; O’Reilly, J.; Gregg, J.L.; Hershberger, P.K.

    2012-01-01

    Viral haemorrhagic septicaemia virus (VHSV) and its associated disease state, viral haemorrhagic septicaemia (VHS), is hypothesized to be a proximate factor accounting for the decline and failed recovery of Pacific herring populations in Prince William Sound, AK (Marty et al. 1998, 2003, 2010). Survivors of laboratory-induced VHSV epizootics develop resistance to subsequent viral exposure (Kocan et al. 2001; Hershberger et al. 2007, 2010), which is likely the result of immune system recognition of the viral glycoprotein (G) (Lecocq-Xhonneux et al. 1994), a surface antigen that contains neutralizing epitopes (Lorenzen, Olesen & Jorgensen 1990; Jørgensen et al. 1995) and cell attachment domains (Lecocq-Xhonneux et al. 1994; Estepa & Coll 1996). These properties have proven useful in the development of G-gene-based DNA vaccines for VHSV and a related rhabdovirus, infectious haematopoietic necrosis virus (IHNV) (Anderson et al. 1996; Heppell et al. 1998; Corbeil et al. 1999; Einer-Jensen et al. 2009). Rainbow trout fingerlings, Oncorhynchus mykiss (Walbaum), vaccinated with 1 µg of either the VHS or IHN vaccine are protected from VHS when exposed to virus as early as 4 days (44 degree days) post-vaccination (p.v.) (Lorenzen et al. 2002). At later time points (80 days p.v.; 880 degree days), the level of cross-protection against VHS by IHN vaccination is either completely lost (60 days p.v.; 660 degree days) (3 g rainbow trout; 1 µg vaccine dose) (Lorenzen et al. 2002) or present at intermediate levels (6.5 g rainbow trout; 1 µg vaccine dose) (Einer-Jensen et al. 2009). Comparatively, VHS vaccination remains effective as long as 9 months (2520 degree days) p.v. (100 g rainbow trout; 0.5 µg vaccine dose) (McLauchlan et al. 2003). These results suggest that IHN and VHS vaccination activate a rapid transitory innate immune response against VHSV that is followed by long-term adaptive immunity in VHS-vaccinated trout (Lorenzen et al. 2002).

  18. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R.; Anderson, Dwight L.; Jardine, Paul J.; Grimes, Shelley; Ke, Ailong (Cornell); (UMM)

    2011-07-25

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 {angstrom} resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of {psi}29 DNA.

  19. cDNA clone of hepatitis A virus encoding a virulent virus: induction of viral hepatitis by direct nucleic acid transfection of marmosets.

    OpenAIRE

    Emerson, S U; Lewis, M; Govindarajan, S. (Srinath); M. Shapiro(Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America); Moskal, T; Purcell, R. H.

    1992-01-01

    Direct inoculation of marmoset livers with an in vitro transcription mixture containing cDNA and full-length genomic RNA transcripts of hepatitis A virus resulted in acute viral hepatitis. Elevations in serum levels of liver enzymes were correlated with appearance of antibody to hepatitis A virus. Genomes of infectious hepatitis A virus isolated from the feces of transfected marmosets contained the same mutation as the cDNA template used for transfection. Liver biopsies confirmed that the vir...

  20. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    Science.gov (United States)

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

  1. Microfluidic cartridge with integrated array of amorphous silicon photosensors for chemiluminescence detection of viral DNA

    Directory of Open Access Journals (Sweden)

    M. Zangheri

    2016-03-01

    With this system, taking advantage of oligoprobes specificity, chemiluminescence detectability, and photosensor sensitivity, accurate quantification of target analytes down to 70 pmol L−1 was obtained for each B19 DNA genotype, with high specificity and multiplexing ability. Results confirm the good detection capabilities and assay applicability of the proposed system, prompting the development of innovative portable analytical devices with enhanced sensitivity and multiplexed capabilities.

  2. Ricerca di HPV-DNA e tipizzazione virale nella diagnostica di prevenzione del carcinoma della cervice uterina

    Directory of Open Access Journals (Sweden)

    Mauro Carcheri

    2003-03-01

    Full Text Available Papillomaviruses are relatively ubiquitous and have been described as causative agents for epithelial lesions in a wide variety of animals as well as in humans. Approximately 30 HPV types have been isolated from anogenital epithelium (cervix, vagina, vulva, rectum and penis. HPVs induce a variety of proliferative lesions, but only the “high-risk” types are associated with anogenital cancers. “Low-risk” viral types include HPV-6 and HPV-11; “high-risk” types include HPV-16, 18, 31 and 56. HPVs “high-risk” types are more strongly associated with high-grade lesions (HSIL than they are with low-grade lesions (LSIL. The traditional process for cervical cancer screening programs (PAP test is vulnerable to air drying artifacts and has limits to sensitivity, since as many as 90 percent of collectet cells can be discarted with collection device. The present study was undertaken to assess the screening performance of HPV-DNA typing in a sample of 142 women drawn from a routine screening for the prevention of cervical cancer. The results indicate that HPV-DNA screening and typing, used together with PAP test, can improve the detection of patients with cervical disease and can serve as a quality assurance indicator in cervical cancer screening programs.

  3. Condylomata acuminata of the urinary bladder. Natural history, viral typing, and DNA content.

    Science.gov (United States)

    Del Mistro, A; Koss, L G; Braunstein, J; Bennett, B; Saccomano, G; Simons, K M

    1988-03-01

    Three patients with condylomata acuminata of the urinary bladder are reported. Two of the patients were immunosuppressed, and one had longstanding extensive condylomata acuminata of the external genitalia and adjacent areas. All lesions recurred at least once and were difficult to treat. The diagnosis was confirmed by in situ hybridization on archival material with human papillomavirus (HPV) DNA probes under stringent conditions. In two of the patients, probes for HPV types 6 and 11 were positive; HPV 11 only was identified in one patient. Probes for HPV types 16 and 18 and pBR322 vector controls were negative. In one patient with a strong hybridization signal, the lesion was also positive for common papillomavirus antigen. DNA content measured by cytophotometry of Feulgen-stained whole nuclei isolated from lesions in two patients revealed a markedly aneuploid DNA pattern. Whether this is a factor in the behavior of the lesions is not known at this time. Although rare, HPV infection of the urinary bladder may result in widespread condylomatosis and may mimic giant condylomas of Buschke-Löwenstein or even verrucous carcinomas, sometimes necessitating radical treatment. Nevertheless, until there is proof to the contrary, the lesions must be considered benign and should not be confused with squamous cancer of the bladder.

  4. A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

    Directory of Open Access Journals (Sweden)

    Hammond Richard

    2010-02-01

    Full Text Available Abstract Background Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor. Results In the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10-12 M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification. Conclusions An analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters

  5. Massive APOBEC3 editing of hepatitis B viral DNA in cirrhosis.

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Vartanian

    2010-05-01

    Full Text Available DNA viruses, retroviruses and hepadnaviruses, such as hepatitis B virus (HBV, are vulnerable to genetic editing of single stranded DNA by host cell APOBEC3 (A3 cytidine deaminases. At least three A3 genes are up regulated by interferon-alpha in human hepatocytes while ectopic expression of activation induced deaminase (AICDA, an A3 paralog, has been noted in a variety of chronic inflammatory syndromes including hepatitis C virus infection. Yet virtually all studies of HBV editing have confined themselves to analyses of virions from culture supernatants or serum where the frequency of edited genomes is generally low (HBV>alcoholic cirrhosis. A3C and A3D were up regulated for all groups while the interferon inducible A3G was over expressed in virus associated cirrhosis, as was AICDA in approximately 50% of these HBV/HCV samples. While AICDA can indeed edit HBV DNA ex vivo, A3G is the dominant deaminase in vivo with up to 35% of HBV genomes being edited. Despite these highly deleterious mutant spectra, a small fraction of genomes survive and contribute to loss of HBeAg antigenemia and possibly HBsAg immune escape. In conclusion, the cytokine storm associated with chronic inflammatory responses to HBV and HCV clearly up regulates a number of A3 genes with A3G clearly being a major restriction factor for HBV. Although the mutant spectrum resulting from A3 editing is highly deleterious, a very small part, notably the lightly edited genomes, might help the virus evolve and even escape immune responses.

  6. Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.

    Science.gov (United States)

    Migliori, Amy D; Smith, Douglas E; Arya, Gaurav

    2014-12-12

    Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations.

  7. Development and use of an efficient DNA-based viral gene silencing vector for soybean.

    Science.gov (United States)

    Zhang, Chunquan; Yang, Chunling; Whitham, Steven A; Hill, John H

    2009-02-01

    Virus-induced gene silencing (VIGS) is increasingly being used as a reverse genetics tool to study functions of specific plant genes. It is especially useful for plants, such as soybean, that are recalcitrant to transformation. Previously, Bean pod mottle virus (BPMV) was shown to be an effective VIGS vector for soybean. However, the reported BPMV vector requires in vitro RNA transcription and inoculation, which is not reliable or amenable to high-throughput applications. To increase the efficiency of the BPMV vector for soybean functional genomics, a DNA-based version was developed. Reported here is the construction of a Cauliflower mosaic virus 35S promoter-driven BPMV vector that is efficient for the study of soybean gene function. The selection of a mild rather than a severe BPMV strain greatly reduced the symptom interference caused by virus infection. The DNA-based BPMV vector was used to silence soybean homologues of genes involved in plant defense, translation, and the cytoskeleton in shoots and in roots. VIGS of the Actin gene resulted in reduced numbers of Soybean mosaic virus infection foci. The results demonstrate the utility of this new vector as an efficient tool for a wide range of applications for soybean functional genomics.

  8. The varicella-zoster virus ORFS/L (ORF0) gene is required for efficient viral replication and contains an element involved in DNA cleavage.

    Science.gov (United States)

    Kaufer, Benedikt B; Smejkal, Benjamin; Osterrieder, Nikolaus

    2010-11-01

    The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U(L)) and unique short (U(S)), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U(L) and U(S). VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U(L). An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U(L), directly adjacent to the a-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5' region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5' terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U(L), we concluded that our results can provide an explanation for the almost exclusive orientation of the U(L) seen in packaged VZV DNA.

  9. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    Science.gov (United States)

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  10. Frequent Hypermethylation of RASSF1A, TSLC1, High Viral Load of Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma, Matched Tumor-Adjacent Tissues

    Directory of Open Access Journals (Sweden)

    Liang Zhou

    2005-09-01

    Full Text Available We examined the promoter hypermethylation of tumorsuppressor genes RASSF1A, TSLC1, quantitated EBV DNA load in nasopharyngeal carcinoma (NPC tissues (T tissues, matched tumor-adjacent tissues outside 0.5 cm (P tissues, outside 1.0 cm (Z tissues to evaluate the role of promoter hypermethylation of RASSF1A, TSLC1 as well as viral load in the pathogenesis of NPC. Methylation-specific polymerase chain reaction (PCR for RASSF1A, TSLC1, quantitative real-time PCR analysis of EBV DNA were performed on matched T, P, Z tissues (n = 28 as well as chronic nasopharyngitis tissues (n = 8. Hypermethylated RASSF1A was frequently detected in the T (82%, P tissues (75%, but less frequently in Z tissues (46%. The average quantities of EBV DNA (copies/μg DNA in matched T, P, Z tissues were 673,000, 90,000, 7000. The differences of promoter hypermethylation of RASSF1A, EBV viral load among T, P, Z tissues were statistically significant, with more frequent methylation, higher viral load detected when tissues examined were nearer to the NPC tissues. Our results suggest that aberrant hypermethylation of RASSF1A, high EBV load might be important events in NPC pathogenesis, they may be useful molecular diagnostic markers for this cancer.

  11. Expression of the late cytomegalovirus (CMV) pp150 transcript in leukocytes of AIDS patients is associated with a high viral DNA load in leukocytes and presence of CMV DNA in plasma.

    Science.gov (United States)

    Boivin, G; Handfield, J; Toma, E; Lalonde, R; Bergeron, M G

    1999-05-01

    The expression of a late cytomegalovirus (CMV) transcript (pp150) was sought in peripheral blood leukocytes (PBL) of subjects with AIDS and correlated with the amounts of CMV DNA in PBL and plasma, by means of quantitative polymerase chain reaction (PCR). The detection of the late CMV transcript was associated with a high number of CMV DNA copies in PBL (P=.0015) and with a positive CMV PCR assay in plasma (P<.001). Expression of CMV pp150 mRNA was best predicted by viral DNA thresholds corresponding to 7058 and 30 copies in PBL and plasma, respectively. The detection of CMV pp150 mRNA was associated with the presence of CMV disease in a univariate analysis but not in a multivariate analysis after controlling for the viral DNA load in PBL. Thus, active viral replication as determined by a high CMV DNA load in PBL is reflected by expression of the late CMV transcript in the same cells and by the presence of CMV DNA in plasma.

  12. Dexamethasone-induced reactivation of bovine herpesvirus type 5 latent infection in experimentally infected rabbits results in a broader distribution of latent viral DNA in the brain

    Directory of Open Access Journals (Sweden)

    S.V. Mayer

    2006-03-01

    Full Text Available Bovine herpesvirus type 5 (BHV-5 is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8 or submitted to dexamethasone treatment (2.6 mg kg-1 day-1, im, for 5 days and euthanized 60 days later (group B, N = 7 for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3 submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8, frequently in cerebellum (5/8, anterior cerebral cortex and pons-medulla (3/8 and occasionally in dorsolateral (2/8, ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8. Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7 and posterior cerebral cortices (5/7, pons-medulla (6/7, thalamus (4/7, and midbrain (3/7. In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.

  13. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

    Directory of Open Access Journals (Sweden)

    Sandra Arenhart

    Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

  14. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones.

    Science.gov (United States)

    Arenhart, Sandra; Silva, José Valter Joaquim; Flores, Eduardo Furtado; Weiblen, Rudi; Gil, Laura Helena Vega Gonzales

    The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  15. cDNA microarray analysis of disk abalone genes in gills and hemocytes after viral hemorrhagic septicemia virus (VHSV) challenge.

    Science.gov (United States)

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Oh, Chulhong; Whang, Ilson; Shin, Hyun-Jin; Lee, Jehee

    2012-06-01

    A disk abalone Haliotis discus discus 4.2 K cDNA microarray was designed by selecting abalone expressed sequence tags (ESTs). Transcriptional profiles in gills and hemocytes were analyzed upon abalone challenged with viral hemorrhagic septicemia virus (VHSV) in order to select candidates for screening of immune response genes. Among the 4188 genes analyzed, 280 (6.6%) transcripts were changed their expression level in gills and hemocytes against VHSV challenge compared to control animals. Total of 88 and 65 genes were up-regulated in gills and hemocytes, respectively. These genes can be grouped under various immune-functional categories such as transcription factors (Krüppell-like factor; ETS-family transcription factor), inflammatory and apoptosis related genes (TNF superfamily members, Fas ligand), IFN regulatory proteins (IFN-44 like, interferon gamma-inducible lysosomal thiol reductase) and detoxification proteins (glutathione peroxidase). In contrast, 25 and 102 genes were shown down-regulation in gills and hemocytes, respectively. Among the differentially expressed transcripts, considerably higher numbers of ESTs were represented as either hypothetical (unknown) proteins or no GenBank match suggesting those may be novel genes associated with internal defense of abalone.

  16. Viral epigenetics.

    Science.gov (United States)

    Milavetz, Barry I; Balakrishnan, Lata

    2015-01-01

    DNA tumor viruses including members of the polyomavirus, adenovirus, papillomavirus, and herpes virus families are presently the subject of intense interest with respect to the role that epigenetics plays in control of the virus life cycle and the transformation of a normal cell to a cancer cell. To date, these studies have primarily focused on the role of histone modification, nucleosome location, and DNA methylation in regulating the biological consequences of infection. Using a wide variety of strategies and techniques ranging from simple ChIP to ChIP-chip and ChIP-seq to identify histone modifications, nuclease digestion to genome wide next generation sequencing to identify nucleosome location, and bisulfite treatment to MeDIP to identify DNA methylation sites, the epigenetic regulation of these viruses is slowly becoming better understood. While the viruses may differ in significant ways from each other and cellular chromatin, the role of epigenetics appears to be relatively similar. Within the viral genome nucleosomes are organized for the expression of appropriate genes with relevant histone modifications particularly histone acetylation. DNA methylation occurs as part of the typical gene silencing during latent infection by herpesviruses. In the simple tumor viruses like the polyomaviruses, adenoviruses, and papillomaviruses, transformation of the cell occurs via integration of the virus genome such that the virus's normal regulation is disrupted. This results in the unregulated expression of critical viral genes capable of redirecting cellular gene expression. The redirected cellular expression is a consequence of either indirect epigenetic regulation where cellular signaling or transcriptional dysregulation occurs or direct epigenetic regulation where epigenetic cofactors such as histone deacetylases are targeted. In the more complex herpersviruses transformation is a consequence of the expression of the viral latency proteins and RNAs which again can

  17. Single-epitope DNA vaccination prevents exhaustion and facilitates a broad antiviral CD8+ T cell response during chronic viral infection

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Stryhn, Anette; Christensen, Jan Pravsgaard

    2004-01-01

    Induction of a monospecific antiviral CD8+ T cell response may pose a risk to the host due to the narrow T cell response induced. At the individual level, this may result in selection of CD8+ T cell escape variants, particularly during chronic viral infection. Second, prior immunization toward...... a single dominant epitope may suppress the response to other viral epitopes, and this may lead to increased susceptibility to reinfection with escape variants circulating in the host population. To address these issues, we induced a memory response consisting solely of monospecific, CD8+ T cells by use...... acute LCMV infection, DNA vaccination did not significantly impair naturally induced immunity. Thus, the response to the other immunogenic epitopes was not dramatically suppressed in DNA-immunized mice undergoing normal immunizing infection, and the majority of mice were protected against rechallenge...

  18. The AT-hook DNA binding ability of the Epstein Barr virus EBNA1 protein is necessary for the maintenance of viral genomes in latently infected cells.

    Science.gov (United States)

    Chakravorty, Adityarup; Sugden, Bill

    2015-10-01

    Epstein Barr Virus (EBV) is a human tumor virus that is causally linked to malignancies such as Burkitt׳s lymphoma, and gastric and nasopharyngeal carcinomas. Tethering of EBV genomes to cellular chromosomes is required for the synthesis and persistence of viral plasmids in tumor cells. However, it is not established how EBV genomes are tethered to cellular chromosomes. We test the hypothesis that the viral protein EBNA1 tethers EBV genomes to chromosomes specifically through its N-terminal AT-hook DNA-binding domains by using a small molecule, netropsin, that has been shown to inhibit the AT-hook DNA-binding of EBNA1 in vitro. We show that netropsin forces the loss of EBV genomes from epithelial and lymphoid cells in an AT-hook dependent manner and that EBV-positive lymphoma cells are significantly more inhibited in their growth by netropsin than are corresponding EBV-negative cells.

  19. Modification of a viral satellite DNA-based gene silencing vector and its application to leaf or flower color change in Petunia hybrida

    Institute of Scientific and Technical Information of China (English)

    TAO Xiaorong; QIAN Yajuan; ZHOU Xueping

    2006-01-01

    Virus-induced gene silencing offers a powerful reverse-genetic tool for the study of gene function in plants. We have previously reported effective gene silencing of plant genes using a viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV). In this study, we further modified the viral satellite DNA-based vector. The modified vector can induce sulfu (Su) gene silencing as effective as the original vector in Nicotiana benthamiana plants, but the new system simplifies procedures for construction of vector derivative. Furthermore, a fragment of petunia Su or chalcone synthase (CHS) endogenous gene was inserted into the modified vector. When petunia plants were agro- inoculated with the modified vector carrying a Su or CHS gene, the Su silenced plants started to appear yellowing in veins of systemically infected upper leaves two weeks after agroinoculation, while the CHS silenced plants started to show flower color change one month after agroinoculation and later single-color flowers became mosaic.

  20. Discovery of a Novel Single-Stranded DNA Virus from a Sea Turtle Fibropapilloma by Using Viral Metagenomics▿

    OpenAIRE

    Ng, Terry Fei Fan; Manire, Charles; Borrowman, Kelly; Langer, Tammy; Ehrhart, Llewellyn; Breitbart, Mya

    2008-01-01

    Viral metagenomics, consisting of viral particle purification and shotgun sequencing, is a powerful technique for discovering viruses associated with diseases with no definitive etiology, viruses that share limited homology with known viruses, or viruses that are not culturable. Here we used viral metagenomics to examine viruses associated with sea turtle fibropapillomatosis (FP), a debilitating neoplastic disease affecting sea turtles worldwide. By means of purifying and shotgun sequencing t...

  1. Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core-related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment.

    Science.gov (United States)

    Rokuhara, A; Tanaka, E; Matsumoto, A; Kimura, T; Yamaura, T; Orii, K; Sun, X; Yagi, S; Maki, N; Kiyosawa, K

    2003-07-01

    We aimed to assess the clinical performance of a newly developed chemiluminescence enzyme immunoassay (CLEIA) for the detection of hepatitis B virus (HBV) core-related antigen (HBcrAg) in patients with chronic HBV infection. A total of 82 patients with chronic HBV infection and 167 HBV-negative controls were studied. HBcrAg was measured by CLEIA with monoclonal antibodies to hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg), and HBV DNA was measured by transcription-mediated amplification assay (TMA) and in-house real-time detection polymerase chain reaction (RTD-PCR). The HBcrAg assay detected viremia in 189 of 216 samples (88%) collected from 72 patients whilst the TMA assay detected viremia in 178 of the 216 samples (82%) (P = 0.019). The HBcrAg concentration correlated linearly with the HBV DNA concentration (P HBcrAg assay was not affected by the absence of hepatitis B e antigen from the serum or the presence of precore mutations in the HBV genome. In patients without anti-viral drugs, changes in their serum HBcrAg concentration over time corresponded to their HBV DNA concentration. In six additional patients who were later treated with lamivudine, HBV DNA concentration declined more rapidly than their HBcrAg concentration. Three months after treatment commenced, the ratio of HBcrAg: HBV DNA had increased in all six patients (P = 0.031). The HBcrAg assay is a sensitive and useful test for the assessment of a patient's HBV load. When monitoring the anti-viral effect of lamivudine, HBcrAg provides a viral marker which is independent of HBV DNA.

  2. Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII.

    Science.gov (United States)

    Summers, J

    1975-04-01

    Digestion of polyoma viral DNA with a restriction enzyme from Haemophilus aegyptius generates at least 22 unique fragments. The fragments have been characterized with respect to size and physical order on the polyoma genome, and the 5' to 3' orientation of the (+) and (-) strands has been determined. A method for specific radiolabeling of adjacent fragments was employed to establish the fragment order. This technique may be useful for ordering the fragments produced by digestion of complex DNAs.

  3. Low concentrations of HIV-1 DNA at birth delays diagnosis, complicating identification of infants for antiretroviral therapy to potentially prevent the establishment of viral reservoirs.

    Science.gov (United States)

    Mitchell, Caroline; Dross, Sandra; Beck, Ingrid A; Micek, Mark A; Frenkel, Lisa M

    2014-04-01

    Among infants exposed to human immunodeficiency virus type 1 (HIV-1), detection of viral infection at birth was increased by 39% (95% confidence interval, 19%-47%) by increasing DNA input from dried blood spots into polymerase chain reaction. Infants with low concentrations of HIV-1 at birth may be the best target population to evaluate whether immediate antiretroviral therapy can prevent long-term infection.

  4. Determination of HPV DNA viral load by hybrid capture assay and its association with cytological findings Determinação da carga viral de DNA de HPV pelo ensaio de captura híbrida e sua associação com achados citológicos

    Directory of Open Access Journals (Sweden)

    Inês Aparecida Tozetti

    2006-12-01

    Full Text Available OBJECTIVE: To compare the relation between HPV viral load by hybrid capture II test (HCII and cytological findings. METHODS: Three hundred sixty-two reagent samples to HPV DNA by HCII had their viral loads classified in four categories and correlated to cytological results. RESULTS: Twenty-two samples (6.1% were reagent only to low-risk oncogenic types (group A and 340 (93.9% were reagent to high-risk oncogenic types (group B. The correlation between viral load for the reagent samples to group A and cytological results showed low-grade squamous intraepithelial lesion (LSIL predominance (50%. Most of this group samples had viral load between 1 to OBJETIVO: Comparar a relação entre a carga viral do HPV por captura híbrida II (HCII e os achados citológicos. METODOS: Trezentas e sessenta e duas amostras reagentes para DNA de HPV por HCII tiveram suas cargas virais classificadas em quatro categorias e correlacionadas aos resultados citológicos. RESULTADOS: Vinte e duas amostras (6,1% foram reagentes somente para os tipos de baixo risco oncogênico (grupo A e 340 (93,9% foram reagentes para os tipos de alto risco oncogênico (grupo B. A correlação entre carga viral das amostras reagentes para o grupo A e resultados citológicos mostrou predominância (50% de lesão escamosa intraepitelial de baixo grau (LSIL. A maioria das amostras desse grupo teve carga viral entre 1 e < 10RLU/PCA. Nos pacientes reagentes para o grupo B observamos que 52,1% tiveram citologia LSIL e 38,2% tiveram citologia negativa para lesão intraepitelial e malignidade (NILM. Os pacientes com LSIL tiveram a carga viral bem distribuída, com ligeira predominância da categoria de 100 a < 1.000RLU/PCB. As amostras com carga viral entre 1 e < 10RLU/PCB mostraram predominância de citologia NILM (48.1%. Lesões escamosas de alto grau (3,4% foram presentes nas amostras com carga viral entre 100 e < 1.000RLU/PCB (p = 0,023. Houve correlação entre a mediana da carga viral para o

  5. Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size.

    Science.gov (United States)

    De-Donatis, Gian Marco; Zhao, Zhengyi; Wang, Shaoying; Huang, Lisa P; Schwartz, Chad; Tsodikov, Oleg V; Zhang, Hui; Haque, Farzin; Guo, Peixuan

    2014-01-01

    Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is

  6. Relationship between serum HBV RNA levels and intrahepatic viral as well as histologic activity markers in entecavir-treated patients.

    Science.gov (United States)

    Wang, Jing; Yu, Yiqi; Li, Guojun; Shen, Chuan; Meng, Zhefeng; Zheng, Jianming; Jia, Yanhong; Chen, Shaolong; Zhang, Xiao; Zhu, MengQi; Zheng, Jiangjiang; Song, Zhangzhang; Wu, Jing; Shao, Lingyun; Qian, Peiyu; Mao, Xiaona; Wang, Xuanyi; Huang, Yuxian; Zhao, Caiyan; Zhang, Jiming; Qiu, Chao; Zhang, Wenhong

    2017-09-01

    In diagnostics, serum hepatitis B virus (HBV)-RNA levels are valuable when the HBV-DNA load in circulation is effectively suppressed by nucleos(t)ide analogue (NUC) therapy. This study aimed to determine the intrahepatic viral replication activity reflected in serum HBV-RNA and whether HBV-RNA contributes to liver histological changes in NUC-treated patients. A cross-sectional set of serum and liver biopsy samples was obtained from entecavir-treated patients with undetectable levels of serum HBV-DNA. The correlations between HBV-RNA concentration in serum and levels of peripheral and intrahepatic viral replicative forms and histological scores were analyzed. Quasispecies of serum HBV-RNA and intrahepatic viral replicative forms were examined by deep sequencing. HBV-RNA-positive hepatocytes were visualized by in situ hybridization. Serum HBV-RNA was detected in 35 of 47 patients (74.47%, 2.33-4.80 log10 copies/mL). These levels correlated not only with the intrahepatic HBV-RNA level and the ratio of intrahepatic HBV-RNA to covalently closed circular DNA (cccDNA), but also with the histological scores for grading and staging. From the view of quasispecies, serum HBV-RNA was more genetically homogenous with contemporaneously sampled intrahepatic HBV-RNA relative to cccDNA pool and dynamically changed over time in consecutive samples. In situ histology study revealed that HBV-RNA-positive hepatocytes were clustered in foci, sporadically distributed across the lobules, and co-localized with hepatitis B surface antigen. Serum HBV-RNA levels reflect intrahepatic viral transcriptional activity and are associated with liver histopathology in patients receiving NUC therapy. Our study sheds light on the nature of HBV-RNA in the pathogenesis of chronic HBV infection and has implications for the management of chronic hepatitis B during NUC therapy. Serum HBV-RNA levels are indicative of the intrahepatic transcriptional activity of covalently closed circular DNA and are

  7. The dual CCR5 and CCR2 inhibitor cenicriviroc does not redistribute HIV into extracellular space: implications for plasma viral load and intracellular DNA decline.

    Science.gov (United States)

    Kramer, Victor G; Hassounah, Said; Colby-Germinario, Susan P; Oliveira, Maureen; Lefebvre, Eric; Mesplède, Thibault; Wainberg, Mark A

    2015-03-01

    Cenicriviroc is a potent antagonist of the chemokine coreceptors 5 and 2 (CCR5/CCR2) and blocks HIV-1 entry. The CCR5 inhibitor maraviroc has been shown in tissue culture to be able to repel cell-free virions from the cell surface into extracellular space. We hypothesized that cenicriviroc might exhibit a similar effect, and tested this using clinical samples from the Phase IIb study 652-2-202, by measuring rates of intracellular DNA decline. We also monitored viral RNA levels in culture fluids. We infected PM-1 cells with CCR5-tropic HIV-1 BaL in the presence or absence of inhibitory concentrations of cenicriviroc (20 nM) or maraviroc (50 nM) or controls. Viral load levels and p24 were measured by ELISA, quantitative PCR and quantitative real-time reverse transcription PCR at 4 h post-infection. Frozen PBMC DNA samples from 30 patients with virological success in the Phase IIb study were studied, as were early and late reverse transcript levels. Docking studies compared binding between cenicriviroc/CCR5 and maraviroc/CCR5. Unlike maraviroc, cenicriviroc did not cause an increase in the amount of virus present in culture fluids at 4 h compared with baseline. The use of cenicriviroc did, however, result in lower levels of intracellular viral DNA after 4 h. Structural modelling indicates that cenicriviroc binds more deeply than maraviroc to the hydrophobic pocket of CCR5, providing an explanation for the absence of viral rebound with cenicriviroc. In contrast to maraviroc, cenicriviroc does not repel virus back into extracellular space. Differences in results may be due to superior binding of cenicriviroc to CCR5 compared with maraviroc. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Delivering the goods: viral and non-viral gene therapy systems and the inherent limits on cargo DNA and internal sequences.

    Science.gov (United States)

    Atkinson, Helen; Chalmers, Ronald

    2010-05-01

    Viruses have long been considered to be the most promising tools for human gene therapy. However, the initial enthusiasm for the use of viruses has been tarnished in the light of potentially fatal side effects. Transposons have a long history of use with bacteria in the laboratory and are now routinely applied to eukaryotic model organisms. Transposons show promise for applications in human genetic modification and should prove a useful addition to the gene therapy tool kit. Here we review the use of viruses and the limitations of current approaches to gene therapy, followed by a more detailed analysis of transposon length and the physical properties of internal sequences, which both affect transposition efficiency. As transposon length increases, transposition decreases: this phenomenon is known as length-dependence, and has implications for vector cargo capacity. Disruption of internal sequences, either via deletion of native DNA or insertion of exogenous DNA, may reduce or enhance genetic mobility. These effects may be related to host factor binding, essential spacer requirements or other influences yet to be elucidated. Length-dependence is a complex phenomenon driven not simply by the distance between the transposon ends, but by host proteins, the transposase and the properties of the DNA sequences encoded within the transposon.

  9. Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

    Science.gov (United States)

    Norkin, L C; Steinberg, V I; Kosz-Vnenchak, M

    1985-02-01

    A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.

  10. The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

    Energy Technology Data Exchange (ETDEWEB)

    Hipp, Katharina; Rau, Peter; Schäfer, Benjamin [Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart (Germany); Gronenborn, Bruno [Institut des Sciences du Végétal, CNRS, 91198 Gif-sur-Yvette (France); Jeske, Holger, E-mail: holger.jeske@bio.uni-stuttgart.de [Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart (Germany)

    2014-08-15

    Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.

  11. Integration-free reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) without viral vectors, recombinant DNA, and genetic modification.

    Science.gov (United States)

    Heng, Boon Chin; Fussenegger, Martin

    2014-01-01

    Stem cells are envisaged to be integral components of multicellular systems engineered for therapeutic applications. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) via recombinant expression of a limited number of transcription factors, which was first achieved by Yamanaka and colleagues in 2007, heralded a major breakthrough in the stem cell field. Since then, there has been rapid progress in the field of iPSC generation, including the identification of various small molecules that can enhance reprogramming efficiency and reduce the number of different transcription factors required for reprogramming. Nevertheless, the major obstacles facing clinical applications of iPSCs are safety concerns associated with the use of viral vectors and recombinant DNA for expressing the appropriate transcription factors during reprogramming. In particular, permanent genetic modifications to newly reprogrammed iPSCs have to be avoided in order to meet stringent safety requirements for clinical therapy. These safety challenges can be overcome by new technology platforms that enable cellular reprogramming to iPSCs without the need to utilize either recombinant DNA or viral vectors. The use of recombinant cell-penetrating peptides and direct transfection of synthetic mRNA encoding appropriate transcription factors have both been shown to successfully reprogram somatic cells to iPSCs. It has also been shown more recently that the direct transfection of certain miRNA species can reprogram somatic cells to pluripotency without the need for any of the transcription factors commonly utilized for iPSC generation. This chapter describes protocols for iPSC generation with these new techniques, which would obviate the use of recombinant DNA and viral vectors in cellular reprogramming, thus avoiding permanent genetic modification to the reprogrammed cells.

  12. In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

    Science.gov (United States)

    Moens, U; Seternes, O M; Hey, A W; Silsand, Y; Traavik, T; Johansen, B; Rekvig, O P

    1995-01-01

    Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus. PMID:8618908

  13. Revolution rather than rotation of AAA+ hexameric phi29 nanomotor for viral dsDNA packaging without coiling☆

    Science.gov (United States)

    Schwartz, Chad; De Donatis, Gian Marco; Zhang, Hui; Fang, Huaming; Guo, Peixuan

    2013-01-01

    It has long been believed that the DNA-packaging motor of dsDNA viruses utilizes a rotation mechanism. Here we report a revolution rather than rotation mechanism for the bacteriophage phi29 DNA packaging motor. The phi29 motor contains six copies of the ATPase (Schwartz et al., this issue); ATP binding to one ATPase subunit stimulates the ATPase to adopt a conformation with a high affinity for dsDNA. ATP hydrolysis induces a new conformation with a lower affinity, thus transferring the dsDNA to an adjacent subunit by a power stroke. DNA revolves unidirectionally along the hexameric channel wall of the ATPase, but neither the dsDNA nor the ATPase itself rotates along its own axis. One ATP is hydrolyzed in each transitional step, and six ATPs are consumed for one helical turn of 360°. Transition of the same dsDNA chain along the channel wall, but at a location 60° different from the last contact, urges dsDNA to move forward 1.75 base pairs each step (10.5 bp per turn/6ATP=1.75 bp per ATP). Each connector subunit tilts with a left-handed orientation at a 30° angle in relation to its vertical axis that runs anti-parallel to the right-handed dsDNA helix, facilitating the one-way traffic of dsDNA. The connector channel has been shown to cause four steps of transition due to four positively charged lysine rings that make direct contact with the negatively charged DNA phosphate backbone. Translocation of dsDNA into the procapsid by revolution avoids the difficulties during rotation that are associated with DNA supercoiling. Since the revolution mechanism can apply to any stoichiometry, this motor mechanism might reconcile the stoichiometry discrepancy in many phage systems where the ATPase has been found as a tetramer, hexamer, or nonamer. PMID:23763768

  14. Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus).

    Science.gov (United States)

    Pereiro, P; Martinez-Lopez, A; Falco, A; Dios, S; Figueras, A; Coll, J M; Novoa, B; Estepa, A

    2012-06-01

    Turbot (Scophthalmus maximus) is a high-value farmed marine flatfish with growing demand and production levels in Europe susceptible to turbot-specific viral haemorrhagic septicaemia virus (VHSV) strains. To evaluate the possibility of controlling the outbreaks of this infectious disease by means of DNA vaccination, the gpG of a VHSV isolated from farmed turbot (VHSV(860)) was cloned into an expression plasmid containing the human cytomegalovirus (CMV) promoter (pMCV1.4-G(860)). In our experimental conditions, DNA immunised turbots were more than 85% protected against VHSV(860) lethal challenge and showed both VHSV-gpG specific and neutralizing antibodies. To our knowledge this is the first report showing the efficacy of turbot genetic immunisation against a VHSV. Work is in progress to determine the contribution of innate and adaptive immunity to the protective response elicited by the immunization.

  15. Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

    Science.gov (United States)

    Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

    2017-02-01

    The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

  16. HIV-1 viral infectivity factor (Vif) alters processive single-stranded DNA scanning of the retroviral restriction factor APOBEC3G.

    Science.gov (United States)

    Feng, Yuqing; Love, Robin P; Chelico, Linda

    2013-03-01

    APOBEC3G is a retroviral restriction factor that can inhibit the replication of human immunodeficiency virus, type 1 (HIV-1) in the absence of the viral infectivity factor (Vif) protein. Virion-encapsidated APOBEC3G can deaminate cytosine to uracil in viral (-)DNA, which leads to hypermutation and inactivation of the provirus. APOBEC3G catalyzes these deaminations processively on single-stranded DNA using sliding and jumping movements. Vif is thought to primarily overcome APOBEC3G through an interaction that mediates APOBEC3G ubiquitination and results in its proteasomal degradation. However, Vif may also inhibit APOBEC3G mRNA translation, virion encapsidation, and deamination activity. Here we investigated the molecular mechanism of VifIIIB- and VifHXB2-mediated inhibition of APOBEC3G deamination activity. Biochemical assays using a model HIV-1 replication assay and synthetic single-stranded or partially double-stranded DNA substrates demonstrated that APOBEC3G has an altered processive mechanism in the presence of Vif. Specifically, VifHXB2 inhibited the jumping and VifIIIB inhibited the sliding movements of APOBEC3G. The absence of such an effect by Vif on degradation-resistant APOBEC3G D128K indicates that a Vif-APOBEC3G interaction mediates this effect. That the partially processive APOBEC3G was less effective at inducing mutagenesis in a model HIV-1 replication assay suggests that Vif co-encapsidation with APOBEC3G can promote sublethal mutagenesis of HIV-1 proviral DNA.

  17. BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses.

    Directory of Open Access Journals (Sweden)

    Dipanjan Dutta

    2015-06-01

    Full Text Available The innate immune system pattern recognition receptors (PRR are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β. NOD-like receptors (NLRs and AIM2-like receptors (ALRs are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by

  18. Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

    Science.gov (United States)

    Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

    2011-02-20

    MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Min Zhang

    Full Text Available 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.

  1. The Frequency of Cytidine Editing of Viral DNA Is Differentially Influenced by Vpx and Nucleosides during HIV-1 or SIVMAC Infection of Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Xuan-Nhi Nguyen

    Full Text Available Two cellular factors are currently known to modulate lentiviral infection specifically in myeloid cells: SAMHD1 and APOBEC3A (A3A. SAMHD1 is a deoxynucleoside triphosphohydrolase that interferes with viral infection mostly by limiting the intracellular concentrations of dNTPs, while A3A is a cytidine deaminase that has been described to edit incoming vDNA. The restrictive phenotype of myeloid cells can be alleviated through the direct degradation of SAMHD1 by the HIV-2/SIVSM Vpx protein or else, at least in the case of HIV-1, by the exogenous supplementation of nucleosides that artificially overcome the catabolic activity of SAMHD1 on dNTPs. Here, we have used Vpx and dNs to explore the relationship existing between vDNA cytidine deamination and SAMHD1 during HIV-1 or SIVMAC infection of primary dendritic cells. Our results reveal an interesting inverse correlation between conditions that promote efficient infection of DCs and the extent of vDNA editing that may reflect the different susceptibility of vDNA to cytoplasmic effectors during the infection of myeloid cells.

  2. Viral marketing

    OpenAIRE

    Král, Jiří

    2015-01-01

    Bachelor's Thesis deals with effective promotional tools called viral marketing. The main contribution of the thesis is the definition and history of viral marketing, making analysis of process of viral marketing, progresses definition and rules for creating a viral campaign. And also aspects are necessary for a successful viral spread. There are analysis of the characteristics of social media which are dividing according to the orientation and marketing tactics. Thesis is especially about so...

  3. The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes : A comparative study

    NARCIS (Netherlands)

    Kneuer, Carsten; Ehrhardt, Carsten; Bakowsky, Heike; Kumar, M. N. V. Ravi; Oberle, Volker; Lehr, Claus M.; Hoekstra, Dick; Bakowsky, Udo

    2006-01-01

    Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class o

  4. RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA.

    Science.gov (United States)

    Kabir, M S; Clements, M O; Kimmitt, P T

    2015-01-01

    The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, an RT-Bst method was developed for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single-tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of polymerase chain reaction (PCR)-amplified products. A superior performance was observed when amplifying MS2 cDNA with random primers following RT-Bst compared to RT alone, indicating greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify eight target regions spanning the respiratory syncytial virus (RSV) genome. Six out of eight targets were amplified consistently by PCR subsequent to RT-Bst amplification, whereas only three out of eight targets could be amplified after RT alone. The RSV sequences were selectively amplified using RSV-specific primers from a mixed template containing an excess of MS2 RNA without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence-specific primers, and enhances the yield of cDNA when compared to RT alone.

  5. Revolution rather than rotation of AAA+ hexameric phi29 nanomotor for viral dsDNA packaging without coiling.

    Science.gov (United States)

    Schwartz, Chad; De Donatis, Gian Marco; Zhang, Hui; Fang, Huaming; Guo, Peixuan

    2013-08-15

    It has long been believed that the DNA-packaging motor of dsDNA viruses utilizes a rotation mechanism. Here we report a revolution rather than rotation mechanism for the bacteriophage phi29 DNA packaging motor. The phi29 motor contains six copies of the ATPase (Schwartz et al., this issue); ATP binding to one ATPase subunit stimulates the ATPase to adopt a conformation with a high affinity for dsDNA. ATP hydrolysis induces a new conformation with a lower affinity, thus transferring the dsDNA to an adjacent subunit by a power stroke. DNA revolves unidirectionally along the hexameric channel wall of the ATPase, but neither the dsDNA nor the ATPase itself rotates along its own axis. One ATP is hydrolyzed in each transitional step, and six ATPs are consumed for one helical turn of 360°. Transition of the same dsDNA chain along the channel wall, but at a location 60° different from the last contact, urges dsDNA to move forward 1.75 base pairs each step (10.5bp per turn/6ATP=1.75bp per ATP). Each connector subunit tilts with a left-handed orientation at a 30° angle in relation to its vertical axis that runs anti-parallel to the right-handed dsDNA helix, facilitating the one-way traffic of dsDNA. The connector channel has been shown to cause four steps of transition due to four positively charged lysine rings that make direct contact with the negatively charged DNA phosphate backbone. Translocation of dsDNA into the procapsid by revolution avoids the difficulties during rotation that are associated with DNA supercoiling. Since the revolution mechanism can apply to any stoichiometry, this motor mechanism might reconcile the stoichiometry discrepancy in many phage systems where the ATPase has been found as a tetramer, hexamer, or nonamer. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Loss of retinoblastoma protein, but not p53, is associated with the presence of papillomaviral DNA in feline viral plaques, Bowenoid in situ carcinomas, and squamous cell carcinomas.

    Science.gov (United States)

    Munday, J S; Aberdein, D

    2012-05-01

    Although papillomaviral (PV) DNA is frequently present in feline cutaneous squamous cell carcinomas (SCCs), a causative association cannot be proven. Oncogenic human PVs cause neoplastic transformation by inhibiting retinoblastoma (pRb) and p53 activity. Therefore, absence of pRb and p53 immunostaining, along with increased p16 immunostaining, indicates a PV cause in some human SCCs. If PVs cause cutaneous feline SCCs, it was hypothesized that a similar immunohistochemistry profile, along with PV DNA, would be detectable. This was investigated using 5 feline viral plaques, 10 Bowenoid in situ carcinomas, 19 SCCs from ultraviolet-exposed (UV-exposed) skin, and 11 SCCs from UV-protected skin. Papillomaviral DNA was amplified by polymerase chain reaction from 30 of 45 lesions. Reduced pRb immunostaining was present in 26 of 45; increased p16 immunostaining was in 30; and p53 immunostaining was in 19. Both reduced pRb immunostaining and increased p16 immunostaining were more frequent in lesions containing PV DNA. In contrast, no association was observed between p53 immunostaining and the presence of PV DNA. SCCs from UV-protected skin more frequently contained PV DNA, reduced pRb, and increased p16 than UV-exposed SCCs. UV exposure was not associated with p53 immunostaining within the SCCs. These results suggest that feline PVs alter cell regulation by degrading pRb. Unlike oncogenic human PVs, there was no evidence that feline PVs degrade p53. These results provide further evidence that PVs may cause feline cutaneous SCCs, especially those in UV-protected skin, and they suggest a possible mechanism of this oncogenic action.

  7. Viral Disease Networks?

    Science.gov (United States)

    Gulbahce, Natali; Yan, Han; Vidal, Marc; Barabasi, Albert-Laszlo

    2010-03-01

    Viral infections induce multiple perturbations that spread along the links of the biological networks of the host cells. Understanding the impact of these cascading perturbations requires an exhaustive knowledge of the cellular machinery as well as a systems biology approach that reveals how individual components of the cellular system function together. Here we describe an integrative method that provides a new approach to studying virus-human interactions and its correlations with diseases. Our method involves the combined utilization of protein - protein interactions, protein -- DNA interactions, metabolomics and gene - disease associations to build a ``viraldiseasome''. By solely using high-throughput data, we map well-known viral associated diseases and predict new candidate viral diseases. We use microarray data of virus-infected tissues and patient medical history data to further test the implications of the viral diseasome. We apply this method to Epstein-Barr virus and Human Papillomavirus and shed light into molecular development of viral diseases and disease pathways.

  8. Viral DNA load of high-risk human papilloma virus is closely associated with the grade of cervical lesions

    OpenAIRE

    Shen, Guqun; Cheng, Jingxin; Wang, Yan; Zhou, Ping; Zhang, Guoqing

    2014-01-01

    This study is to explore the correlation between the viral load of high-risk human papilloma virus (HPV) and the degree of cervical lesions, as well as the follow-up monitoring role of high-risk HPV measurements in the treatment of patients with cervical lesions. Hybrid capture-2 method was used to measure the amount of high-risk HPV load of 361 patients who were enrolled from January 2009 to December 2010 at the Affiliated Tumor Hospital of Xinjiang Medical University, including 76 cases of ...

  9. Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Deborah Heydenburg Fuller

    Full Text Available Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT, during antiretroviral therapy (ART induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.

  10. In addition to its antiviral and immunomodulatory properties, the zebrafish β-defensin 2 (zfBD2) is a potent viral DNA vaccine molecular adjuvant.

    Science.gov (United States)

    García-Valtanen, P; Martinez-Lopez, A; Ortega-Villaizan, M; Perez, L; Coll, J M; Estepa, A

    2014-01-01

    It is well known that β-defensins are key components of the host innate immune response against pathogens and potentially provide a link between innate and adaptive immunity. In zebrafish (Danio rerio), a vertebrate model species in numerous biomedical fields, three β-defensin isoforms were recently identified. To our knowledge, however, studies describing antimicrobial or immunomodulatory properties of any of the zebrafish β-defensins isoforms are absent today. Since it is indubitable that deepening the study of zebrafish β-defensins would be of interest in this work we investigated whether or not the zebrafish β-defensin 2 (zfBD2) has the antiviral properties described for their vertebrate counterparts. Our in vitro and in vivo studies showed that zfBD2 has antiviral activity, immunomodulatory properties and, most importantly, is a potent viral DNA vaccine molecular adjuvant. In addition, a potential relationship between zfBD2 activity and the NF-κB signaling pathway is suggested. Altogether these results show that the zebrafish could be a suitable in vivo animal model to study the roles played by β-defensin 2 in viral diseases, vaccinology and even in clinical dermatology. To note that psoriasis can be induced in zebrafish and the over-expression of β-defensin 2 is implicated in the inflammatory response associated with this human skin disorder.

  11. Viral marketing

    OpenAIRE

    BLÁHOVÁ, Adéla

    2012-01-01

    The aim of my thesis is to provide a comprehensive overview of the viral marketing and to analyze selected viral campaigns. There is a description of advantages and disadvantages of this marketing tool. In the end I suggest for which companies viral marketing is an appropriate form of the promotion.

  12. Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation.

    Directory of Open Access Journals (Sweden)

    Vanessa C McFadden

    2017-02-01

    Full Text Available The FoxA family of pioneer transcription factors regulates hepatitis B virus (HBV transcription, and hence viral replication. Hepatocyte-specific FoxA-deficiency in the HBV transgenic mouse model of chronic infection prevents the transcription of the viral DNA genome as a result of the failure of the developmentally controlled conversion of 5-methylcytosine residues to cytosine during postnatal hepatic maturation. These observations suggest that pioneer transcription factors such as FoxA, which mark genes for expression at subsequent developmental steps in the cellular differentiation program, mediate their effects by reversing the DNA methylation status of their target genes to permit their ensuing expression when the appropriate tissue-specific transcription factor combinations arise during development. Furthermore, as the FoxA-deficient HBV transgenic mice are viable, the specific developmental timing, abundance and isoform type of pioneer factor expression must permit all essential liver gene expression to occur at a level sufficient to support adequate liver function. This implies that pioneer transcription factors can recognize and mark their target genes in distinct developmental manners dependent upon, at least in part, the concentration and affinity of FoxA for its binding sites within enhancer and promoter regulatory sequence elements. This selective marking of cellular genes for expression by the FoxA pioneer factor compared to HBV may offer the opportunity for the specific silencing of HBV gene expression and hence the resolution of chronic HBV infections which are responsible for approximately one million deaths worldwide annually due to liver cirrhosis and hepatocellular carcinoma.

  13. PP2A targeting by viral proteins: a widespread biological strategy from DNA/RNA tumor viruses to HIV-1.

    Science.gov (United States)

    Guergnon, Julien; Godet, Angélique N; Galioot, Amandine; Falanga, Pierre Barthélémy; Colle, Jean-Hervé; Cayla, Xavier; Garcia, Alphonse

    2011-11-01

    Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as constitutive housekeeping enzymes, it is now well established that PP2A proteins represent a family of highly and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A targeting for research in human diseases, and in further therapeutic developments, is also discussed.

  14. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    Science.gov (United States)

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  15. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    Directory of Open Access Journals (Sweden)

    Chih-Lang Lin

    Full Text Available Occult hepatitis B virus (HBV infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg. Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2. Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  16. SERS detection of indirect viral DNA capture using colloidal gold and methylene blue as a Raman label

    Science.gov (United States)

    An indirect capture model assay using colloidal Au nanoparticles is demonstrated for surface enhanced Raman scattering (SERS) spectroscopy detection of DNA. The sequence targeted for capture is derived from the West Nile Virus (WNV) RNA genome and was selected on the basis of exhibiting minimal seco...

  17. Impedimetric genosensor for detection of hepatitis C virus (HCV1) DNA using viral probe on methylene blue doped silica nanoparticles.

    Science.gov (United States)

    Singhal, Chaitali; Ingle, Aviraj; Chakraborty, Dhritiman; Pn, Anoop Krishna; Pundir, C S; Narang, Jagriti

    2017-05-01

    An impedimetric genosensor was fabricated for detection of hepatitis C virus (HCV) genotype 1 in serum, based on hybridization of the probe with complementary target cDNA from sample. To achieve it, probe DNA complementary to HCVgene was immobilized on the surface of methylene blue (MB) doped silica nanoparticles MB@SiNPs) modified fluorine doped tin oxide (FTO) electrode. The synthesized MB@SiNPs was characterized using scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) pattern. This modified electrode (ssDNA/MB@SiNPs/FTO) served both as a signal amplification platform (due to silica nanoparticles (SiNPs) as well as an electrochemical indicator (due to methylene blue (MB)) for the detection of the HCV DNA in patient serum sample. The genosensor was optimized and evaluated. The sensor showed a dynamic linear range 100-10(6) copies/mL, with a detection limit of 90 copies/mL. The sensor was applied for detection of HCV in sera of hepatitis patient and could be renewed. The half life of the sensor was 4 weeks. The MB@SiNPs/FTO electrode could be used for preparation of other gensensors also. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.

    2011-01-01

    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...

  19. Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands

    Energy Technology Data Exchange (ETDEWEB)

    Bates, R.C.; Snyder, C.E.; Banerjee, P.T.; Mitra, S.

    1984-02-01

    Autonomous parvoviruses are thought to uniquely encapsidate single-stranded DNA of minus polarity. In contrast, the defective adeno-associated viruses separately encapsidate equal amounts of plus and minus DNA strands. The uniqueness of minus strand encapsidation is reexamined for the autonomous parvoviruses. Although it was found that Kilham rat virus and H-1 virus encapsidate varying but small amounts of complementary-strand DNA, it was unexpected to find that LuIII virus encapsidated equal amounts of plus and minus DNA. The extracted LuIII DNA possessed properties of double-stranded replicative-form DNA, including insensitivity to S1 endonuclease, cleavage by restriction enzymes, and conversion to unit-length, single-stranded DNA when electrophoresed under denaturing conditions. However, the inability of this DNA to form single-stranded DNA circles when denatured and then renatured in the presence of formamide and the lack of double-stranded DNA circle formation after treatment with exonuclease III and reannealing shows a lack of sequence homology of the 3' and 5' termini of LuIII DNA, in contrast to adeno-associated virus DNA. Digestion of LuIII double-stranded DNA with EcoRI and HincII and separation of plus and minus DNA strands on composite agarose-acrylamide gels identified a heterogeneity present only in the plus DNA strand. These results suggest that strand specificity of viral DNA encapsidation is not a useful property for differentiation between the autonomous and defective parvoviruses. Furthermore, encapsidation by LuIII of equal amounts of complementary DNA strands in contrast to encapsidation of minus strands by H-1 virus, when propagated in the same host cell type, suggests that selection of strands for encapsidation is a virus-coded rather than host-controlled event.

  20. Poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine]:A Non-viral Polymer with Potential for DNA Delivery

    Institute of Scientific and Technical Information of China (English)

    Zhi YANG; Gu Ping TANG

    2004-01-01

    A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than 600) was grafted to the backbone. The polymer was characterized by 1H NMR. It appeared lower cytotoxity compared to poly(ethylenimine) (25KDa), which was quantified by MTT assay. Electrophoresis indicated that the polymer could retardate DNA at N/P ratio 1.2-1.8 (w/w). Transfection efficiency of the complexes was studied in NT2 cell lines. It was 1.5 fold higher than molecular weight PEI (Mw = 25KDa).

  1. The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

    Science.gov (United States)

    Cao, Maohua; You, Hong; Hermonat, Paul L

    2014-01-01

    Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  2. JC virus small T antigen binds phosphatase PP2A and Rb family proteins and is required for efficient viral DNA replication activity.

    Directory of Open Access Journals (Sweden)

    Brigitte Bollag

    Full Text Available BACKGROUND: The human polyomavirus, JC virus (JCV produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg and three T' proteins, but little is known about small tumor antigen (tAg functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. METHODOLOGY AND FINDINGS: We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A, and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. CONCLUSIONS: JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions.

  3. Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

    Directory of Open Access Journals (Sweden)

    Irena Zurnic

    2016-08-01

    Full Text Available Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H screen with prototype FV (PFV Gag protein as bait we identified human polo-like kinase 2 (hPLK2, a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells.

  4. Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

    Science.gov (United States)

    Zurnic, Irena; Hütter, Sylvia; Rzeha, Ute; Stanke, Nicole; Reh, Juliane; Müllers, Erik; Hamann, Martin V.; Kern, Tobias; Gerresheim, Gesche K.; Serrao, Erik; Lesbats, Paul; Engelman, Alan N.; Cherepanov, Peter; Lindemann, Dirk

    2016-01-01

    Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. PMID:27579920

  5. Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California.

    Science.gov (United States)

    Pusterla, Nicola; Wilson, W David; Mapes, Samantha; Finno, Carrie; Isbell, Diane; Arthur, Rick M; Ferraro, Gregory L

    2009-02-01

    The objective of this study was to determine viral loads, strain (neuropathogenic versus non-neuropathogenic) and state (lytic, non-replicating, latent) of equine herpesvirus-1 (EHV-1) by real-time polymerase chain reaction (PCR) in the blood and nasopharyngeal secretions of adult horses following natural exposure. The index case, a 4-year-old Thoroughbred gelding with confirmed EHV-1 myeloencephalopathy, as well as potentially exposed horses, were sampled over a period of 3 weeks. The study population comprised of 39 adult Thoroughbred horses and 35 adult "pony" and outrider horses of various breeds housed at a racetrack in Northern California. Blood samples and nasopharyngeal secretions (NPS) from all horses were tested on several occasions for EHV-1 DNA viral loads, targeting the glycoprotein B (gB) gene, viral strain, targeting the ORF 30 gene, and transcriptional activity of EHV-1, targeting the gB gene and latency-associated transcripts (LATs). Viral loads and transcriptional activity of the gB gene declined rapidly in the index case following antiviral treatment. The prevalence of EHV-1 infection in NPS determined by PCR slowly decreased over the 22 day study period from 25% to 14%. The initial surveillance showed multiple clusters of exposure, one associated with the index case and two related to horses that had recently returned from a different racetrack. Viral strain differentiation showed that only two horses (the index case and a neighboring horse) were infected with only a neuropathogenic strain, while all other horses were infected with either a non-neuropathogenic strain or were dually infected with both neuropathogenic and non-neuropathogenic strains. In most cases, the virus was present in either a lytic or a non-replicating form, while latent virus was found in blood and NPS much less frequently. The molecular approach used in this study showed promise for assessing the risk of exposing other horses to EHV-1 and for studying viral kinetics in

  6. Mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change DNA translocation velocity and processivity.

    Science.gov (United States)

    Tsay, James M; Sippy, Jean; DelToro, Damian; Andrews, Benjamin T; Draper, Bonnie; Rao, Venigalla; Catalano, Carlos E; Feiss, Michael; Smith, Douglas E

    2010-07-30

    Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage lambda gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.

  7. Viral information.

    Science.gov (United States)

    Rohwer, Forest; Barott, Katie

    2013-03-01

    Viruses are major drivers of global biogeochemistry and the etiological agents of many diseases. They are also the winners in the game of life: there are more viruses on the planet than cellular organisms and they encode most of the genetic diversity on the planet. In fact, it is reasonable to view life as a viral incubator. Nevertheless, most ecological and evolutionary theories were developed, and continue to be developed, without considering the virosphere. This means these theories need to be to reinterpreted in light of viral knowledge or we need to develop new theory from the viral point-of-view. Here we briefly introduce our viral planet and then address a major outstanding question in biology: why is most of life viral? A key insight is that during an infection cycle the original virus is completely broken down and only the associated information is passed on to the next generation. This is different for cellular organisms, which must pass on some physical part of themselves from generation to generation. Based on this premise, it is proposed that the thermodynamic consequences of physical information (e.g., Landauer's principle) are observed in natural viral populations. This link between physical and genetic information is then used to develop the Viral Information Hypothesis, which states that genetic information replicates itself to the detriment of system energy efficiency (i.e., is viral in nature). Finally, we show how viral information can be tested, and illustrate how this novel view can explain existing ecological and evolutionary theories from more fundamental principles.

  8. VIRAL MARKETING

    OpenAIRE

    OLENTSOVA Y.A.

    2016-01-01

    Abstract This project seeks to investigate how the company Gitz can create awareness towards their brand by using viral marketing. To do this we analyze which elements of viral marketing the company can use, to reach their goal. In order to utilize the selected tools of viral marketing best possible, we need to figure out the company’s customer segment and figure out how to reach that segment. This has been done with the use of Henrik Dahl’s Minerva-model that divides the population into f...

  9. Viral arthritis

    Science.gov (United States)

    Infectious arthritis - viral ... Arthritis may be a symptom of many virus-related illnesses. It usually disappears on its own without ... the rubella vaccine, only a few people develop arthritis. No risk factors are known.

  10. Kaposi's Sarcoma-Associated Herpesvirus K-bZIP Protein Is Necessary for Lytic Viral Gene Expression, DNA Replication, and Virion Production in Primary Effusion Lymphoma Cell Lines▿ †

    OpenAIRE

    Lefort, Sylvain; Flamand, Louis

    2009-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human proliferative disorders, namely, Kaposi's sarcoma, primary effusion lymphomas (PEL), and multicentric Castleman's disease. Lytic DNA replication of KSHV, which is essential for viral propagation, requires the binding of at least two KSHV proteins, replication and transactivation activator (RTA) and K-bZIP, on the lytic origin of replication. Moreover, K-bZIP physically interacts with RTA and represses its tra...

  11. Viral induced demyelination.

    Science.gov (United States)

    Stohlman, S A; Hinton, D R

    2001-01-01

    Viral induced demyelination, in both humans and rodent models, has provided unique insights into the cell biology of oligodendroglia, their complex cell-cell interactions and mechanisms of myelin destruction. They illustrate mechanisms of viral persistence, including latent infections in which no infectious virus is readily evident, virus reactivation and viral-induced tissue damage. These studies have also provided excellent paradigms to study the interactions between the immune system and the central nervous system (CNS). Although of interest in their own right, an understanding of the diverse mechanisms used by viruses to induce demyelination may shed light into the etiology and pathogenesis of the common demyelinating disorder multiple sclerosis (MS). This notion is supported by the persistent view that a viral infection acquired during adolescence might initiate MS after a long period of quiescence. Demyelination in both humans and rodents can be initiated by infection with a diverse group of enveloped and non-enveloped RNA and DNA viruses (Table 1). The mechanisms that ultimately result in the loss of CNS myelin appear to be equally diverse as the etiological agents capable of causing diseases which result in demyelination. Although demyelination can be a secondary result of axonal loss, in many examples of viral induced demyelination, myelin loss is primary and associated with axonal sparing. This suggests that demyelination induced by viral infections can result from: 1) a direct viral infection of oligodendroglia resulting in cell death with degeneration of myelin and its subsequent removal; 2) a persistent viral infection, in the presence or absence of infectious virus, resulting in the loss of normal cellular homeostasis and subsequent oligodendroglial death; 3) a vigorous virus-specific inflammatory response wherein the virus replicates in a cell type other than oligodendroglia, but cytokines and other immune mediators directly damage the

  12. MicroRNA expression in rainbow trout (Oncorhynchus mykiss) vaccinated with a DNA vaccine encoding the glycoprotein gene of Viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Lorenzen, Niels

    Viral hemorrhagic septicemia caused by a fish rhabdovirus, Viral hemorrhagic septicemia virus (VHSV), results in significant mortality in farmed rainbow trout (Oncorhynchus mykiss Walbaum). Although the disease had been eradicated in Denmark, wildlife marine reservoir of VHSV poses a threat parti...

  13. Serum HBV DNA level at week 12 is superior to viral response at week 24 in predicting long-term treatment outcome of telbivudine for chronic hepatitis B patients

    Institute of Scientific and Technical Information of China (English)

    L(U) Wei; YANG Hai-hong; FAN Yun-ming; LI Takming; ZHANG Li-fan; MUI Chongseong; FAN Hong-wei

    2013-01-01

    Background Telbivudine,one of the five nucleos(t)ide antiviral drugs,was reported to be superior to lamivudine in a better biochemical,virological,and histological response for treatment-naive patients in the GLOBE trial.The aim of this study was to determine the antiviral potency,viral resistance,and the significance of early response for long-term telbivudine treatment.Methods We recruited 161 patients of chronic hepatitis B (CHB) on telbivudine between January 2009 and September 2011 in Macau,China.The serum hepatitis B virus DNA levels,hepatitis B e antigen (HBeAg) seroconversion,alanine aminotransferese (ALT) normalization,and viral resistance were analyzed.Results The median age and follow-up duration were 48 years and 16.9 months.All patients were followed up for at least 6 months,while data were collected for 132,120,95,and 53 patients at 12,24,48,and 96 weeks respectively.The cumulative HBeAg seroconversion rate was 20.8% and only three patients (1.9%) presented with telbivudine low level resistance.The ALT normalization rates were 76.9% at 48 weeks and 77.6% at 96 weeks.Undetectable HBV DNA was achieved by 1.8%,31.6%,60%,and 74.1% in HBeAg positive patients and 29.3%,60.3%,84%,and 84.6% in HBeAg negative patients at each time point.Week 12 HBV DNA level <1000 copies/ml (<200 IU/ml) was a better predictor of viral suppression at 2-year follow-up (P=-0.001,OR=27.00) than undetectable HBV DNA level at week 24 (P=0.120,OR=4.81).Conclusions Two-year telbivudine treatment yielded high rates of viral suppression and ALT normalization.Serum HBV DNA level at week 12 is a superior predictor for long-term viral suppression.

  14. Viral quasispecies.

    Science.gov (United States)

    Andino, Raul; Domingo, Esteban

    2015-05-01

    New generation sequencing is greatly expanding the capacity to examine the composition of mutant spectra of viral quasispecies in infected cells and host organisms. Here we review recent progress in the understanding of quasispecies dynamics, notably the occurrence of intra-mutant spectrum interactions, and implications of fitness landscapes for virus adaptation and de-adaptation. Complementation or interference can be established among components of the same mutant spectrum, dependent on the mutational status of the ensemble. Replicative fitness relates to an optimal mutant spectrum that provides the molecular basis for phenotypic flexibility, with implications for antiviral therapy. The biological impact of viral fitness renders particularly relevant the capacity of new generation sequencing to establish viral fitness landscapes. Progress with experimental model systems is becoming an important asset to understand virus behavior in the more complex environments faced during natural infections.

  15. RNA silencing and plant viral diseases.

    Science.gov (United States)

    Wang, Ming-Bo; Masuta, Chikara; Smith, Neil A; Shimura, Hanako

    2012-10-01

    RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.

  16. Human cytomegalovirus glycoprotein B genotypes in blood of AIDS patients: lack of association with either the viral DNA load in leukocytes or presence of retinitis.

    Science.gov (United States)

    Gilbert, C; Handfield, J; Toma, E; Lalonde, R; Bergeron, M G; Boivin, G

    1999-09-01

    It has been suggested that human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes could be used as a marker for viral virulence in patients with AIDS. The present study was designed to evaluate a possible association between specific gB genotypes, the presence of HCMV retinitis, and the HCMV viral load. Fifty-four blood samples were obtained from 54 HIV- and HCMV-infected patients. Twenty-seven of these patients were asymptomatic for HCMV, whereas the other 27 patients had been diagnosed recently with HCMV retinitis. HCMV gB genotyping was carried out by using restriction enzyme analysis of PCR-amplified PMNL extracts. Determination of the HCMV viral load in the same specimens was carried out using a quantitative-PCR. HCMV gB genotype 2 was found more frequently than other genotypes in PCR-amplified polymorphonuclear leukocytes (PMNL) of patients with AIDS (P < 0.05) but not more frequently in samples from patients with HCMV retinitis. No significant association was found between any HCMV gB genotypes and the viral load in blood. In conclusion, the actual HCMV gB genotyping system using PMNL provides no additional benefit over the viral load in blood for identification of HIV-infected subjects at risk of HCMV disease.

  17. Correlation of mRNA Profiles, miRNA Profiles, and Functional Immune Response in Rainbow Trout (Oncorrhynkus Mykiss) Infected With Viral Hemorrhagic Septicemia Virus (VHSV) and in Fish Vaccinated With a DNA Vaccine Against VHSV

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Jørgensen, Hanne

    2011-01-01

    and are incorporated into the RNA-Induced Silencing Complex (RISC), which target specific mRNA sequences, causing either mRNA degradation or translation repression. This results in altered mRNA and protein profiles characteristic of a particular cellular phenotype or physiological state. By targeting immune relevant m...... with the highly pathogenic fish rhabdovirus Viral hemorrhagic septicemia virus (VHSV). This talk will discuss our overall strategy and present preliminary data on the expression of miRNAs and the type I interferon-inducible Mx gene in the liver and the skeletal muscle tissue of fish injected with a DNA vaccine...

  18. Anatomic viral detection is automated: the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates, using immunocytochemical and nucleic acid hybridization techniques.

    Science.gov (United States)

    Montone, K. T.; Brigati, D. J.; Budgeon, L. R.

    1989-01-01

    This paper presents the first automated system for simultaneously detecting human papilloma, herpes simplex, adenovirus, or cytomegalovirus viral antigens and gene sequences in standard formalin-fixed, paraffin-embedded tissue substrates and tissue culture. These viruses can be detected by colorimetric in situ nucleic acid hybridization, using biotinylated DNA probes, or by indirect immunoperoxidase techniques, using polyclonal or monoclonal antibodies, in a 2.0-hour assay performed at a single automated robotic workstation. Images FIG. 1 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 10 FIG. 11 PMID:2773514

  19. PEG-b-PPS-b-PEI micelles and PEG-b-PPS/PEG-b-PPS-b-PEI mixed micelles as non-viral vectors for plasmid DNA: tumor immunotoxicity in B16F10 melanoma.

    Science.gov (United States)

    Velluto, Diana; Thomas, Susan N; Simeoni, Eleonora; Swartz, Melody A; Hubbell, Jeffrey A

    2011-12-01

    Cationic micelles formed from poly(ethylene glycol)-bl-poly(propylene sulfide)-bl-poly(ethylene imine) (PEG-b-PPS-b-PEI) and from mixtures of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-b-PPS) with PEG-b-PPS-b-PEI were explored as non-viral vectors for plasmid DNA (pDNA) transfection in a tumor immunotoxicity model. Complexes with pDNA were found to be templated exclusively by the size of the pDNA-free micelles and ranged from 240 nm (for PEG-b-PPS-b-PEI) to 30 nm (for mixed micelles of PEG-b-PPS/PEG-b-PPS-b-PEI). Both formulations transfected melanoma cells well in vitro. As a model with a functional read-out of tumor cell death, one with likely only small bystander effects, tumors were transfected with an antigen transgene, using an antigen to which the recipient animals had been previously vaccinated with a Th1-biasing adjuvant. Reduction in tumor growth, increase in intratumoral infiltration of cytotoxic T lymphocytes and accumulation of Th1-biasing cytokines indicated that both micelle formulations transfected efficiently compared with naked pDNA and with low cytotoxicity.

  20. [The identification of viruses of human papilloma of high carcinogenic risk and evaluation of physical status of viral DNA using technique of polymerase-chain reaction under affection of cervical epithelium].

    Science.gov (United States)

    Viazovaia, A A; Kuevda, D A; Trofimova, O B; Shipulina, O Iu; Ershov, V A; Lialina, L V; Narvskaia, O V

    2013-08-01

    The DNA of virus of human papilloma of high carcinogenic risk was detected in 116 cervical samples. At that, the morphological symptoms of background processes are detected in 19 samples, CIN 1 in 9, CIN 2 in 23, CIN 3 in 54 (and out of them carcinoma in situ in 13), epidermoid cancer (squamous cell carcinoma) in 11 cases. The viral load of human papilloma of high carcinogenic risk in all samples of DNA exceeded threshold of clinical value (3 lg copies of DNA of human papilloma/105 cells). The genetic typing of human papilloma of high carcinogenic risk revealed the dominance of human papilloma of type 16 in 49.7%, type 33 in 15.3%, type 31 in 12.3% and type 45 in 5.5%. In women with background processes in cervix of the uterus DNA of human papilloma type 16 was detected more often in episome form. In case of dysplastic alterations of epithelium and cervical cancer DNA of human papilloma type 16 is detected in mixt form with different degree of integration into cell genome.

  1. APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA

    Directory of Open Access Journals (Sweden)

    Pathak Vinay K

    2009-02-01

    Full Text Available Abstract Background Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown. Results To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y40RHHY44, were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA, and the levels of G-to-A mutations in virion RNA (vRNA were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA. Conclusion These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual

  2. Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection.

    Science.gov (United States)

    Pérez-Santiago, Josué; Schrier, Rachel D; de Oliveira, Michelli F; Gianella, Sara; Var, Susanna R; Day, Tyler R C; Ramirez-Gaona, Miguel; Suben, Jesse D; Murrell, Ben; Massanella, Marta; Cherner, Mariana; Smith, Davey M; Ellis, Ronald J; Letendre, Scott L; Mehta, Sanjay R

    2016-04-01

    Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, we measured mtDNA levels by droplet digital PCR, and soluble CD14 and CD163, neurofilament light, and neopterin by ELISA. In blood and CSF, we measured soluble IP-10, MCP-1, TNF-α, and IL-6 by ELISA, and intracellular expression of IL-2, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells by flow cytometry. We also evaluated the relationship between CSF pleocytosis and mtDNA longitudinally in another set of five individuals participating in an antiretroviral treatment (ART) interruption study. Cell-free CSF mtDNA levels strongly correlated with neurocognitive performance among individuals with neurocognitive impairment (NCI) (r = 0.77, p = 0.001). CSF mtDNA also correlated with levels of IP-10 in CSF (r = 0.70, p = 0.007) and MCP-1 in blood plasma (r = 0.66, p = 0.01) in individuals with NCI. There were no significant associations between inflammatory markers and mtDNA in subjects without NCI, and levels of mtDNA did not differ between subjects with and without NCI. MtDNA levels preceded pleocytosis and HIV RNA following ART interruption. Cell-free mtDNA in CSF was strongly associated with the severity of neurocognitive dysfunction and inflammation only in individuals with NCI. Our findings suggest that within a subset of subjects cell-free CSF mtDNA is associated with inflammation and degree of NCI.

  3. Advances in intracellular DNA sensors of PYHIN family and mechanisms of viral evasion of innate immune responses%DNA 识别受体 PYHIN 家族及相关病毒免疫逃逸机制的研究

    Institute of Scientific and Technical Information of China (English)

    魏巍; 汪速飞; 倪明; 余冰

    2016-01-01

    宿主细胞内的DNA识别受体可识别病毒核酸分子并激活细胞天然免疫反应,从而产生抗病毒效应;同时,病毒也进化出相应机制来逃避或抑制这种免疫反应。本文总结了宿主细胞内 DNA识别受体PYHIN家族识别病毒核酸并激活细胞天然免疫反应的特点和分子机制,并讨论了病毒逃避宿主天然免疫应答的方式。%The intracellular DNA sensors in host cells recognize viral nucleic acid and activate the innate immunity , then resulting in antiviral effects . However , the viral pathogens have also evolved with appropriate strategies to evade or suppress the innate immune responses .In this review ,the characteristics and molecular mechanisms of the immune effects of PYHIN family ,one of the DNA sensors in host cells that could recognize viral DNA and activate innate immunity are discussed .The strategies of how viruses escape from the immune surveillance are also summarized .

  4. The Distribution of HIV DNA and RNA in Cell Subsets Differs in Gut and Blood of HIV-Positive Patients on ART: Implications for Viral Persistence

    Science.gov (United States)

    Yukl, Steven A.; Shergill, Amandeep K.; Ho, Terence; Killian, Maudi; Girling, Valerie; Epling, Lorrie; Li, Peilin; Wong, Lisa K.; Crouch, Pierre; Deeks, Steven G.; Havlir, Diane V.; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K.

    2013-01-01

    Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir. PMID:23852128

  5. Fusion of a viral antigen to invariant chain leads to augmented T-cell immunity and improved protection in gene-gun DNA-vaccinated mice

    DEFF Research Database (Denmark)

    Grujic, Mirjana; Holst, Peter J; Christensen, Jan P

    2009-01-01

    against lethal peripheral challenge. The current study questioned whether the same strategy, i.e. linkage of GP to an Ii chain, could be applied to a naked DNA vaccine. Following gene-gun immunization with the linked construct (DNA-IiGP), GP-specific CD4(+) T cells could not be detected by flow cytometry...

  6. High Viral Loads of Epstein-Barr Virus DNA in Peripheral Blood of Patients with Chronic Lymphocytic Leukemia Associated with Unfavorable Prognosis.

    Science.gov (United States)

    Grywalska, Ewelina; Roliński, Jacek; Pasiarski, Marcin; Korona-Glowniak, Izabela; Maj, Maciej; Surdacka, Agata; Grafka, Agnieszka; Stelmach-Gołdyś, Agnieszka; Zgurski, Michał; Góźdź, Stanisław; Malm, Anna; Grabarczyk, Piotr; Starosławska, Elżbieta

    2015-01-01

    Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. The potential involvement of EBV in the clinical course of chronic lymphocytic leukemia (CLL) remains unexplained. The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs) of CLL patients may influence heterogeneity in the course of the disease. The study included peripheral blood samples from 115 previously untreated patients with CLL (54 women and 61 men) and 40 healthy controls (16 women and 24 men). We analyzed the association between the EBV-DNA load in PBMCs and the stage of the disease, adverse prognostic factors, and clinical outcome. Detectable numbers of EBV-DNA copies in PBMCs were found in 62 out of 115 CLL patients (53.91%). The EBV-DNA copy number/μg DNA was significantly higher in patients who required early implementation of treatment, presented with lymphocyte count doubling time DNA copy number/μg DNA showed significant positive correlation with the concentrations of lactate dehydrogenase (LDH) and beta-2-microglobulin. We have shown that in CLL patients, higher EBV-DNA copy number predicted shorter survival and shorter time to disease progression, and it was associated with other established unfavorable prognostic factors. This suggests that EBV may negatively affect the outcome of CLL.

  7. Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/Anti-HBe system, viral dna polymerase and HBV-DNA Comparação dos marcadores sorológicos da replicação do vírus da hepatite B: estudos sobre o sistema AgHBe/anti-HBe, DNA polimerase virai e HBV-DNA

    Directory of Open Access Journals (Sweden)

    João Renato Rebello Pinho

    1989-10-01

    Full Text Available The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker of replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV - related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.A detecção do genoma do HBV no soro por hibridização molecular é o mais sensível e específico marcador da replicação e infectividade do HBV, sendo sua utilização proposta como técnica rotineira no acompanhamento de doenças relacionadas a este vírus. Comparando diferentes técnicas descritas anteriormente, escolhemos a deposição direta das amostras séricas sobre a membrana de nitrocelulose sob filtração a vácuo, seguida de banhos desnaturantes e neutralisantes como mais prática e simples, com sensibilidade equivalente. O ensaio da DNA polimerase usando ácido fosfonofórmico como inibidor virai específico mostrou 86.8% de concordância com a detecção direta do DNA viral, sendo, portanto, uma alternativa viável no acompanhamento de pacientes com hepatite crônica B. Encontramos 19,2% das amostras AgHBe positivos sem outros marcadores de replica

  8. Strongly correlated electrostatics of viral genome packaging.

    Science.gov (United States)

    Nguyen, Toan T

    2013-03-01

    The problem of viral packaging (condensation) and ejection from viral capsid in the presence of multivalent counterions is considered. Experiments show divalent counterions strongly influence the amount of DNA ejected from bacteriophage. In this paper, the strong electrostatic interactions between DNA molecules in the presence of multivalent counterions is investigated. It is shown that experiment results agree reasonably well with the phenomenon of DNA reentrant condensation. This phenomenon is known to cause DNA condensation in the presence of tri- or tetra-valent counterions. For divalent counterions, the viral capsid confinement strongly suppresses DNA configurational entropy, therefore the correlation between divalent counterions is strongly enhanced causing similar effect. Computational studies also agree well with theoretical calculations.

  9. A nuclear-replicating viroid antagonizes infectivity and accumulation of a geminivirus by upregulating methylation-related genes and inducing hypermethylation of viral DNA

    Science.gov (United States)

    Torchetti, Enza Maria; Pegoraro, Mattia; Navarro, Beatriz; Catoni, Marco; Di Serio, Francesco; Noris, Emanuela

    2016-01-01

    DNA methylation and post-transcriptional gene silencing play critical roles in controlling infection of single-stranded (ss) DNA geminiviruses and ssRNA viroids, respectively, but both pathogens can counteract these host defense mechanisms and promote their infectivity. Moreover, a specific role of DNA methylation in viroid-host interactions is not yet confirmed. Here, using an experimental system where two nuclear-replicating agents, the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV) and potato spindle tuber viroid (PSTVd), co-infect their common host tomato, we observed that PSTVd severely interferes with TYLCSV infectivity and accumulation, most likely as a consequence of strong activation of host DNA methylation pathways. In fact, PSTVd alone or in co-infection with TYLCSV significantly upregulates the expression of key genes governing DNA methylation in plants. Using methylation-sensitive restriction and bisulfite conversion assays, we further showed that PSTVd infection promotes a strong hypermethylation of TYLCSV DNA, thus supporting a mechanistic link with the antagonism of the viroid on the virus in co-infected tomato plants. These results describe the interaction between two nuclear-replicating pathogens and show that they differentially interfere with DNA methylation pathways. PMID:27739453

  10. Innate immune response to viral infection.

    Science.gov (United States)

    Koyama, Shohei; Ishii, Ken J; Coban, Cevayir; Akira, Shizuo

    2008-09-01

    In viral infections the host innate immune system is meant to act as a first line defense to prevent viral invasion or replication before more specific protection by the adaptive immune system is generated. In the innate immune response, pattern recognition receptors (PRRs) are engaged to detect specific viral components such as viral RNA or DNA or viral intermediate products and to induce type I interferons (IFNs) and other pro-inflammatory cytokines in the infected cells and other immune cells. Recently these innate immune receptors and their unique downstream pathways have been identified. Here, we summarize their roles in the innate immune response to virus infection, discrimination between self and viral nucleic acids and inhibition by virulent factors and provide some recent advances in the coordination between innate and adaptive immune activation.

  11. High Viral Loads of Epstein-Barr Virus DNA in Peripheral Blood of Patients with Chronic Lymphocytic Leukemia Associated with Unfavorable Prognosis.

    Directory of Open Access Journals (Sweden)

    Ewelina Grywalska

    Full Text Available Epstein-Barr virus (EBV is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. The potential involvement of EBV in the clinical course of chronic lymphocytic leukemia (CLL remains unexplained. The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs of CLL patients may influence heterogeneity in the course of the disease. The study included peripheral blood samples from 115 previously untreated patients with CLL (54 women and 61 men and 40 healthy controls (16 women and 24 men. We analyzed the association between the EBV-DNA load in PBMCs and the stage of the disease, adverse prognostic factors, and clinical outcome. Detectable numbers of EBV-DNA copies in PBMCs were found in 62 out of 115 CLL patients (53.91%. The EBV-DNA copy number/μg DNA was significantly higher in patients who required early implementation of treatment, presented with lymphocyte count doubling time <12 months, displayed CD38-positive or ZAP-70-positive phenotype, and with the del(11q22.3 cytogenetic abnormality. Furthermore, the EBV-DNA copy number/μg DNA showed significant positive correlation with the concentrations of lactate dehydrogenase (LDH and beta-2-microglobulin. We have shown that in CLL patients, higher EBV-DNA copy number predicted shorter survival and shorter time to disease progression, and it was associated with other established unfavorable prognostic factors. This suggests that EBV may negatively affect the outcome of CLL.

  12. Identification of a provirally activated c-Ha-ras oncogene in an avian nephroblastoma via a novel procedure: cDNA cloning of a chimaeric viral-host transcript.

    Science.gov (United States)

    Westaway, D; Papkoff, J; Moscovici, C; Varmus, H E

    1986-01-01

    Retrovirus without oncogenes often exert their neoplastic potential as insertional mutagens of cellular proto-oncogenes. This may be associated with the production of chimaeric viral-host transcripts; in these cases; activated cellular genes can be identified by obtaining cDNA clones of bipartite RNAs. This approach was used in the analysis of chicken nephroblastomas induced by myeloblastosis-associated virus (MAV). One tumor contained a novel mRNA species initiated within a MAV LTR. cDNA cloning revealed that this mRNA encodes a protein of 189 amino acids, identical to that of normal human Ha-ras-1 at 185 positions, including positions implicated in oncogenic activation of ras proto-oncogenes; there are no differences between the coding sequences of presumably normal Ha-ras cDNA clones from chicken lymphoma RNA and the tumor-derived cDNAs. The chimaeric mRNA in the nephroblastoma is at least 25-fold more abundant than c-Ha-ras mRNA in normal kidney tissue, and a 21-kd ras-related protein is present in relatively large amounts in the tumor. We conclude that a quantitative change in c-Ha-ras gene expression results from an upstream insertion mutation and presumably contributes to tumorigenesis in this single case. Little or no increase in c-Ha-ras RNA or protein was observed in other nephroblastomas. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 10. PMID:3011401

  13. DNA

    Science.gov (United States)

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  14. A comparison of the effectiveness of cationic polymers poly-L-lysine (PLL) and polyethylenimine (PEI) for non-viral delivery of plasmid DNA to bone marrow stromal cells (BMSC).

    Science.gov (United States)

    Farrell, Laura-Lee; Pepin, Joel; Kucharski, Cezary; Lin, Xiaoyue; Xu, Zhenghe; Uludag, Hasan

    2007-03-01

    Bone marrow stromal cells (BMSC) represent an important cell phenotype for pursuit of successful gene therapy. Non-viral methods to enable expression of exogenous genes in BMSC will accelerate clinical application of gene therapy, without the concerns associated with the viral means of gene transfer. Towards this end, this study investigated the potential of cationic polymers poly-L-lysine (PLL) and branched polyethylenimine (PEI) as gene carriers for modification of BMSC. Both polymers rapidly (approximately 30 min) condensed a 4.2 kb Enhanced Green Fluorescent Protein (pEGFP-N2) plasmid into 100-200 nm particles. PLL and PEI were both readily internalized with BMSC with >80% of BMSC exhibiting polymer uptake by flow cytometric analysis. The relative uptake of PEI, however, was significantly higher as compared to the PLL. The majority of the BMSC (>60%) exhibited nuclear presence of the polymers as analyzed by fluorescent microscopy. Although both polymers were able to deliver the pEGFP-N2 into the cells under microscopic evaluation, only a small fraction of the cells (PLL and PEI readily displayed cellular uptake, but PEI was more effective in delivering plasmid DNA intracellularly, which was likely the underlying basis for a more sustained gene expression.

  15. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Koyama

    2015-07-01

    Full Text Available We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”, is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

  16. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine.

    Science.gov (United States)

    Koyama, Yoshiyuki; Sugiura, Kikuya; Yoshihara, Chieko; Inaba, Toshio; Ito, Tomoko

    2015-07-23

    We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine "Max" (PEI "Max"), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI "Max"/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI "Max"/polyanion small ternary complexes with high transfection efficiency. DNA/PEI "Max"/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

  17. Analysis of Epstein-Barr viral DNA load, EBV-LMP2 specific cytotoxic T-lymphocytes and levels of CD4+CD25+T cells in patients with nasopharyngeal carcinomas positive for IgA antibody to EBV viral capsid antigen

    Institute of Scientific and Technical Information of China (English)

    MO Wu-ning; TANG An-zhou; ZHOU Ling; HUANG Guang-wu; WANG Zhan; ZENG Yi

    2009-01-01

    Background Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignant diseases including nasopharyngeal carcinoma (NPC), which is a common cancer in Southeastem Asia. Previous studies showed that plasma levels of EBV-DNA might be a sensitive and reliable biomarker for the diagnosis, staging and evaluating of therapy for NPC. There are a few analyses of the levels of EBV-latent membrane protein 2 (LMP2)-specific cytotoxic T-lymphocytes (CTLs) in patients with NPC. This study was conducted to investigate the levels of EBV-LMP2-specific CTLs, EBV-DNA load and the level of CD4+CD25+T cells in such patients.Methods From February 2006 to April 2006, 62 patients with NPC, 40 healthy virus carders positive for EBV viral capsid antigen (EBV-IgA-VCA) and 40 controls were enrolled in the study. We used a highly sensitive ELISPOT assay,real-time polymerase chain reaction (PCR) and flow cytometry to measure the EBV-LMP2-specific CTL response, the EBV DNA load and the level of CD4+CD25+T cells, respectively.Results The EBV-LMP2-specific CTL responses of the samples from the control, healthy virus carders and patients with NPC were significantly different from the LMP2 epitopes, with the control and healthy virus carder samples displaying a stronger response in three cases. There were significant differences in EBV DNA load in serum between NPC and the healthy groups; patients with NPC at stages Ⅲ or Ⅳ had significantly higher viral loads compared with those at stages Ⅰ or Ⅱ. A significantly higher percentage of CD4+CD25+ T lymphocytes were detected in the patients, compared with healthy virus carriers and healthy controls. Moreover, patients with advanced stages of NPC (Ⅲ and Ⅲ) had significantly higher percentages than the patients with early stages (Ⅰ and Ⅱ).Conclusions Patients with NPC are frequently unable to establish or maintain sufficient immunosurveillance to control proliferating B cells harboring EBV and to destroy the tumor

  18. Sequence-specific cleavage of BM2 gene transcript of influenza B virus by 10-23 catalytic motif containing DNA enzymes significantly inhibits viral RNA translation and replication.

    Science.gov (United States)

    Kumar, Binod; Kumar, Prashant; Rajput, Roopali; Saxena, Latika; Daga, Mradul K; Khanna, Madhu

    2013-10-01

    One of the hallmarks of progression of influenza virus replication is the step involving the virus uncoating that occurs in the host cytoplasm. The BM2 ion channel protein of influenza B virus is highly conserved and is essentially required during the uncoating processes of virus, thus an attractive target for designing antiviral drugs. We screened several DNA enzymes (Dzs) containing the 10-23 catalytic motif against the influenza B virus BM2 RNA. Dzs directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. The Dz209 not only showed significant intracellular reduction of BM2 gene expression in transient-expression system but also provided considerable protection against influenza B virus challenge in MDCK cells. Our findings suggest that the Dz molecule can be used as selective and effective inhibitor of viral RNA replication, and can be explored further for development of a potent therapeutic agent against influenza B virus infection.

  19. Non-viral gene delivery carrier of probe type host molecule --Interactions between DNA and β-cyclodextrin derivative complexes (Ⅰ)

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A host type non-virus gene delivery carrier, phenanthroline-β-cyclodextrin derivative host molecule, was produced which can be used as molecular probe. Interactions between DZY-1 and DNA were investigated by electrophoresis assay. Hind III enzyme inhibition assay was carried out using DNA condensates induced by host molecules or host- guest molecule complexes to explore their ability to inhibit enzyme digestion. Micro-structure of DNA condensates induced by host molecules and host-guest molecule complexes was observed by scanning electron microscope (SEM). Our work indicates the delivery mechanism of DZY-1 used as a gene delivery carrier and also provides a method to design and produce non-virus gene delivery carriers.

  20. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    Science.gov (United States)

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  1. Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41.

    Science.gov (United States)

    Lazarte, Jassy Mary S; Kim, Young Rim; Lee, Jung Seok; Im, Se Pyeong; Kim, Si Won; Jung, Jae Wook; Kim, Jaesung; Lee, Woo Jai; Jung, Tae Sung

    2017-03-01

    The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been thoroughly studied in recent years. Glycoprotein (G)-based DNA vaccines had been proven to be effective in combating infection against Rhabdovirus (especially infectious hematopoietic necrosis virus, IHNV) in salmonids. DDX41 is a helicase known to induce antiviral and inflammatory responses by inducing a type I IFN innate immune response. To gain more information regarding G-based DNA vaccines in olive flounder (Paralicthys olivaceus), we tried to develop a more efficient G-based DNA vaccine by adding a molecular adjuvant, DDX41. We designed a DNA vaccine in which the VHSV glycoprotein (G-protein) and DDX41 were driven by the EF-1α and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 μg of plasmids encoding the G-based DNA vaccine alone (pEF-G), the molecular adjuvant alone (pEF-D), or the vaccine-adjuvant construct (pEF-GD). At two different time points, 15 and 30 days later, the fish were intraperitoneally infected with VHSV (100 μL; 1 × 10(6) TCID50/mL). Our assays revealed that the plasmid constructs showed up-regulated expression of IFN-1 and its associated genes at day 3 post-vaccination in both kidney and spleen samples. Specifically, pEF-GD showed statistically higher expression of immune response genes than pEF-G and pEF-D treated group (p fish group treated with pEF-GD showed higher survival rate than the pEF-G treated group, though difference was not statistically significant in the 15 dpv challenged group however in the 30 dpv challenged group, the difference was statistically significant (p < 0.05). Together, these results clearly demonstrate that DDX41 is an effective adjuvant for the G-based DNA vaccine in olive flounder. Our novel findings could facilitate the development of more effective DNA vaccines for the aquaculture industry.

  2. Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: I. binding to DNA AND to hexon of the precursor to protein VI, pVI, of human adenovirus.

    Science.gov (United States)

    Graziano, Vito; McGrath, William J; Suomalainen, Maarit; Greber, Urs F; Freimuth, Paul; Blainey, Paul C; Luo, Guobin; Xie, X Sunney; Mangel, Walter F

    2013-01-18

    The precursor to adenovirus protein VI, pVI, is a multifunctional protein with different roles early and late in virus infection. Here, we focus on two roles late in infection, binding of pVI to DNA and to the major capsid protein hexon. pVI bound to DNA as a monomer independent of DNA sequence with an apparent equilibrium dissociation constant, K(d)((app)), of 46 nm. Bound to double-stranded DNA, one molecule of pVI occluded 8 bp. Upon the binding of pVI to DNA, three sodium ions were displaced from the DNA. A ΔG(0)(0) of -4.54 kcal/mol for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy resulted from nonspecific interactions between pVI and DNA. The proteolytically processed, mature form of pVI, protein VI, also bound to DNA; its K(d)((app)) was much higher, 307 nm. The binding assays were performed in 1 mm MgCl(2) because in the absence of magnesium, the binding to pVI or protein VI to DNA was too tight to determine a K(d)((app)). Three molecules of pVI bound to one molecule of the hexon trimer with an equilibrium dissociation constant K(d)((app)) of 1.1 nm.

  3. DNA病毒准种研究的临床意义%Clinical significance of the study on DNA viral quasispecies

    Institute of Scientific and Technical Information of China (English)

    章莉; 季育华; 胡炯

    2010-01-01

    @@ 病毒准种(viral quasispecies),是指一群在"突变-选择压力"环境中相互竞争且核酸序列上高度相似但不完全相同的病毒群体(异质性2%~5%)."准种"的概念是1971年Eigen在阐述超循环理论中首次提出,主要是用于描述微观整体分子水平上进化规律的模型.随后,准种理论被应用于病毒学研究,并受到了广泛的关注.

  4. HC-Pro viral suppressor from tobacco vein banding mosaic virus interferes with DNA methylation and activates the salicylic acid pathway.

    Science.gov (United States)

    Yang, Liping; Xu, Yanan; Liu, Yuqing; Meng, Dawei; Jin, Taicheng; Zhou, Xiaofu

    2016-10-01

    Salicylic acid (SA) is an important signalling molecule that is synthesized by plants and induces the expression of resistance genes. The SA pathway is typically activated by DNA viruses as well as RNA viruses. Here, we demonstrated that heper-component protease (HC-Pro) encoded by tobacco vein banding mosaic virus (TVBMV) decreases in DNA methylation at the promoters of the regulators ACD6 and NPR1 in the SA pathway. We found that the overexpression of HC-Pro increases the expression of components in the SA pathway in plants. The results revealed that HC-Pro interferes in DNA methylation and activates the SA pathway in the HC-Pro transgenic plants and TVBMV-infected plants. We further found that the accumulation of siRNAs derived from the promoter repeats of ACD6 and NPR1 is greatly reduced in the HC-Pro plants. Our results suggested that HC-Pro-mediated interference with DNA methylation is likely caused by a reduction in accumulation of siRNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Sequencing Needs for Viral Diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, S N; Lam, M; Mulakken, N J; Torres, C L; Smith, J R; Slezak, T

    2004-01-26

    We built a system to guide decisions regarding the amount of genomic sequencing required to develop diagnostic DNA signatures, which are short sequences that are sufficient to uniquely identify a viral species. We used our existing DNA diagnostic signature prediction pipeline, which selects regions of a target species genome that are conserved among strains of the target (for reliability, to prevent false negatives) and unique relative to other species (for specificity, to avoid false positives). We performed simulations, based on existing sequence data, to assess the number of genome sequences of a target species and of close phylogenetic relatives (''near neighbors'') that are required to predict diagnostic signature regions that are conserved among strains of the target species and unique relative to other bacterial and viral species. For DNA viruses such as variola (smallpox), three target genomes provide sufficient guidance for selecting species-wide signatures. Three near neighbor genomes are critical for species specificity. In contrast, most RNA viruses require four target genomes and no near neighbor genomes, since lack of conservation among strains is more limiting than uniqueness. SARS and Ebola Zaire are exceptional, as additional target genomes currently do not improve predictions, but near neighbor sequences are urgently needed. Our results also indicate that double stranded DNA viruses are more conserved among strains than are RNA viruses, since in most cases there was at least one conserved signature candidate for the DNA viruses and zero conserved signature candidates for the RNA viruses.

  6. Population Dynamics of Viral Inactivation

    Science.gov (United States)

    Freeman, Krista; Li, Dong; Behrens, Manja; Streletzky, Kiril; Olsson, Ulf; Evilevitch, Alex

    We have investigated the population dynamics of viral inactivation in vitrousing time-resolved cryo electron microscopy combined with light and X-ray scattering techniques. Using bacteriophage λ as a model system for pressurized double-stranded DNA viruses, we found that virions incubated with their cell receptor eject their genome in a stochastic triggering process. The triggering of DNA ejection occurs in a non synchronized manner after the receptor addition, resulting in an exponential decay of the number of genome-filled viruses with time. We have explored the characteristic time constant of this triggering process at different temperatures, salt conditions, and packaged genome lengths. Furthermore, using the temperature dependence we determined an activation energy for DNA ejections. The dependences of the time constant and activation energy on internal DNA pressure, affected by salt conditions and encapsidated genome length, suggest that the triggering process is directly dependent on the conformational state of the encapsidated DNA. The results of this work provide insight into how the in vivo kinetics of the spread of viral infection are influenced by intra- and extra cellular environmental conditions. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1252522.

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

    Science.gov (United States)

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

    2015-03-01

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

  8. Testing the ability of viral haemorrhagic septicaemia virus to evade the protective immune response induced in rainbow trout by DNA vaccination

    DEFF Research Database (Denmark)

    Sepulveda, Dagoberto; Lorenzen, Niels

    2013-01-01

    glycoprotein, the only surface protein of the VHSV, has been successful as an experimental prophylactic treatment against this disease, because it induces a strong innate (interferon) and adaptive (cellular and humoral) immune response. However, since RNA viruses are known to possess high variability......, this work aims to evaluate whether VHSV is able to evade the protective immune response induced by the DNA vaccination. Earlier studies have demonstrated that VHSV can evade the neutralizing effect of monoclonal antibodies by mutations in the glycoprotein gene. One approach of the present study is therefore...... to try to isolate VHSV variants which can escape the neutralizing activity of serum from fish immunized with the DNA vaccine. To do so, a highly pathogenic VHSV isolate (DK3592B) will be repeatedly passaged in fish cell cultures in the presence of neutralizing fish serum. Another approach comprises...

  9. Integration host factor assembly at the cohesive end site of the bacteriophage lambda genome: implications for viral DNA packaging and bacterial gene regulation.

    Science.gov (United States)

    Sanyal, Saurarshi J; Yang, Teng-Chieh; Catalano, Carlos Enrique

    2014-12-09

    Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF-cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

  10. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation

    OpenAIRE

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-01-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21–22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, di...

  11. Human T-Cell Leukemia Virus Type I-Mediated Repression of PDZ-LIM Domain-Containing Protein 2 Involves DNA Methylation But Independent of the Viral Oncoprotein Tax

    Directory of Open Access Journals (Sweden)

    Pengrong Yan

    2009-10-01

    Full Text Available Human T-cell leukemia virus type I (HTLV-I is the etiological agent of adult T-cell leukemia (ATL. Our recent studies have shown that one important mechanism of HTLV-I-Mediated tumorigenesis is through PDZ-LIM domain-containing protein 2 (PDLIM2 repression, although the involved mechanism remains unknown. Here, we further report that HTLV-I-Mediated PDLIM2 repression was a pathophysiological event and the PDLIM2 repression involved DNA methylation. Whereas DNA methyltransferases 1 and 3b but not 3a were upregulated in HTLV-I-transformed T cells, the hypomethylating agent 5-aza-2′-deoxycytidine (5-aza-dC restored PDLIM2 expression and induced death of these malignant cells. Notably, the PDLIM2 repression was independent of the viral regulatory protein Tax because neither short-term induction nor long-term stable expression of Tax could downregulate PDLIM2 expression. These studies provide important insights into PDLIM2 regulation, HTLV-I leukemogenicity, long latency, and cancer health disparities. Given the efficient antitumor activity with no obvious toxicity of 5-aza-dC, these studies also suggest potential therapeutic strategies for ATL.

  12. Use of the modified viral satellite DNA vector to silence mineral nutrition-related genes in plants: silencing of the tomato ferric chelate reductase gene, FRO1, as an example

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Virus-induced gene silencing (VIGS) is potentially an attractive reverse-genetics tool for studies of plant gene function, but whether it is effective in silencing mineral nutritional-related genes in roots has not been demonstrated. Here we report on an efficient VIGS system that functions in tomato roots using a modified viral satellite DNA (DNAmβ) associated with Tomato yellow leaf curl China virus (TYLCCNV). A cDNA fragment of the ferric chelate reductase gene (FRO1) from tomato was inserted into the DNAmβ vector. Tomato roots agro-inoculated with DNAmβ carrying both a fragment of FRO1 and TYLCCNV used as a helper virus exhibited a significant reduction at the FRO1 mRNA level. As a consequence, ferric chelate reductase activity, as determined by visualization of the pink FeBPDS3 complex was significantly decreased. Our results clearly demonstrated that VIGS system can be employed to investigate gene function associated with plant nutrient uptake in roots.

  13. Use of the modified viral satellite DNA vector to silence mineral nutrition-related genes in plants: silencing of the tomato ferric chelate reductase gene, FRO1, as an example

    Institute of Scientific and Technical Information of China (English)

    HE XiuXia; JIN ChongWei; LI GuiXin; YOU GuangYi; ZHOU XuePing; ZHENG ShaoJian

    2008-01-01

    Virus-induced gene silencing (VIGS) is potentially an attractive reverse-genetics tool for studies of plant gene function, but whether it is effective in silencing mineral nutritional-related genes in roots has not been demonstrated. Here we report on an efficient VIGS system that functions in tomato roots using a modified viral satellite DNA (DNAmβ) associated with Tomato yellow leaf curl China virus (TYLCCNV). A cDNA fragment of the ferric chelate reductase gene (FRO1) from tomato was inserted into the DNAmβ vector. Tomato roots agro-inoculated with DNAmβ carrying both a fragment of FRO1 and TYLCCNV used as a helper virus exhibited a significant reduction at the FRO1 mRNA level. As a consequence, ferric chelate reductase activity, as determined by visualization of the pink FeBPDS3complex was significantly decreased. Our results clearly demonstrated that VIGS system can be employed to investigate gene function associated with plant nutrient uptake in roots.

  14. DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size, dose, route of injection and duration of protection-early protection correlates with Mx expression

    DEFF Research Database (Denmark)

    McLauchlan, P.E.; Collet, B.; Ingerslev, Esben;

    2003-01-01

    , or with inactivated VHS virus. Fish were challenged at different times post-vaccination (p.v.) to assess protection. At certain times p.v., serum samples were analysed for neutralising antibody and liver tissue was analysed for Mx mRNA expression.A DNA dose of 0.5 mug injected by the i.m. route induced protection...... in fish of all sizes in challenges performed either I or 4 weeks p.v. This dose also conferred effective protection up to 9 months p.v. in fish >100 g. With lower doses of DNA (0.1 and 0.01 mug) and challenge at 4 weeks p.v., 10 g fish were partially protected but protection was not observed in 100 g fish....... Vaccination by the i.p. route induced no or lower levels of protection compared with the i.m. route.Fish vaccinated with 0.5 mug DNA i.m. had no detectable serum neutralising antibody (NAb) at 4 weeks p.v. (with the exception of a single 10 g fish) but antibody was detected at 8 weeks and 6 months p.v...

  15. Small molecule inhibition of Epstein-Barr virus nuclear antigen-1 DNA binding activity interferes with replication and persistence of the viral genome.

    Science.gov (United States)

    Lee, Eun Kyung; Kim, Sun Young; Noh, Ka-Won; Joo, Eun Hye; Zhao, Bo; Kieff, Elliott; Kang, Myung-Soo

    2014-04-01

    The replication and persistence of extra chromosomal Epstein-Barr virus (EBV) episome in latently infected cells are primarily dependent on the binding of EBV-encoded nuclear antigen 1 (EBNA1) to the cognate EBV oriP element. In continuation of the previous study, herein we characterized EBNA1 small molecule inhibitors (H20, H31) and their underlying inhibitory mechanisms. In silico docking analyses predicted that H20 fits into a pocket in the EBNA1 DNA binding domain (DBD). However, H20 did not significantly affect EBNA1 binding to its cognate sequence. A limited structure-relationship study of H20 identified a hydrophobic compound H31, as an EBNA1 inhibitor. An in vitro EBNA1 EMSA and in vivo EGFP-EBNA1 confocal microscopy analysis showed that H31 inhibited EBNA1-dependent oriP sequence-specific DNA binding activity, but not sequence-nonspecific chromosomal association. Consistent with this, H31 repressed the EBNA1-dependent transcription, replication, and persistence of an EBV oriP plasmid. Furthermore, H31 induced progressive loss of EBV episome. In addition, H31 selectively retarded the growth of EBV-infected LCL or Burkitt's lymphoma cells. These data indicate that H31 inhibition of EBNA1-dependent DNA binding decreases transcription from and persistence of EBV episome in EBV-infected cells. These new compounds might be useful probes for dissecting EBNA1 functions in vitro and in vivo.

  16. Detection of Porcine Circovirus Type 2 and Viral Replication by In Situ Hybridization in Primary Lymphoid Organs From Naturally and Experimentally Infected Pigs

    DEFF Research Database (Denmark)

    Hansen, Mette Sif; Segalés, J.; Fernandes, L.;

    2013-01-01

    affected by PMWS (n = 33), and age-matched healthy control animals (n = 29). In situ hybridization (ISH) techniques were used to detect PCV2 nucleic acid irrespective of replicative status (complementary probe, CP) or to detect only the replicative form of the virus (replicative form probe, RFP). PCV2...... was not detected in the experimentally PCV2-inoculated pigs or the control animals. Among the PMWS-affected pigs, 19 of 20 (95%) thymuses were positive for PCV2 by CP ISH, and 7 of 19 (37%) of these also supported viral replication. By CP ISH, PCV2 was detected in 16 of 33 (48%) bone marrow samples, and 5 of 16...... of PMWS. The aim of this study was to determine if primary lymphoid organ cells support viral replication during PCV2 infection. This was done by histopathological examination of thymus and bone marrow from pigs experimentally inoculated with PCV2 (n = 24), mock-infected pigs (n = 12), pigs naturally...

  17. Rapid and highly fieldable viral diagnostic

    Science.gov (United States)

    McKnight, Timothy E.

    2016-12-20

    The present invention relates to a rapid, highly fieldable, nearly reagentless diagnostic to identify active RNA viral replication in a live, infected cells, and more particularly in leukocytes and tissue samples (including biopsies and nasal swabs) using an array of a plurality of vertically-aligned nanostructures that impale the cells and introduce a DNA reporter construct that is expressed and amplified in the presence of active viral replication.

  18. Rapid and highly fieldable viral diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    McKnight, Timothy E.

    2016-12-20

    The present invention relates to a rapid, highly fieldable, nearly reagentless diagnostic to identify active RNA viral replication in a live, infected cells, and more particularly in leukocytes and tissue samples (including biopsies and nasal swabs) using an array of a plurality of vertically-aligned nanostructures that impale the cells and introduce a DNA reporter construct that is expressed and amplified in the presence of active viral replication.

  19. Sulfated small molecules targeting eBV in Burkitt lymphoma: from in silico screening to the evidence of in vitro effect on viral episomal DNA.

    Science.gov (United States)

    Lima, Raquel T; Seca, Hugo; Palmeira, Andreia; Fernandes, Miguel X; Castro, Felipe; Correia-da-Silva, Marta; Nascimento, Maria S J; Sousa, Emília; Pinto, Madalena; Vasconcelos, M Helena

    2013-05-01

    Epstein-Barr virus (EBV) infects more than 90% of the world population. Following primary infection, Epstein-Barr virus persists in an asymptomatic latent state. Occasionally, it may switch to lytic infection. Latent EBV infection has been associated with several diseases, such as Burkitt lymphoma (BL). To date, there are no available drugs to target latent EBV, and the existing broad-spectrum antiviral drugs are mainly active against lytic viral infection. Thus, using computational molecular docking, a virtual screen of a library of small molecules, including xanthones and flavonoids (described with potential for antiviral activity against EBV), was carried out targeting EBV proteins. The more interesting molecules were selected for further computational analysis, and subsequently, the compounds were tested in the Raji (BL) cell line, to evaluate their activity against latent EBV. This work identified three novel sulfated small molecules capable of decreasing EBV levels in a BL. Therefore, the in silico screening presents a good approach for the development of new anti-EBV agents.

  20. Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens.

    LENUS (Irish Health Repository)

    Keegan, Helen

    2012-02-01

    Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6\\/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205\\/299) of the cases were positive by hc2 and 38% (112\\/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.

  1. Perioperative entecavir for patients with HBV-related hepatocellular carcinoma and low levels of viral DNA: analysis using propensity score matching.

    Science.gov (United States)

    Yuan, Bao-Hong; Li, Ru-Hong; Yuan, Wei-Ping; Xiang, Bang-De; Zheng, Ming-Hua; Yang, Tian; Zhong, Jian-Hong; Li, Le-Qun

    2017-08-01

    The safety and efficacy of perioperative antiviral therapy for patients with hepatitis B virus related hepatocellular carcinoma and low serum levels of hepatitis B virus DNA are unknown. This retrospective study compared serum levels of hepatitis B virus DNA, liver function, morbidity, and length of hospital stay between patients who underwent hepatic resection alone and patients who received entecavir therapy before and after resection (n = 44 in each group). Propensity score matching was used to reduce confounding due to baseline differences between the groups. Hepatitis B virus reactivation during follow-up, which lasted a median of 6.1 months, occurred in one patient in the entecavir group (2.3%) and 11 patients in the resection-only group (25%; P = 0.02). Liver function, especially alanine aminotransferase levels, recovered much faster in the entecavir group. This group also showed a slightly lower rate of morbidity (P = 0.081) as well as significantly shorter overall hospital stay (20.1 ± 4.9 vs 24.9 ± 13.2 days; P = 0.028) and postoperative hospital stay (11.4 ± 1.9 vs 16.8 ± 13.1 days; P = 0.008). These results from this pilot study suggest that patients with hepatitis B virus related hepatocellular carcinoma and low levels of hepatitis B virus DNA are at risk of hepatitis B virus reactivation following resection, and that perioperative entecavir therapy can safely and effectively reduce this risk. Such therapy also appears to improve liver function and shorten hospitalization.

  2. Physical state and expression of HPV DNA in benign and dysplastic cervical tissue: different levels of viral integration are correlated with lesion grade.

    Science.gov (United States)

    Hudelist, Gernot; Manavi, Mahmood; Pischinger, Kerstin I D; Watkins-Riedel, Thomas; Singer, Christian F; Kubista, E; Czerwenka, Klaus F

    2004-03-01

    Human papillomavirus (HPV) infection is the most important event in the malignant transformation of human cervical epithelium. Several high-risk (HR-)HPV subtypes have been identified, which lead to CIN and subsequently to invasive carcinoma. The reason for this phenomenon is still unknown, but it seems to be related to the physical state of HPV DNA. Digene HC II test was used to identify HR- and/or low-risk (LR-)HPV infections in cervical swabs of 275 women attending our clinic for routine cytological screening and/or colposcopy because of an abnormal Pap smear comprising low-grade squamous intraepithelial lesions (LGSIL) and high-grade SIL (HGSIL). Specific HR (16, 18, 31, 33, 52b, 58) and LR (6, 11) subtypes were characterized in cervical biopsies of 10 women with benign cellular changes and of 68 women with CIN I-III by the PCR-restriction enzyme method. The physical state of HPV DNA (episomal, mixed and integrated form) was analyzed by bi-dimensional (2D)-gel electrophoresis. In addition, mRNA expression of E6/E7 genes was analyzed by RT-PCR. Furthermore, the relative virus load was determined in nine selected cases. The physical state and transcriptional activity of HPV DNA were then correlated to histopathological results. LR-HPV infection [27 cases (9.8%)] and HR-HPV infection [121 cases (44%)] of cervical swabs were clearly correlated to the degree of SIL. Further HPV typing in cervical biopsies of 78 women showed that HPV6 and 11 were restricted to benign cellular changes, CIN I and II, whereas HPV16 and 18 were observed predominantly in CIN III/CIS (P=0.01). No clear distribution pattern was observed for HPV31, 33, 52b and 58. Expression of HPV E6 and E7 transcripts was uniformly correlated with the different physical state of HPV DNA. Analyzing the physical state of these HPV subtypes, HPV6 and 11 could only be detected as an episomal form, independent of SIL grade. In normal epithelium and in CIN I and II, HPV16 and 18 were exclusively found in the

  3. Construction of a viral vector based on DNA-A of Tomato rugose mosaic virus (ToRMV) for induction of gene silencing in host plants

    OpenAIRE

    Cardoso, Mariana Santos

    2009-01-01

    Os begomovírus pertencem à família Geminiviridae, que inclui vírus com genoma composto por uma ou duas moléculas de DNA circular de fita simples, encapsidado em partículas icosaédricas geminadas. Os begomovírus são transmitidos pela mosca branca Bemisia tabaci e causam doenças de importância econômica em diversas culturas, principalmente em regiões tropicais e subtropicais. No Brasil, há diversos relatos de begomovírus causando sérias perdas nas culturas do feijoeiro e tomateiro e relatos esp...

  4. Clonal selection for transcriptionally active viral oncogenes during progression to cancer.

    NARCIS (Netherlands)

    Tine, BA Van; Kappes, JC; Banerjee, NS; Knops, J; Lai, L; Steenbergen, R.D.M.; Meijer, C.J.L.M.; Snijders, P.J.F.; Chatis, P; Broker, TR; Moen, PTJr; Chow, L.T.

    2004-01-01

    Primary keratinocytes immortalized by human papillomaviruses (HPVs), along with HPV-induced cervical carcinoma cell lines, are excellent models for investigating neoplastic progression to cancer. By simultaneously visualizing viral DNA and nascent viral transcripts in interphase nuclei, we demonstra

  5. Spontaneous HBsAg loss in Korean patients: relevance of viral genotypes, S gene mutations, and covalently closed circular DNA copy numbers

    Science.gov (United States)

    Chang, Hye-Young; Park, Jun Yong; Park, Eun-Sook; Park, Yong Kwang; Han, Kwang-Hyub

    2014-01-01

    Background/Aims Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. Methods Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. Results Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. Conclusions Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect. PMID:25320728

  6. Viral Marketing Past Present Future

    OpenAIRE

    Nessipbekova, Zarina

    2010-01-01

    The work studies the viral marketing. These are past viral campaigns, viral campaigns today, and evaluates their actuality. The work tries to predict the development of viral marketing on the basis of the research done by the author.

  7. Generating viral metagenomes from the coral holobiont

    Directory of Open Access Journals (Sweden)

    Karen Dawn Weynberg

    2014-05-01

    Full Text Available Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

  8. Latent Herpes Viral Reactivation in Astronauts

    Science.gov (United States)

    Pierson, D. L.; Mehta, S. K.; Stowe, R.

    2008-01-01

    Latent viruses are ubiquitous and reactivate during stressful periods with and without symptoms. Latent herpes virus reactivation is used as a tool to predict changes in the immune status in astronauts and to evaluate associated health risks. Methods: Viral DNA was detected by real time polymerase chain reaction in saliva and urine from astronauts before, during and after short and long-duration space flights. Results and Discussion: EpsteinBarr virus (EBV), cytomegalovirus (CMV), and varicella zoster virus (VZV) reactivated, and viral DNA was shed in saliva (EBV and VZV) or urine (CMV). EBV levels in saliva during flight were 10fold higher than baseline levels. Elevations in EBV specific CD8+ T-cells, viral antibody titers, and specific cytokines were consistent with viral reactivation. Intracellular levels of cytokines were reduced in EBVspecific Tcells. CMV, rarely present in urine of healthy individuals, was shed in urine of 27% of astronauts during all phases of spaceflight. VZV, not found in saliva of asymptomatic individuals, was found in saliva of 50% of astronauts during spaceflight and 35 days after flight. VZV recovered from astronaut saliva was found to be live, infectious virus. DNA sequencing demonstrated that the VZV recovered from astronauts was from the common European strain of VZV. Elevation of stress hormones accompanied viral reactivation indicating involvement of the hypothalmic-pituitary-adrenal and sympathetic adrenal-medullary axes in the mechanism of viral reactivation in astronauts. A study of 53 shingles patients found that all shingles patients shed VZV DNA in their saliva and the VZV levels correlated with the severity of the disease. Lower VZV levels in shingles patients were similar to those observed in astronauts. We proposed a rapid, simple, and cost-effective assay to detect VZV in saliva of patients with suspected shingles. Early detection of VZV infection allows early medical intervention.

  9. Latent Herpes Viral Reactivation in Astronauts

    Science.gov (United States)

    Pierson, D. L.; Mehta, S. K.; Stowe, R.

    2008-01-01

    Latent viruses are ubiquitous and reactivate during stressful periods with and without symptoms. Latent herpes virus reactivation is used as a tool to predict changes in the immune status in astronauts and to evaluate associated health risks. Methods: Viral DNA was detected by real time polymerase chain reaction in saliva and urine from astronauts before, during and after short and long-duration space flights. Results and Discussion: EpsteinBarr virus (EBV), cytomegalovirus (CMV), and varicella zoster virus (VZV) reactivated, and viral DNA was shed in saliva (EBV and VZV) or urine (CMV). EBV levels in saliva during flight were 10fold higher than baseline levels. Elevations in EBV specific CD8+ T-cells, viral antibody titers, and specific cytokines were consistent with viral reactivation. Intracellular levels of cytokines were reduced in EBVspecific Tcells. CMV, rarely present in urine of healthy individuals, was shed in urine of 27% of astronauts during all phases of spaceflight. VZV, not found in saliva of asymptomatic individuals, was found in saliva of 50% of astronauts during spaceflight and 35 days after flight. VZV recovered from astronaut saliva was found to be live, infectious virus. DNA sequencing demonstrated that the VZV recovered from astronauts was from the common European strain of VZV. Elevation of stress hormones accompanied viral reactivation indicating involvement of the hypothalmic-pituitary-adrenal and sympathetic adrenal-medullary axes in the mechanism of viral reactivation in astronauts. A study of 53 shingles patients found that all shingles patients shed VZV DNA in their saliva and the VZV levels correlated with the severity of the disease. Lower VZV levels in shingles patients were similar to those observed in astronauts. We proposed a rapid, simple, and cost-effective assay to detect VZV in saliva of patients with suspected shingles. Early detection of VZV infection allows early medical intervention.

  10. Evaluation for quantitative methods of HBV DNA in serum with very high viral loads%高病毒载量血清HBV DNA定量方法的评价

    Institute of Scientific and Technical Information of China (English)

    李雷; 冯振华; 许碧云; 顾光煜; 张葵; 周乙华

    2012-01-01

    Objective To explore whether quantitative standard curve may be directly extended to determine HBV DNA level in the cases of very high viral loads which was over the upper limit of detection range. Methods A total of 30 serum samples were included. The HBV DNA levels of all the samples were more than 3×107 IU/ml detected by fluorescent quantification kit purchased from Shanghai Shenyou Company. Each samples was quantitatively retested for three times. Both the serum samples and the HBV DNA extracted from serum were further measured in 10-, 100- and 1 000-fold dilution, respectively. After logarithmic conversion the quantitative values were statistically analyzed. Results When the quantitative data of HBV DNA were expressed as logarithm values, the inter-class correlation coefficient was 0.356 (F = 2.66, P 0. 05 ), and the maximum differences were less than 1 in each sample. Conclusion In the determination of HBV DNA by using domestic reagents, direct extension of the quantitative standard curve may be acceptable even if the level of HBV DNA was over the upper limit of detection. For accurate quantification, it is suggested that the serum-extracted DNA with 100-fold dilution should be used as the test materials.%目的 探讨血清HBV DNA定量检测结果高于检测上限时,能否直接延伸标准曲线,用于HBV DNA定量分析.方法 以上海申友生物公司HBV DNA荧光定量检测试剂盒为例,选择HBV DNA>3×107 IU/mL的血清标本30例.每例血清在3个不同时间点提取DNA,或将血清以10、100和1000倍稀释后提取DNA,或将未稀释血清提取的DNA以10、100和1000倍稀释后进行HBV DNA定量检测,检测结果以10为底数进行对数转换后进行统计学分析.结果 经对数转换,3个不同时间点HBV DNA定量检测结果的组间相关系数为0.356(F=2.66,P<0.01);36.7% (11/30)的标本最大差值<0.5,60.0%(18/30)介于0.5~1.0,3.3% (1/30)达1.01;血清稀释后定量检测HBV DNA,96.7%(29/30)

  11. Viral Haemorrhagic Septicaemia Virus

    DEFF Research Database (Denmark)

    Olesen, Niels Jørgen; Skall, Helle Frank

    2013-01-01

    This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus.......This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus....

  12. [Emergent viral infections

    NARCIS (Netherlands)

    Galama, J.M.D.

    2001-01-01

    The emergence and re-emergence of viral infections is an ongoing process. Large-scale vaccination programmes led to the eradication or control of some viral infections in the last century, but new viruses are always emerging. Increased travel is leading to a rise in the importation of exotic infecti

  13. Viral Haemorrhagic Septicaemia Virus

    DEFF Research Database (Denmark)

    Olesen, Niels Jørgen; Skall, Helle Frank

    2013-01-01

    This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus.......This chapter covers the genetics (genotypes and serotypes), clinical signs, host species, transmission, prevalence, diagnosis, control and prevention of viral haemorrhagic septicaemia virus....

  14. Increased fecal viral content associated with obesity in mice

    Institute of Scientific and Technical Information of China (English)

    Hariom; Yadav; Shalini; Jain; Ravinder; Nagpal; Francesco; Marotta

    2016-01-01

    AIM: To investigate the presence of total gut viral content in obese mice, and establish correlation with obesity associated metabolic measures and gut microbiome.METHODS: Fresh fecal samples were collected from normal and obese(Leptin deficient Lepob/ob) mice. Total viral DNA and RNA was isolated and quantified for establishing the correlation with metabolic measures and composition of gut bacterial communities.RESULTS: In this report, we found that obese mice feces have higher viral contents in terms of total viral DNA and RNA(P 0.6), whilst negatively correlated with bacteroidetes and bifidobacteria. CONCLUSION: This study suggests the strong correlation of increased viral population into the gut of obese mice and opens new avenues to explore the role of gut virome in pathophysiology of obesity.

  15. Description of viral assemblages associated with the Gorgonia ventalina holobiont

    Science.gov (United States)

    Hewson, I.; Brown, J. M.; Burge, C. A.; Couch, C. S.; LaBarre, B. A.; Mouchka, M. E.; Naito, M.; Harvell, C. D.

    2012-06-01

    The diversity and function of viruses in coral holobionts has only recently received attention. The non-reef building gorgonian octocoral, Gorgonia ventalina, is a major constituent of Caribbean reefs. We investigated viral communities associated with G. ventalina tissues to understand their role in gorgonian ecology. Pyrosequencing was used to prepare a total of 514,632 sequence reads of DNA- and RNA-based mixed-community viral genomes (metaviromes). RNA viral assemblages were comprised of primarily unidentifiable reads, with most matching host transcripts and other RNA metaviromes. DNA metaviromes were similar between healthy and diseased tissues and comprised of contiguous sequences (contigs) that matched primarily metazoan and bacterial proteins. Only ~5% of contigs matched viral proteins that were primarily cyanophage and viruses of Chlorella and Ostreococcus. Our results confirm that DNA and RNA viruses comprise a component of the gorgonian holobiont, suggesting that they may play a role in the ecology of G. ventalina.

  16. APOBEC3 Interference during Replication of Viral Genomes

    Directory of Open Access Journals (Sweden)

    Luc Willems

    2015-06-01

    Full Text Available Co-evolution of viruses and their hosts has reached a fragile and dynamic equilibrium that allows viral persistence, replication and transmission. In response, infected hosts have developed strategies of defense that counteract the deleterious effects of viral infections. In particular, single-strand DNA editing by Apolipoprotein B Editing Catalytic subunits proteins 3 (APOBEC3s is a well-conserved mechanism of mammalian innate immunity that mutates and inactivates viral genomes. In this review, we describe the mechanisms of APOBEC3 editing during viral replication, the viral strategies that prevent APOBEC3 activity and the consequences of APOBEC3 modulation on viral fitness and host genome integrity. Understanding the mechanisms involved reveals new prospects for therapeutic intervention.

  17. Viral marketing on the Internet

    OpenAIRE

    Štverák, Martin

    2008-01-01

    Thesis provides an overview of viral marketing. It describes the process by which you can be inspired to implement viral campaign. The thesis includes analysis of specific viral Web project. The aim of this thesis is to create a breakdown of the various components of viral marketing, to establish conditions that should be satisfied for the viral marketing to success, suggesting how to use viral marketing on social network Facebook and evaluate the various components of this service for the pr...

  18. Hepatitis viral aguda

    OpenAIRE

    Héctor Rubén Hernández Garcés; René F Espinosa Álvarez

    1998-01-01

    Se realizó una revisión bibliográfica de las hepatitis virales agudas sobre aspectos vinculados a su etiología. Se tuvieron en cuenta además algunos datos epidemiológicos, las formas clínicas más importantes, los exámenes complementarios con especial énfasis en los marcadores virales y el diagnóstico positivoA bibliographical review of acute viral hepatitis was made taking into account those aspects connected with its etiology. Some epidemiological markers, the most important clinical forms, ...

  19. Understanding Image Virality

    Science.gov (United States)

    2015-06-08

    Example non-viral images. Figure 1: Top: Images with high viral scores in our dataset depict internet “celebrity” memes ex. “Grumpy Cat”; Bottom: Images...of images that is most similar to ours is the concurrently introduced viral meme generator of Wang et al., that combines NLP and Computer Vision (low...doing any of our tasks. The test included questions about widely spread Reddit memes and jargon so that anyone familiar with Reddit can easily get a high

  20. Viral markers in HIV infection and AIDS.

    Science.gov (United States)

    Cunningham, A L; Dwyer, D E; Dowton, D N

    1993-01-01

    Viral and immune markers are used for monitoring either progression of human immunodeficiency virus (HIV) disease or response to antiviral therapy. Ideal properties of viral markers are that they are present in all HIV-infected persons at all stages of disease, that they are related to disease pathogenesis, that they can be easily quantitated, that this quantitation correlates rapidly and predictably with both disease stage and response to antivirals, and that they can be developed into rapid, reproducible automated tests. Currently available viral markers include HIV p24 antigenemia (after acid glycine dissociation), anti-p24 antibody titres, quantitative DNA and RNA polymerase chain reaction performed on cells and plasma, and HIV isolate phenotype. In Australia, these markers have been studied in acute HIV seroconversion, in neonatal infection, in body fluids other than blood, and in monitoring of response to antiviral drug therapy.

  1. Viral capsids: Mechanical characteristics, genome packaging and delivery mechanisms

    NARCIS (Netherlands)

    Roos, W.H.; Ivanovska, I.L.; Evilevitch, A.; Wuite, G.J.L.

    2007-01-01

    The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its

  2. The fecal viral flora of wild rodents.

    Directory of Open Access Journals (Sweden)

    Tung G Phan

    2011-09-01

    Full Text Available The frequent interactions of rodents with humans make them a common source of zoonotic infections. To obtain an initial unbiased measure of the viral diversity in the enteric tract of wild rodents we sequenced partially purified, randomly amplified viral RNA and DNA in the feces of 105 wild rodents (mouse, vole, and rat collected in California and Virginia. We identified in decreasing frequency sequences related to the mammalian viruses families Circoviridae, Picobirnaviridae, Picornaviridae, Astroviridae, Parvoviridae, Papillomaviridae, Adenoviridae, and Coronaviridae. Seventeen small circular DNA genomes containing one or two replicase genes distantly related to the Circoviridae representing several potentially new viral families were characterized. In the Picornaviridae family two new candidate genera as well as a close genetic relative of the human pathogen Aichi virus were characterized. Fragments of the first mouse sapelovirus and picobirnaviruses were identified and the first murine astrovirus genome was characterized. A mouse papillomavirus genome and fragments of a novel adenovirus and adenovirus-associated virus were also sequenced. The next largest fraction of the rodent fecal virome was related to insect viruses of the Densoviridae, Iridoviridae, Polydnaviridae, Dicistroviriade, Bromoviridae, and Virgaviridae families followed by plant virus-related sequences in the Nanoviridae, Geminiviridae, Phycodnaviridae, Secoviridae, Partitiviridae, Tymoviridae, Alphaflexiviridae, and Tombusviridae families reflecting the largely insect and plant rodent diet. Phylogenetic analyses of full and partial viral genomes therefore revealed many previously unreported viral species, genera, and families. The close genetic similarities noted between some rodent and human viruses might reflect past zoonoses. This study increases our understanding of the viral diversity in wild rodents and highlights the large number of still uncharacterized viruses in

  3. Viral Gastroenteritis (Stomach Flu)

    Science.gov (United States)

    ... contaminated food or water, although person-to-person transmission also is possible. Rotavirus. Worldwide, this is the ... contaminated drinking water is a cause of viral diarrhea, in many cases the virus is passed through ...

  4. Hepatitis viral aguda

    Directory of Open Access Journals (Sweden)

    Héctor Rubén Hernández Garcés

    1998-10-01

    Full Text Available Se realizó una revisión bibliográfica de las hepatitis virales agudas sobre aspectos vinculados a su etiología. Se tuvieron en cuenta además algunos datos epidemiológicos, las formas clínicas más importantes, los exámenes complementarios con especial énfasis en los marcadores virales y el diagnóstico positivoA bibliographical review of acute viral hepatitis was made taking into account those aspects connected with its etiology. Some epidemiological markers, the most important clinical forms, and the complementary examinations with special emphasis on the viral markers and the positive diagnosis were also considered

  5. Viral quasispecies complexity measures.

    Science.gov (United States)

    Gregori, Josep; Perales, Celia; Rodriguez-Frias, Francisco; Esteban, Juan I; Quer, Josep; Domingo, Esteban

    2016-06-01

    Mutant spectrum dynamics (changes in the related mutants that compose viral populations) has a decisive impact on virus behavior. The several platforms of next generation sequencing (NGS) to study viral quasispecies offer a magnifying glass to study viral quasispecies complexity. Several parameters are available to quantify the complexity of mutant spectra, but they have limitations. Here we critically evaluate the information provided by several population diversity indices, and we propose the introduction of some new ones used in ecology. In particular we make a distinction between incidence, abundance and function measures of viral quasispecies composition. We suggest a multidimensional approach (complementary information contributed by adequately chosen indices), propose some guidelines, and illustrate the use of indices with a simple example. We apply the indices to three clinical samples of hepatitis C virus that display different population heterogeneity. Areas of virus biology in which population complexity plays a role are discussed.

  6. Fragmentation of bacteriophage S13 replicative from DNA by restriction endonucleases from Hemophilus influenzae and Hemophilus aegyptius.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); K.M. Ojamaa; J.H. Spencer

    1976-01-01

    textabstractThe restriction enzymes Hind from Hemophilus influenzae and HaeIII from Hemophilus aegyptius cleave bacteriophage S13 replicative form (RF) DNA into 13 and 10 specific fragments, respectively. The sizes of these fragments were estimated by gel electrophoresis, electron microscopy, and py

  7. Treatment of viral encephalitis.

    Science.gov (United States)

    Domingues, Renan Barros

    2009-03-01

    Several viruses may cause central nervous system diseases with a broad range of clinical manifestations. The time course of the viral encephalitis can be acute, subacute, or chronic. Pathologically there are encephalitis with direct viral entry into the CNS in which brain parenchyma exhibits neuronal damaging and viral antigens and there are postinfectious autoimmune encephalitis associated with systemic viral infections with brain tissue presenting perivascular aggregation of immune cells and myelin damaging. Some virus affect previously healthy individuals while others produce encephalitis among imunocompromised ones. Factors such evolving lifestyles and ecological changes have had a considerable impact on the epidemiology of some viral encephalitis [e.g. West-Nile virus, and Japanese B virus]. Citomegalovirus and JC virus are examples of infections of the brain that have been seen more frequently because they occur in immunocompromised patients. In the other hand many scientific achievements in neuroimaging, molecular diagnosis, antiviral therapy, immunomodulatory treatments, and neurointensive care have allowed more precise and earlier diagnoses and more efficient treatments, resulting in improved outcomes. In this article, we will present the current drug options in the management of the main acute and chronic viral infection of the central nervous system of immunocompetent and immunocompromised adults, focusing on drugs mechanisms of action, efficacy, and side effects. The early diagnosis and correct management of such diseases can reduce mortality and neurological sequelae; however, even with recent treatment advances, potentially devastating outcomes are still possible.

  8. Immigration and viral hepatitis.

    Science.gov (United States)

    Sharma, Suraj; Carballo, Manuel; Feld, Jordan J; Janssen, Harry L A

    2015-08-01

    WHO estimates reveal that the global prevalence of viral hepatitis may be as high as 500 million, with an annual mortality rate of up to 1.3 million individuals. The majority of this global burden of disease is borne by nations of the developing world with high rates of vertical and iatrogenic transmission of HBV and HCV, as well as poor access to healthcare. In 2013, 3.2% of the global population (231 million individuals) migrated into a new host nation. Migrants predominantly originate from the developing countries of the south, into the developed economies of North America and Western Europe. This mass migration of individuals from areas of high-prevalence of viral hepatitis poses a unique challenge to the healthcare systems of the host nations. Due to a lack of universal standards for screening, vaccination and treatment of viral hepatitis, the burden of chronic liver disease and hepatocellular carcinoma continues to increase among migrant populations globally. Efforts to increase case identification and treatment among migrants have largely been limited to small outreach programs in urban centers, such that the majority of migrants with viral hepatitis continue to remain unaware of their infection. This review summarizes the data on prevalence of viral hepatitis and burden of chronic liver disease among migrants, current standards for screening and treatment of immigrants and refugees, and efforts to improve the identification and treatment of viral hepatitis among migrants.

  9. Microvesicles and Viral Infection▿

    Science.gov (United States)

    Meckes, David G.; Raab-Traub, Nancy

    2011-01-01

    Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with enveloped viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. For the most part, these particles have not been analyzed for their content or functions during viral infection. However, early studies of microvesicles (L-particles) secreted from herpes simplex virus-infected cells provided the first evidence of microvesicle-mediated intercellular communication. In the case of Epstein-Barr virus, recent evidence suggests that this tumorigenic herpesvirus also utilizes exosomes as a mechanism of cell-to-cell communication through the transfer of signaling competent proteins and functional microRNAs to uninfected cells. This review focuses on aspects of the biology of microvesicles with an emphasis on their potential contributions to viral infection and pathogenesis. PMID:21976651

  10. Microvesicles and viral infection.

    Science.gov (United States)

    Meckes, David G; Raab-Traub, Nancy

    2011-12-01

    Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with enveloped viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. For the most part, these particles have not been analyzed for their content or functions during viral infection. However, early studies of microvesicles (L-particles) secreted from herpes simplex virus-infected cells provided the first evidence of microvesicle-mediated intercellular communication. In the case of Epstein-Barr virus, recent evidence suggests that this tumorigenic herpesvirus also utilizes exosomes as a mechanism of cell-to-cell communication through the transfer of signaling competent proteins and functional microRNAs to uninfected cells. This review focuses on aspects of the biology of microvesicles with an emphasis on their potential contributions to viral infection and pathogenesis.

  11. NCBI viral genomes resource.

    Science.gov (United States)

    Brister, J Rodney; Ako-Adjei, Danso; Bao, Yiming; Blinkova, Olga

    2015-01-01

    Recent technological innovations have ignited an explosion in virus genome sequencing that promises to fundamentally alter our understanding of viral biology and profoundly impact public health policy. Yet, any potential benefits from the billowing cloud of next generation sequence data hinge upon well implemented reference resources that facilitate the identification of sequences, aid in the assembly of sequence reads and provide reference annotation sources. The NCBI Viral Genomes Resource is a reference resource designed to bring order to this sequence shockwave and improve usability of viral sequence data. The resource can be accessed at http://www.ncbi.nlm.nih.gov/genome/viruses/ and catalogs all publicly available virus genome sequences and curates reference genome sequences. As the number of genome sequences has grown, so too have the difficulties in annotating and maintaining reference sequences. The rapid expansion of the viral sequence universe has forced a recalibration of the data model to better provide extant sequence representation and enhanced reference sequence products to serve the needs of the various viral communities. This, in turn, has placed increased emphasis on leveraging the knowledge of individual scientific communities to identify important viral sequences and develop well annotated reference virus genome sets.

  12. Polymerase Chain Reaction on a Viral Nanoparticle.

    Science.gov (United States)

    Carr-Smith, James; Pacheco-Gómez, Raúl; Little, Haydn A; Hicks, Matthew R; Sandhu, Sandeep; Steinke, Nadja; Smith, David J; Rodger, Alison; Goodchild, Sarah A; Lukaszewski, Roman A; Tucker, James H R; Dafforn, Timothy R

    2015-12-18

    The field of synthetic biology includes studies that aim to develop new materials and devices from biomolecules. In recent years, much work has been carried out using a range of biomolecular chassis including α-helical coiled coils, β-sheet amyloids and even viral particles. In this work, we show how hybrid bionanoparticles can be produced from a viral M13 bacteriophage scaffold through conjugation with DNA primers that can template a polymerase chain reaction (PCR). This unprecedented example of a PCR on a virus particle has been studied by flow aligned linear dichroism spectroscopy, which gives information on the structure of the product as well as a new protototype methodology for DNA detection. We propose that this demonstration of PCR on the surface of a bionanoparticle is a useful addition to ways in which hybrid assemblies may be constructed using synthetic biology.

  13. Viral genome sequencing by random priming methods

    Directory of Open Access Journals (Sweden)

    Zhang Xinsheng

    2008-01-01

    Full Text Available Abstract Background Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing. Results We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000–15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter. Conclusion The method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.

  14. Viral Ancestors of Antiviral Systems

    Directory of Open Access Journals (Sweden)

    Luis P. Villarreal

    2011-10-01

    Full Text Available All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the ‘Big Bang’ theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features.

  15. Viral ancestors of antiviral systems.

    Science.gov (United States)

    Villarreal, Luis P

    2011-10-01

    All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the 'Big Bang' theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features.

  16. Viral Ancestors of Antiviral Systems

    Science.gov (United States)

    Villarreal, Luis P.

    2011-01-01

    All life must survive their corresponding viruses. Thus antiviral systems are essential in all living organisms. Remnants of virus derived information are also found in all life forms but have historically been considered mostly as junk DNA. However, such virus derived information can strongly affect host susceptibility to viruses. In this review, I evaluate the role viruses have had in the origin and evolution of host antiviral systems. From Archaea through bacteria and from simple to complex eukaryotes I trace the viral components that became essential elements of antiviral immunity. I conclude with a reexamination of the ‘Big Bang’ theory for the emergence of the adaptive immune system in vertebrates by horizontal transfer and note how viruses could have and did provide crucial and coordinated features. PMID:22069523

  17. Bile acids for viral hepatitis

    DEFF Research Database (Denmark)

    Chen, Weikeng; Liu, J; Gluud, C

    2003-01-01

    The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness.......The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness....

  18. Bile acids for viral hepatitis

    DEFF Research Database (Denmark)

    Chen, Weikeng; Liu, J; Gluud, C

    2003-01-01

    The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness.......The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness....

  19. Dynamics of viral replication in blood and lymphoid tissues during SIVmac251 infection of macaques

    Directory of Open Access Journals (Sweden)

    Mannioui Abdelkrim

    2009-01-01

    Full Text Available Abstract Background Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied. Results The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT, with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected. Conclusion We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important

  20. Metagenomic analysis of viral communities in (hadopelagic sediments.

    Directory of Open Access Journals (Sweden)

    Mitsuhiro Yoshida

    Full Text Available In this study, we analyzed viral metagenomes (viromes in the sedimentary habitats of three geographically and geologically distinct (hadopelagic environments in the northwest Pacific; the Izu-Ogasawara Trench (water depth = 9,760 m (OG, the Challenger Deep in the Mariana Trench (10,325 m (MA, and the forearc basin off the Shimokita Peninsula (1,181 m (SH. Virus abundance ranged from 10(6 to 10(11 viruses/cm(3 of sediments (down to 30 cm below the seafloor [cmbsf]. We recovered viral DNA assemblages (viromes from the (hadopelagic sediment samples and obtained a total of 37,458, 39,882, and 70,882 sequence reads by 454 GS FLX Titanium pyrosequencing from the virome libraries of the OG, MA, and SH (hadopelagic sediments, respectively. Only 24-30% of the sequence reads from each virome library exhibited significant similarities to the sequences deposited in the public nr protein database (E-value <10(-3 in BLAST. Among the sequences identified as potential viral genes based on the BLAST search, 95-99% of the sequence reads in each library were related to genes from single-stranded DNA (ssDNA viral families, including Microviridae, Circoviridae, and Geminiviridae. A relatively high abundance of sequences related to the genetic markers (major capsid protein [VP1] and replication protein [Rep] of two ssDNA viral groups were also detected in these libraries, thereby revealing a high genotypic diversity of their viruses (833 genotypes for VP1 and 2,551 genotypes for Rep. A majority of the viral genes predicted from each library were classified into three ssDNA viral protein categories: Rep, VP1, and minor capsid protein. The deep-sea sedimentary viromes were distinct from the viromes obtained from the oceanic and fresh waters and marine eukaryotes, and thus, deep-sea sediments harbor novel viromes, including previously unidentified ssDNA viruses.

  1. Relação entre a carga viral de HPV oncogênico determinada pelo método de captura híbrida e o diagnóstico citológico de lesões de alto grau Association between high-risk human papillomavirus DNA load detected by hybrid capture II and high-grade precursor lesions of cervical cancer in women

    Directory of Open Access Journals (Sweden)

    Siumara Tulio

    2007-02-01

    Full Text Available INTRODUÇÃO: O papilomavírus humano (HPV é o principal fator de risco para as neoplasias intra-epiteliais cervicais (NIC e o câncer cervical. OBJETIVO: O objetivo deste estudo é avaliar se há associação entre a carga viral de HPV oncogênico (alto risco, determinada por meio do teste molecular captura híbrida II (CH II, e o diagnóstico de lesões de alto grau (NIC II/III. MATERIAL E MÉTODOS: Foram analisadas 982 amostras cervicovaginais de exames ginecológicos de rotina, obtidas pelos métodos Papanicolaou convencional e/ou citologia em base líquida (DNA-Citoliq-Digene. Os resultados foram confirmados utilizando-se o método de captura híbrida (CH [Digene] para detecção de DNA/HPV de alto grau. Os resultados com valor > 1 pg/ml foram considerados positivos, e esses foram divididos em dois grupos: 1. carga viral 100 pg/ml. RESULTADOS: Dos 210 (21,4% casos diagnosticados como NIC I, 152 (72,4% foram positivos para HPV de alto risco por CH II. Desses, 101 (66,4% apresentaram carga viral > 100 pg/ml. O diagnóstico de NIC II ou III foi confirmado por CH II de alto risco em 86 (43,6% casos, contudo, entre esses, em 53 (61,6% a carga viral detectada foi > 100 pg/ml. DISCUSSÃO E CONCLUSÃO: Nossos resultados demonstram que há uma clara associação entre o valor da carga viral determinada pelo método CH II (versão 1 e o grau das lesões precursoras de câncer. Pacientes com carga viral superior a 100 µg/ml devem ser monitoradas periodicamente.INTRODUCTION: Infection with oncogenic human papilloma virus (HPV has been established as the main etiologic agent for cervical cancer and of cervical intraepithelial neoplasia (CIN. OBJECTIVE: To determine the association between viral loads of the high risk HPV using the hybrid capture II (HC II system and CIN lesion stage. MATERIAL AND METHOD: A total of 982 women with diagnosis of negative or of CIN I-III with Pap or liquid-based cytology (DNA-Citoliq-Digene were included. HC II testing

  2. Viral meningitis and encephalitis.

    Science.gov (United States)

    Tuppeny, Misti

    2013-09-01

    Meningitis is an inflammation of the meninges, whereas encephalitis is inflammation of the parenchymal brain tissue. The single distinguishing element between the 2 diagnoses is the altered state of consciousness, focal deficits, and seizures found in encephalitis. Consequently meningoencephalitis is a term used when both findings are present in the patient. Viral meningitis is not necessarily reported as it is often underdiagnosed, whereas encephalitis cases are on the increase in various areas of North America. Improved imaging and viral diagnostics, as well as enhanced neurocritical care management, have improved patient outcomes to date.

  3. Viral infections in pigeons.

    Science.gov (United States)

    Marlier, D; Vindevogel, H

    2006-07-01

    This review provides a current update on the major viral diseases of the domestic pigeon (Columba livia domestica), based on scientific reports and clinical experience. Paramyxovirus 1, adenovirus, rotavirus, herpesvirus 1, poxvirus and circovirus infections are described according to common clinical signs and target tissues. Since pigeons are sometimes treated as if they were poultry, the review also summarises the common viral infections of poultry for which pigeons are considered resistant. It is hoped that the review will provide a useful reference for veterinarians and others and offer advice on the diagnosis, treatment and prevention of the major infectious diseases of pigeons.

  4. Integrated Evaluation of Latent Viral Reactivation During Spaceflight

    Science.gov (United States)

    Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

    2000-01-01

    This application proposes a continuation of our current effort, which has provided the first demonstration of viral reactivation during space flight. We have used the herpesvirus EBV as a model for latent viral reactivation and have shown that increased amounts of EBV DNA were shed by astronauts during space flight. Analysis of the Antarctic space flight analog indicated that the frequency of viral shedding may also increase (along with the increased numbers of virus) during long periods of isolation. However, a number of critical questions remain before the findings may be considered a significant health risk during extended space flight. These include: Are other latent viruses (e.g., other herpesviruses and polyornaviruses) in addition to EBV also reactivated and shed more frequently and/or in higher numbers during space flight? Is the viral reactivation observed in space flight and ground-based analogs mediated through the hypothalamus-pituitary-adrenal (HPA) axis resulting in a decreased cell-mediated immune response? How does detection of viral DNA by PCR analysis correlate with infectious virus? How does the amount of virus found during flight compare with viral levels observed in acute/chronic viral illnesses and in control individuals? This expanded study will examine the phenomenon of viral reactivation from the initiating stress through the HPA axis with the accompanying suppression of the immune system resulting in viral reactivation. This information is essential to determine if latent viral reactivation among crewmembers represents a sufficient medical risk to space travel to require the development of suitable countermeasures.

  5. Viral assemblage composition in Yellowstone acidic hot springs assessed by network analysis.

    Science.gov (United States)

    Bolduc, Benjamin; Wirth, Jennifer F; Mazurie, Aurélien; Young, Mark J

    2015-10-01

    Understanding of viral assemblage structure in natural environments remains a daunting task. Total viral assemblage sequencing (for example, viral metagenomics) provides a tractable approach. However, even with the availability of next-generation sequencing technology it is usually only possible to obtain a fragmented view of viral assemblages in natural ecosystems. In this study, we applied a network-based approach in combination with viral metagenomics to investigate viral assemblage structure in the high temperature, acidic hot springs of Yellowstone National Park, USA. Our results show that this approach can identify distinct viral groups and provide insights into the viral assemblage structure. We identified 110 viral groups in the hot springs environment, with each viral group likely representing a viral family at the sub-family taxonomic level. Most of these viral groups are previously unknown DNA viruses likely infecting archaeal hosts. Overall, this study demonstrates the utility of combining viral assemblage sequencing approaches with network analysis to gain insights into viral assemblage structure in natural ecosystems.

  6. Viral diseases of the rabbit.

    Science.gov (United States)

    Krogstad, Aric P; Simpson, Janet E; Korte, Scott W

    2005-01-01

    Viral disease in the rabbit is encountered infrequently by the clinical practitioner; however, several viral diseases were reported to occur in this species. Viral diseases that are described in the rabbit primarily may affect the integument, gastrointestinal tract or, central nervous system or maybe multi-systemic in nature. Rabbit viral diseases range from oral papillomatosis, with benign clinical signs, to rabbit hemorrhagic disease and myxomatosis, which may result in significant clinical disease and mortality. The wild rabbit may serve as a reservoir for disease transmission for many of these viral agents. In general, treatment of viral disease in the rabbit is supportive in nature.

  7. Immigration and viral hepatitis

    NARCIS (Netherlands)

    S. Sharma (Suraj); M. Carballo (Manuel); J.J. Feld (Jordan J.); H.L.A. Janssen (Harry)

    2015-01-01

    textabstractWHO estimates reveal that the global prevalence of viral hepatitis may be as high as 500 million, with an annual mortality rate of up to 1.3 million individuals. The majority of this global burden of disease is borne by nations of the developing world with high rates of vertical and

  8. Hepatitis A through E (Viral Hepatitis)

    Science.gov (United States)

    ... Nutrition Clinical Trials Primary Sclerosing Cholangitis Wilson Disease Hepatitis (Viral) View or Print All Sections What is Viral Hepatitis? Viral hepatitis is an infection that causes liver inflammation ...

  9. Bile acids for viral hepatitis

    DEFF Research Database (Denmark)

    Chen, Weikeng; Liu, J; Gluud, C

    2007-01-01

    Trials have assessed bile acids for patients with viral hepatitis, but no consensus has been reached regarding their usefulness.......Trials have assessed bile acids for patients with viral hepatitis, but no consensus has been reached regarding their usefulness....

  10. Viral Marketing and Academic Institution

    OpenAIRE

    Koktová, Silvie

    2010-01-01

    This bachelor thesis examines modern and constantly developing kind of internet marketing -- the so called viral marketing. It deals with its origin, principle, process, advantages and disadvantages, types of viral marketing and presumptions of creating successful viral campaign. The aim of the theoretical part is especially the understanding of viral marketing as one of the effective instruments of contemporary marketing. In this theoretical part the thesis also elaborates a marketing school...

  11. Viral Marketing and Academic Institution

    OpenAIRE

    Koktová, Silvie

    2010-01-01

    This bachelor thesis examines modern and constantly developing kind of internet marketing -- the so called viral marketing. It deals with its origin, principle, process, advantages and disadvantages, types of viral marketing and presumptions of creating successful viral campaign. The aim of the theoretical part is especially the understanding of viral marketing as one of the effective instruments of contemporary marketing. In this theoretical part the thesis also elaborates a marketing school...

  12. [Uracil-DNA glycosylases].

    Science.gov (United States)

    Pytel, Dariusz; Słupianek, Artur; Ksiazek, Dominika; Skórski, Tomasz; Błasiak, Janusz

    2008-01-01

    Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

  13. Epigenetic control of Epstein Barr Virus transcription – relevance to viral life cycle?

    Directory of Open Access Journals (Sweden)

    Alison J. Sinclair

    2013-08-01

    Full Text Available DNA-methylation normally leads to silencing of gene expression but Epstein-Barr virus (EBV provides an exception to the epigenetic paradigm. DNA-methylation is absolutely required for the expression of many viral genes. Although the viral genome is initially unmethylated in newly infected cells, it becomes extensively methylated during the establishment of viral latency. One of the major regulators of EBV gene expression is a viral transcription factor called Zta (BZLF1, ZEBRA, Z that resembles the cellular AP1 transcription factor. Zta recognizes at least 32 variants of a 7-nucleotide DNA sequence element, the Zta-response element (ZRE, some of which contain a CpG motif. Zta only binds to the latter class of ZREs in their DNA-methylated form, whether they occur in viral or cellular promoters and is functionally relevant for the activity of these promoters. The ability of Zta to interpret the differential DNA-methylation of the viral genome is paramount for both the establishment of viral latency and the release from latency to intiate viral replication.

  14. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    Science.gov (United States)

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Immunogenetics of viral infections.

    Science.gov (United States)

    Martin, Maureen P; Carrington, Mary

    2005-10-01

    The HLA class I and II genes encode molecules that lie at the heart of the acquired immune response against infectious diseases. Associations between these polymorphic loci and genetically complex infectious diseases have been historically elusive, in contrast to the more obvious HLA associations with autoimmune diseases. High resolution molecular typing of large, clinically well-defined cohorts has begun to uncover evidence for the influence of HLA diversity on diseases of viral etiology, such as those caused by HIV-1, hepatitis B virus, hepatitis C virus and human papilloma virus. Combinations of HLA and KIR also appear to affect outcome to viral infection, supporting a role for HLA class I diversity in the innate immune response in addition to the acquired immune response.

  16. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple

  17. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  18. Viral quasispecies evolution

    OpenAIRE

    Domingo, Esteban; Sheldon, Julie; Perales, Celia

    2012-01-01

    Summary: Evolution of RNA viruses occurs through disequilibria of collections of closely related mutant spectra or mutant clouds termed viral quasispecies. Here we review the origin of the quasispecies concept and some biological implications of quasispecies dynamics. Two main aspects are addressed: (i) mutant clouds as reservoirs of phenotypic variants for virus adaptability and (ii) the internal interactions that are established within mutant spectra that render a virus ensemble the unit of...

  19. Virion-targeted viral inactivation: new therapy against viral infection.

    Science.gov (United States)

    Okui, N; Kitamura, Y; Kobayashi, N; Sakuma, R; Ishikawa, T; Kitamura, T

    2001-01-01

    Acquired immune deficiency syndrome (AIDS) is resistant to all current therapy. Gene therapy is an attractive alternative or additive to current, unsatisfactory AIDS therapy. To develop an antiviral molecule targeting viral integrase (HIV IN), we generated a single-chain antibody, termed scAb, which interacted with human immunodeficiency virus type 1 (HIV-1) IN and inhibited virus replication at the integration step when expressed intracellularly. To reduce infectivity from within the virus particles, we made expression plasmids (pC-scAbE-Vpr, pC-scAbE-CA, and pC-scAbE-WXXF), which expressed the anti-HIV IN scAb fused to the N-terminus of HIV-1-associated accessory protein R (Vpr), capsid protein (CA), and specific binding motif to Vpr (WXXF), respectively. All fusion proteins were tagged with a nine-amino acid peptide derived from influenza virus hemagglutinin (HA) at the C terminus. The fusion molecules, termed scAbE-Vpr, scAbE-CA, and scAbE-WXXF, interacted specifically with HIV IN immobilized on a nitrocellulose membrane. Immunoblot analysis showed that scAbE-Vpr, scAbE-CA, and scAbE-WXXF were incorporated into the virions produced by cotransfection of 293T cells with HIV-1 infectious clone DNA (pLAI) and pC-scAbE-Vpr, pC-scAbE-WXXF. A multinuclear activation galactosidase indicator (MAGI) assay revealed that the virions released from 293T cells cotransfected with pLAI and pC-scAbE-Vpr, pC-scAbE-WXXF had as little 1000-fold of the infectivity of the control wild-type virions, which were produced from the 293T cells transfected with pLAI alone. Furthermore, the virions produced from the 293T cells cotransfected with pLAI and an scAb expression vector (pC-scAb) showed only 1% of the infectivity of the control HIV-1 in a MAGI assay, although scAb was not incorporated into the virions. In either instance, the total quantity of the progeny virions released from the transfected 293T cells and the patterns of the virion proteins were hardly affected by the presence of

  20. Viral membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

    2015-05-15

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

  1. Hepatitis viral C

    Directory of Open Access Journals (Sweden)

    Pedro A. Poma

    2011-12-01

    Full Text Available El virus de la hepatitis C se trasmite por contacto directo con la sangre de la persona infectada. La mayoría de los pacientes no presenta síntomas en la fase aguda o crónica de la hepatitis. Dos a tres décadas después, algunos pacientes progresan a la cirrosis compensada, que también es asintomática. En un examen de sangre, los anticuerpos se presentan como una sorpresa, porque no se les relaciona con un episodio de contagio. Un embarazo ocasiona la posibilidad de efectos negativos de la infección en la madre o el niño. El tratamiento actual no ofrece la certeza de cura, dependiendo del genotipo viral, y presenta efectos adversos que pueden ser severos. La cirrosis descompensada causa la mayoría de muertes relacionadas con esta infección; algunos de estos pacientes desarrollan carcinoma hepatocelular. La reproducción viral causa partículas virales diferentes del virus original, característica que ha impedido el desarrollo de una vacuna. Actualmente, la prevención consiste en evitar el contacto con sangre infectada. Este artículo revisa la infección con el virus de la hepatitis C, incluyendo los últimos progresos en tratamiento. Es necesario educar a la comunidad acerca de los efectos de este virus en la salud pública.

  2. Viral Vectors for in Vivo Gene Transfer

    Science.gov (United States)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  3. Replication-Uncoupled Histone Deposition during Adenovirus DNA Replication

    OpenAIRE

    Komatsu, Tetsuro; Nagata, Kyosuke

    2012-01-01

    In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of his...

  4. Metagenomic Analysis of the Viral Communities in Fermented Foods▿ †

    Science.gov (United States)

    Park, Eun-Jin; Kim, Kyoung-Ho; Abell, Guy C. J.; Kim, Min-Soo; Roh, Seong Woon; Bae, Jin-Woo

    2011-01-01

    Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae, Podoviridae, and Siphoviridae). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil. PMID:21183634

  5. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus.

    Science.gov (United States)

    Liu, Yingjie; Wu, Jinlu; Chen, Hu; Hew, Choy Leong; Yan, Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  6. Intrahepatic immune response in chronic viral hepatitis : an immunohistochemical study

    NARCIS (Netherlands)

    T.J. Tang (Thjon J.)

    2004-01-01

    textabstractThe hepatitis B virus (HBV) is a 42 nm viral particle and belongs to a family of closely related DNA viruses called the hepadnaviruses. All the hepadnaviruses have similar hepatotropism and life cycles in their hosts. It is an enveloped small circular, partially double stranded

  7. Clinical utility of viral quantification as a tool for disease monitoring

    NARCIS (Netherlands)

    Schutten, Martin; Niesters, H G

    2001-01-01

    The possibility to detect viral DNA or RNA in a quantitative manner has already contributed significantly to the management and diagnosis of viral infections, as well as to the understanding of virus-host interactions. New developments in amplification techniques based on real-time detection, as wel

  8. VIRAL HEPATITIS E DIAGNOSTICS

    Directory of Open Access Journals (Sweden)

    E. Yu. Malinnikova

    2013-01-01

    Full Text Available Abstract. The results of clinical and epidemiological studies conducted in the M.P. Chumakov’ Research Institute of Poliomyelitis and Viral Encephalitis and in the different research institutions of the world have been summarized in the current article. Data on etiology, pathogenesis, clinical symptoms, epidemiology and prevention of hepatitis E are presented. Increasing of significance of this infection for health care system in Russia is emphasized . The actual problems of hepatitis E (autochthonic hepatitis E, hepatitis E as zoonosis, chronic hepatitis E are discussed.

  9. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    OpenAIRE

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-01-01

    A. polyphaga mimivirus, the largest known double-stranded DNA virus, is the first virus to exhibit a nucleoside diphosphate kinase gene. The expression and crystallization of the viral NDK are reported.

  10. Structural organization of DNA in chlorella viruses.

    Directory of Open Access Journals (Sweden)

    Timo Wulfmeyer

    Full Text Available 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.

  11. Viral infections of rabbits.

    Science.gov (United States)

    Kerr, Peter J; Donnelly, Thomas M

    2013-05-01

    Viral diseases of rabbits have been used historically to study oncogenesis (e.g. rabbit fibroma virus, cottontail rabbit papillomavirus) and biologically to control feral rabbit populations (e.g. myxoma virus). However, clinicians seeing pet rabbits in North America infrequently encounter viral diseases although myxomatosis may be seen occasionally. The situation is different in Europe and Australia, where myxomatosis and rabbit hemorrhagic disease are endemic. Advances in epidemiology and virology have led to detection of other lapine viruses that are now recognized as agents of emerging infectious diseases. Rabbit caliciviruses, related to rabbit hemorrhagic disease, are generally avirulent, but lethal variants are being identified in Europe and North America. Enteric viruses including lapine rotavirus, rabbit enteric coronavirus and rabbit astrovirus are being acknowledged as contributors to the multifactorial enteritis complex of juvenile rabbits. Three avirulent leporid herpesviruses are found in domestic rabbits. A fourth highly pathogenic virus designated leporid herpesvirus 4 has been described in Canada and Alaska. This review considers viruses affecting rabbits by their clinical significance. Viruses of major and minor clinical significance are described, and viruses of laboratory significance are mentioned. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. RUNX1 Permits E4orf6-Directed Nuclear Localization of the Adenovirus E1B-55K Protein and Associates with Centers of Viral DNA and RNA Synthesis▿

    Science.gov (United States)

    Marshall, Leslie J.; Moore, Amy C.; Ohki, Misao; Kitabayashi, Issay; Patterson, David; Ornelles, David A.

    2008-01-01

    The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-ΔN variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirus-infected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1. PMID:18417565

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

    Science.gov (United States)

    Goic, Bertsy; Stapleford, Kenneth A.; Frangeul, Lionel; Doucet, Aurélien J.; Gausson, Valérie; Blanc, Hervé; Schemmel-Jofre, Nidia; Cristofari, Gael; Lambrechts, Louis; Vignuzzi, Marco; Saleh, Maria-Carla

    2016-01-01

    Mosquitoes develop long-lasting viral infections without substantial deleterious effects, despite high viral loads. This makes mosquitoes efficient vectors for emerging viral diseases with enormous burden on public health. How mosquitoes resist and/or tolerate these viruses is poorly understood. Here we show that two species of Aedes mosquitoes infected with two arboviruses from distinct families (dengue or chikungunya) generate a viral-derived DNA (vDNA) that is essential for mosquito survival and viral tolerance. Inhibition of vDNA formation leads to extreme susceptibility to viral infections, reduction of viral small RNAs due to an impaired immune response, and loss of viral tolerance. Our results highlight an essential role of vDNA in viral tolerance that allows mosquito survival and thus may be important for arbovirus dissemination and transmission. Elucidating the mechanisms of mosquito tolerance to arbovirus infection paves the way to conceptualize new antivectorial strategies to selectively eliminate arbovirus-infected mosquitoes. PMID:27580708

  14. Cytokine determinants of viral tropism

    OpenAIRE

    McFadden, Grant; Mohamed, Mohamed R.; Rahman, Masmudur M.; Bartee, Eric

    2009-01-01

    The specificity of a given virus for a ceil type, tissue or species — collectively known as viral tropism — is an important factor in determining the outcome of viral infection in any particular host. Owing to the increased prevalence of zoonotic infections and the threat of emerging and re-emerging pathogens, gaining a better understanding of the factors that determine viral tropism has become particularly important. In this Review, we summarize our current understanding of the central role ...

  15. Viral infections as controlling factors for the deep biosphere? (Invited)

    Science.gov (United States)

    Engelen, B.; Engelhardt, T.; Sahlberg, M.; Cypionka, H.

    2009-12-01

    The marine deep biosphere represents the largest biotope on Earth. Throughout the last years, we have obtained interesting insights into its microbial community composition. However, one component that was completely overlooked so far is the viral inventory of deep-subsurface sediments. While viral infections were identified to have a major impact on the benthic microflora of deep-sea surface sediments (Danavaro et al. 2008), no studies were performed on deep-biosphere samples, so far. As grazers probably play only a minor role in anoxic and highly compressed deep sediments, viruses might be the main “predators” for indigenous microorganisms. Furthermore, the release of cell components, called “the viral shunt”, could have a major impact on the deep biosphere in providing labile organic compounds to non-infected microorganisms in these generally nutrient depleted sediments. However, direct counting of viruses in sediments is highly challenging due to the small size of viruses and the high background of small particles. Even molecular surveys using “universal” PCR primers that target phage-specific genes fail due to the vast phage diversity. One solution for this problem is the lysogenic viral life cycle as many bacteriophages integrate their DNA into the host genome. It is estimated that up to 70% of cultivated bacteria contain prophages within their genome. Therefore, culture collections (Batzke et al. 2007) represent an archive of the viral composition within the respective habitat. These prophages can be induced to become free phage particles in stimulation experiments in which the host cells are set under certain stress situations such as a treatment with UV exposure or DNA-damaging antibiotics. The study of the viral component within the deep biosphere offers to answer the following questions: To which extent are deep-biosphere populations controlled by viral infections? What is the inter- and intra-specific diversity and the host-specific viral

  16. DNA UPTAKE BY TRANSFORMABLE BACTERIA

    Energy Technology Data Exchange (ETDEWEB)

    LACKS,S.A.

    1999-09-07

    The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

  17. DNA Uptake by Transformable Bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, Sanford A.

    1999-03-31

    The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

  18. Viral causes of diarrhea.

    Science.gov (United States)

    Goodgame, R W

    2001-09-01

    Viruses are important causes of diarrhea. In healthy adults, the main clinical manifestation is acute, self-limited gastroenteritis. Advances in molecular diagnostics have shown that epidemics of acute gastroenteritis most frequently are due to caliciviruses spread through contaminated food or through person-to-person contact. Application of similar technology is needed to make a definitive statement about the role of such candidate viruses as rotavirus, astrovirus, and adenovirus as the cause of nonepidemic acute gastroenteritis in adults. Rarely a previously healthy adult gets acute CMV colitis. CMV and EBV mainly cause diarrhea in immunocompromised patients, however. Advances in prophylaxis and treatment have reduced the frequency and severity of these diseases. Acute infantile gastroenteritis is caused by rotavirus, calcivirus, astrovirus, and adenovirus. These viral diseases of the gut are seen by the physician as routine and rare clinical problems.

  19. Viral quasispecies evolution.

    Science.gov (United States)

    Domingo, Esteban; Sheldon, Julie; Perales, Celia

    2012-06-01

    Evolution of RNA viruses occurs through disequilibria of collections of closely related mutant spectra or mutant clouds termed viral quasispecies. Here we review the origin of the quasispecies concept and some biological implications of quasispecies dynamics. Two main aspects are addressed: (i) mutant clouds as reservoirs of phenotypic variants for virus adaptability and (ii) the internal interactions that are established within mutant spectra that render a virus ensemble the unit of selection. The understanding of viruses as quasispecies has led to new antiviral designs, such as lethal mutagenesis, whose aim is to drive viruses toward low fitness values with limited chances of fitness recovery. The impact of quasispecies for three salient human pathogens, human immunodeficiency virus and the hepatitis B and C viruses, is reviewed, with emphasis on antiviral treatment strategies. Finally, extensions of quasispecies to nonviral systems are briefly mentioned to emphasize the broad applicability of quasispecies theory.

  20. Viral diagnostics in the era of digital PCR

    Science.gov (United States)

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  1. Towards Viral Genome Annotation Standards, Report from the 2010 NCBI Annotation Workshop.

    Science.gov (United States)

    Brister, James Rodney; Bao, Yiming; Kuiken, Carla; Lefkowitz, Elliot J; Le Mercier, Philippe; Leplae, Raphael; Madupu, Ramana; Scheuermann, Richard H; Schobel, Seth; Seto, Donald; Shrivastava, Susmita; Sterk, Peter; Zeng, Qiandong; Klimke, William; Tatusova, Tatiana

    2010-10-01

    Improvements in DNA sequencing technologies portend a new era in virology and could possibly lead to a giant leap in our understanding of viral evolution and ecology. Yet, as viral genome sequences begin to fill the world's biological databases, it is critically important to recognize that the scientific promise of this era is dependent on consistent and comprehensive genome annotation. With this in mind, the NCBI Genome Annotation Workshop recently hosted a study group tasked with developing sequence, function, and metadata annotation standards for viral genomes. This report describes the issues involved in viral genome annotation and reviews policy recommendations presented at the NCBI Annotation Workshop.

  2. Comparison of specific binding sites for Escherichia coli RNA polymerase with naturally occurring hairpin regions in single-stranded DNA of coliphage M13. [Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Niyogi, S.K.; Mitra, S.

    1978-08-25

    Escherichia coli RNA polymerase binds specifically to the single-stranded circular DNA of coliphage M13 in the presence of a saturating concentration of the bacterial DNA binding protein presumably as an essential step in the synthesis of the RNA primer required for synthesizing the complementary DNA strand in parental replicative-form DNA. The RNA polymerase-protected DNA regions were isolated after extensive digestion with pancreatic DNase, S1 endonuclease of Aspergillus oryzae, and exonuclease I of E. coli. The physicochemical properties of the RNA polymerase-protected segments (called PI and PII) were compared with those of the naturally occurring hairpin regions.

  3. bDNA和NASBA法定量检测HIV-1病毒载量的比较研究%A comparative study on testing methods for HIV-1 viral load by bDNA and NASBA

    Institute of Scientific and Technical Information of China (English)

    郭秋艳; 吴亚松; 张彤; 陈德喜; 吴昊; 黄晓婕; 代丽丽; 汪习成; 魏飞力; 计云霞; 石英; 夏伟; 郭彩萍

    2007-01-01

    目的 比较分支DNA杂交实验(branched DNA,bDNA)和核酸序列扩增实验(Nucleic acid sequence-based amplification,NASBA) 两种方法检测人类免疫缺陷病毒1型病毒(HIV-1) 病毒载量间的一致性.方法 对25例HIV 感染者/艾滋病(Acquired immune deficiency syndrome,AIDS) 患者血浆标本同时用bDNA 法和NASBA 法检测HIV-1病毒载量,并用流式细胞术检测患者外周血CD4+、CD8+T淋巴细胞.结果 bDNA法及NASBA法测得HIV-1病毒载量平均值分别为(4.398±0.580)log 拷贝数/ ml和(4.488±0.602)log 拷贝数/ ml,差异无统计学意义(t=1.210,P>0.05);两种方法检测出的病毒载量呈显著直线相关(r= 0.8004,P<0.001),且与患者的CD4+T细胞数及CD4+/ CD8+比值均呈显著直线负相关.结论 bDNA 法和NASBA 法检测HIV-1 病毒载量具有高度一致性,在实际工作中均可选用.

  4. Function of Membrane Rafts in Viral Lifecycles and Host Cellular Response

    Directory of Open Access Journals (Sweden)

    Tadanobu Takahashi

    2011-01-01

    Full Text Available Membrane rafts are small (10–200 nm sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Membrane rafts play an important role in viral infection cycles and viral virulence. Viruses are divided into four main classes, enveloped DNA virus, enveloped RNA virus, nonenveloped DNA virus, and nonenveloped RNA virus. General virus infection cycle is also classified into two sections, the early stage (entry process and the late stage (assembly, budding, and release processes of virus particles. In the viral cycle, membrane rafts act as a scaffold of many cellular signal transductions, which are associated with symptoms caused by viral infections. In this paper, we describe the functions of membrane rafts in viral lifecycles and host cellular response according to each virus classification, each stage of the virus lifecycle, and each virus-induced signal transduction.

  5. Tandem Oligonucleotide Probe Annealing and Elongation To Discriminate Viral Sequence

    DEFF Research Database (Denmark)

    Taskova, Maria; Uhd, Jesper; Miotke, Laura

    2017-01-01

    followed by click assembly and analysis of the read sequence by various techniques. As we demonstrate in this paper, using our new approach, a viral RNA sequence can be detected in less than 2 h without the need for cDNA synthesis or any other enzymatic reactions and with a sensitivity of ... opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA...

  6. Liposomal nanocontainers as models for viral infection: monitoring viral genomic RNA transfer through lipid membranes.

    Science.gov (United States)

    Bilek, Gerhard; Matscheko, Nena M; Pickl-Herk, Angela; Weiss, Victor U; Subirats, Xavier; Kenndler, Ernst; Blaas, Dieter

    2011-08-01

    After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.

  7. Murine cytomegalovirus protein pM79 is a key regulator for viral late transcription.

    Science.gov (United States)

    Chapa, Travis J; Johnson, L Steven; Affolter, Christopher; Valentine, Mark C; Fehr, Anthony R; Yokoyama, Wayne M; Yu, Dong

    2013-08-01

    Herpesvirus genes are temporally expressed during permissive infections, but how their expression is regulated at late times is poorly understood. Previous studies indicate that the human cytomegalovirus (CMV) gene, UL79, is required for late gene expression. However, the mechanism remains to be fully elucidated, and UL79 homologues in other CMVs have not been studied. Here, we characterized the role of the conserved murine CMV (MCMV) gene M79. We showed that M79 encoded a protein (pM79) which was expressed with early-late kinetics and localized to nuclear viral replication compartments. M79 transcription was significantly decreased in the absence of viral DNA synthesis but markedly stimulated by pM79. To investigate its role, we created the recombinant virus SMin79, in which pM79 expression was disrupted. While marker-rescued virus grew efficiently in fibroblasts, SMin79 failed to produce infectious progeny but was rescued by pM79 expression in trans. During SMin79 infection, representative viral immediate-early and early gene products as well as viral DNA accumulated sufficiently. Formation of viral replication compartments also appeared normal. Pulsed-field gel electrophoresis analysis indicated that the overall structure of replicating viral DNA was indistinguishable between wild-type and SMin79 infection. Viral tiled array and quantitative PCR analysis revealed that many late transcripts sensitive to a viral DNA synthesis inhibitor (phosphonoacetic acid) were markedly reduced by pM79 mutation. This study indicates that cytomegaloviruses use a conserved mechanism to promote transcription at late stages of infection and that pM79 is a critical regulator for at least a subset of viral DNA synthesis-dependent transcripts.

  8. Dengue viral infections

    Directory of Open Access Journals (Sweden)

    Gurugama Padmalal

    2010-01-01

    Full Text Available Dengue viral infections are one of the most important mosquito-borne diseases in the world. Presently dengue is endemic in 112 countries in the world. It has been estimated that almost 100 million cases of dengue fever and half a million cases of dengue hemorrhagic fever (DHF occur worldwide. An increasing proportion of DHF is in children less than 15 years of age, especially in South East and South Asia. The unique structure of the dengue virus and the pathophysiologic responses of the host, different serotypes, and favorable conditions for vector breeding have led to the virulence and spread of the infections. The manifestations of dengue infections are protean from being asymptomatic to undifferentiated fever, severe dengue infections, and unusual complications. Early recognition and prompt initiation of appropriate supportive treatment are often delayed resulting in unnecessarily high morbidity and mortality. Attempts are underway for the development of a vaccine for preventing the burden of this neglected disease. This review outlines the epidemiology, clinical features, pathophysiologic mechanisms, management, and control of dengue infections.

  9. Emerging zoonotic viral diseases.

    Science.gov (United States)

    Wang, L-F; Crameri, G

    2014-08-01

    Zoonotic diseases are infectious diseases that are naturally transmitted from vertebrate animals to humans and vice versa. They are caused by all types of pathogenic agents, including bacteria, parasites, fungi, viruses and prions. Although they have been recognised for many centuries, their impact on public health has increased in the last few decades due to a combination of the success in reducing the spread of human infectious diseases through vaccination and effective therapies and the emergence of novel zoonotic diseases. It is being increasingly recognised that a One Health approach at the human-animal-ecosystem interface is needed for effective investigation, prevention and control of any emerging zoonotic disease. Here, the authors will review the drivers for emergence, highlight some of the high-impact emerging zoonotic diseases of the last two decades and provide examples of novel One Health approaches for disease investigation, prevention and control. Although this review focuses on emerging zoonotic viral diseases, the authors consider that the discussions presented in this paper will be equally applicable to emerging zoonotic diseases of other pathogen types.

  10. Sphingolipids in viral infection.

    Science.gov (United States)

    Schneider-Schaulies, Jürgen; Schneider-Schaulies, Sibylle

    2015-06-01

    Viruses exploit membranes and their components such as sphingolipids in all steps of their life cycle including attachment and membrane fusion, intracellular transport, replication, protein sorting and budding. Examples for sphingolipid-dependent virus entry are found for: human immunodeficiency virus (HIV), which besides its protein receptors also interacts with glycosphingolipids (GSLs); rhinovirus, which promotes the formation of ceramide-enriched platforms and endocytosis; or measles virus (MV), which induces the surface expression of its own receptor CD150 via activation of sphingomyelinases (SMases). While SMase activation was implicated in Ebola virus (EBOV) attachment, the virus utilizes the cholesterol transporter Niemann-Pick C protein 1 (NPC1) as 'intracellular' entry receptor after uptake into endosomes. Differential activities of SMases also affect the intracellular milieu required for virus replication. Sindbis virus (SINV), for example, replicates better in cells lacking acid SMase (ASMase). Defined lipid compositions of viral assembly and budding sites influence virus release and infectivity, as found for hepatitis C virus (HCV) or HIV. And finally, viruses manipulate cellular signaling and the sphingolipid metabolism to their advantage, as for example influenza A virus (IAV), which activates sphingosine kinase 1 and the transcription factor NF-κB.

  11. Viral infection, inflammation and schizophrenia.

    Science.gov (United States)

    Kneeland, Rachel E; Fatemi, S Hossein

    2013-04-05

    Schizophrenia is a severe neurodevelopmental disorder with genetic and environmental etiologies. Prenatal viral/bacterial infections and inflammation play major roles in the genesis of schizophrenia. In this review, we describe a viral model of schizophrenia tested in mice whereby the offspring of mice prenatally infected with influenza at E7, E9, E16, and E18 show significant gene, protein, and brain structural abnormalities postnatally. Similarly, we describe data on rodents exposed to bacterial infection or injected with a synthetic viral mimic (PolyI:C) also demonstrating brain structural and behavioral abnormalities. Moreover, human serologic data has been indispensible in supporting the viral theory of schizophrenia. Individuals born seropositive for bacterial and viral agents are at a significantly elevated risk of developing schizophrenia. While the specific mechanisms of prenatal viral/bacterial infections and brain disorder are unclear, recent findings suggest that the maternal inflammatory response may be associated with fetal brain injury. Preventive and therapeutic treatment options are also proposed. This review presents data related to epidemiology, human serology, and experimental animal models which support the viral model of schizophrenia. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Enfermedades virales emergentes y reemergentes

    OpenAIRE

    Jorge Eliécer Ossa Londoño; Ana Isabel Toro Montoya

    1996-01-01

    Los virus no son una excepción al principio de que toda forma de vida de hoyes el producto de la evolución de información gen ética preexistente. Tradicionalmente se ha reconocido que ta expresión clínica de las enfermedades virales cambia con el tiempo; molecularmente se ha demostrado que esos cambios fenotípicos son el producto de variaciones en el genoma viral. La tasa de cambio
    gen ético y fenotípico no es la misma en todos los agentes virales y ello está determinado, principal...

  13. Encefalitis virales en la infancia

    OpenAIRE

    2013-01-01

    La encefalitis viral es una enfermedad grave que implica el compromiso inflamatorio del parénquima cerebral. Las infecciones virales del SNC ocurren con frecuencia como complicación de infecciones virales sistémicas. Más de 100 virus están implicados como agentes causales, entre los cuales el virus Herpes simplex tipo I, es el agente causal más frecuente de encefalitis no epidémica en todos los grupos poblacionales del mundo; es el responsable de los casos más graves en todas las edades. Much...

  14. Circular Herpesvirus sylvilagus DNA in spleen cells of experimentally infected cottontail rabbits.

    Science.gov (United States)

    Medveczky, P; Kramp, W J; Sullivan, J L

    1984-01-01

    Cottontail rabbits (Sylvilagus floridanus) were infected with Herpesvirus sylvilagus, and spleen cells were analyzed for the presence of virus-specific, covalently closed circular, and linear DNA molecules by a simple electrophoretic technique, followed by transfer to nitrocellulose filters and hybridization with cloned viral DNA (Gardella et al., J. Virol. 50:248-254, 1984). Approximately 0.2 copies per cell of circular DNA and 0.2 copies per cell of linear DNA were detected by hybridization with a cloned viral DNA fragment. The size of the viral DNA was estimated at ca. 158 kilobase pairs. Restriction endonuclease patterns suggested structural similarities to cottontail herpesvirus DNA. Images PMID:6092696

  15. Combined macroscopic and microscopic detection of viral genes in tissues

    Energy Technology Data Exchange (ETDEWEB)

    Haase, A.T.; Gantz, D.; Blum, H.; Stowring, L.; Ventura, P.; Geballe, A.; Moyer, B.; Brahic, M.

    1985-01-15

    A hybridization technique has been devised for detecting and quantitating viral genes in tissues that combines macroscopic and microscopic analyses in the same section. The method is based on dual labeling virus-specific probes with /sup 125/I and /sup 35/S to generate signals that can be detected both with X-ray films and nuclear track emulsions. The regions of increased hybridization evident in the X-ray film serve as a guide to the portion of the section that warrants microscopic examination. Detection of viral RNA in tissues with Visna virus and viral DNA with hepatitis B virus are illustrated, and potential applications of this technique in virology and other disciplines are discussed.

  16. Neuroanatomy goes viral!

    Directory of Open Access Journals (Sweden)

    Jonathan eNassi

    2015-07-01

    Full Text Available The nervous system is complex not simply because of the enormous number of neurons it contains but by virtue of the specificity with which they are connected. Unraveling this specificity is the task of neuroanatomy. In this endeavor, neuroanatomists have traditionally exploited an impressive array of tools ranging from the Golgi method to electron microscopy. An ideal method for studying anatomy would label neurons that are interconnected, and, in addition, allow expression of foreign genes in these neurons. Fortuitously, nature has already partially developed such a method in the form of neurotropic viruses, which have evolved to deliver their genetic material between synaptically connected neurons while largely eluding glia and the immune system. While these characteristics make some of these viruses a threat to human health, simple modifications allow them to be used in controlled experimental settings, thus enabling neuroanatomists to trace multi-synaptic connections within and across brain regions. Wild-type neurotropic viruses, such as rabies and alpha-herpes virus, have already contributed greatly to our understanding of brain connectivity, and modern molecular techniques have enabled the construction of recombinant forms of these and other viruses. These newly engineered reagents are particularly useful, as they can target genetically defined populations of neurons, spread only one synapse to either inputs or outputs, and carry instructions by which the targeted neurons can be made to express exogenous proteins, such as calcium sensors or light-sensitive ion channels, that can be used to study neuronal function. In this review, we address these uniquely powerful features of the viruses already in the neuroanatomist's toolbox, as well as the aspects of their biology that currently limit their utility. Based on the latter, we consider strategies for improving viral tracing methods by reducing toxicity, improving control of transsynaptic

  17. FastStats: Viral Hepatitis

    Science.gov (United States)

    ... Submit What's this? Submit Button NCHS Home Viral Hepatitis Recommend on Facebook Tweet Share Compartir Data are for the U.S. Morbidity Number of new hepatitis A cases: 1,781 (2013) Number of new ...

  18. Viral marketing as epidemiological model

    CERN Document Server

    Rodrigues, Helena Sofia

    2015-01-01

    In epidemiology, an epidemic is defined as the spread of an infectious disease to a large number of people in a given population within a short period of time. In the marketing context, a message is viral when it is broadly sent and received by the target market through person-to-person transmission. This specific marketing communication strategy is commonly referred as viral marketing. Due to this similarity between an epidemic and the viral marketing process and because the understanding of the critical factors to this communications strategy effectiveness remain largely unknown, the mathematical models in epidemiology are presented in this marketing specific field. In this paper, an epidemiological model SIR (Susceptible- Infected-Recovered) to study the effects of a viral marketing strategy is presented. It is made a comparison between the disease parameters and the marketing application, and simulations using the Matlab software are performed. Finally, some conclusions are given and their marketing impli...

  19. Statistical mechanics of viral entry.

    Science.gov (United States)

    Zhang, Yaojun; Dudko, Olga K

    2015-01-09

    Viruses that have lipid-membrane envelopes infect cells by fusing with the cell membrane to release viral genes. Membrane fusion is known to be hindered by high kinetic barriers associated with drastic structural rearrangements-yet viral infection, which occurs by fusion, proceeds on remarkably short time scales. Here, we present a quantitative framework that captures the principles behind the invasion strategy shared by all enveloped viruses. The key to this strategy-ligand-triggered conformational changes in the viral proteins that pull the membranes together-is treated as a set of concurrent, bias field-induced activated rate processes. The framework results in analytical solutions for experimentally measurable characteristics of virus-cell fusion and enables us to express the efficiency of the viral strategy in quantitative terms. The predictive value of the theory is validated through simulations and illustrated through recent experimental data on influenza virus infection.

  20. Viral Evolution Core | FNLCR Staging

    Science.gov (United States)

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

  1. Aseptic meningitis and viral myelitis.

    Science.gov (United States)

    Irani, David N

    2008-08-01

    Meningitis and myelitis represent common and very infrequent viral infections of the central nervous system, respectively. The number of cases of viral meningitis that occurs annually exceeds the total number of meningitis cases caused by all other etiologies combined. Focal central nervous system infections, such as occur in the spinal cord with viral myelitis, are much less common and may be confused with noninfectious disorders that cause acute flaccid paralysis. This article reviews some of the important clinical features, epidemiology, diagnostic approaches, and management strategies for patients with aseptic meningitis and viral myelitis. Particular focus is placed on the diseases caused by enteroviruses, which as a group account for most aseptic meningitis cases and many focal infections of the spinal cord.

  2. Mechanism for CCC DNA synthesis in hepadnaviruses.

    Directory of Open Access Journals (Sweden)

    Ji A Sohn

    Full Text Available Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC DNA from the relaxed circular (RC viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT, or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1 invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2 predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

  3. Viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in China.

    Science.gov (United States)

    Zhang, Bin; Tang, Cheng; Yue, Hua; Ren, Yupeng; Song, Zhigang

    2014-07-01

    To investigate the diversity of viral flora, we used metagenomics to study the viral communities in a pooled faecal sample of 27 diarrhoeic piglets from intensive commercial farms in China. The 15 distinct mammalian viruses identified in the pooled diarrhoeic sample were, in order of abundance of nucleic acid sequence, Porcine epidemic diarrhea virus (PEDV), sapovirus, porcine bocavirus-4 (PBoV-4), sapelovirus, torovirus, coronavirus, PBoV-2, stool-associated single-stranded DNA virus (poSCV), astrovirus (AstV), kobuvirus, posavirus-1, porcine enterovirus-9 (PEV-9), porcine circovirus-like (po-circo-like) virus, picobirnavirus (PBV) and Torque teno sus virus 2 (TTSuV-2). The prevalence rate of each virus was verified from diarrhoeic and healthy piglets by PCR assay. A mean of 5.5 different viruses were shed in diarrhoeic piglets, and one piglet was in fact co-infected with 11 different viruses. By contrast, healthy piglets shed a mean of 3.2 different viruses. Compared with samples from healthy piglets, the co-infection of PEDV and PBoV had a high prevalence rate in diarrhoea samples, suggesting a correlation with the appearance of diarrhoea in piglets. Furthermore, we report here for the first time the presence of several recently described viruses in China, and the identification of novel genotypes. Therefore, our investigation results provide an unbiased survey of viral communities and prevalence in faecal samples of piglets.

  4. Exploring Viral Genomics at Lawrence Livermore National Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Kilpatrick, K; Hiddessen, A

    2007-08-22

    This summer I had the privilege of working at Lawrence Livermore National Laboratory under the Nonproliferation, Homeland and International Security Directorate in the Chemical and Biological Countermeasures Division. I worked exclusively on the Viral Identification and Characterization Initiative (VICI) project focusing on the development of multiplexed polymerase chain reaction (PCR) assays. The goal of VICI is to combine several disciplines such as molecular biology, microfluidics, and bioinformatics in order to detect viruses and identify them in order to effectively and quickly counter infectious disease, natural or engineered. The difficulty in such a countermeasure is that little is known about viral diversity due to the ever changing nature of these organisms. In response, VICI is developing a new microfluidic bioanalytical platform to detect known and unknown viruses by analyzing every virus in a sample by isolating them into picoliter sized droplets on a microchip and individually analyzing them. The sample will be injected into a channel of oil to form droplets that will contain viral nucleic acids that will be amplified using PCR. The multiplexed PCR assay will produce a series of amplicons for a particular virus genome that provides an identifying signature. A device will then detect whether or not DNA is present in the droplet and will sort the empty droplets from the rest. From this point, the amplified DNA is released from the droplets and analyzed using capillary gel electrophoresis in order to read out the series of amplicons and thereby determine the identity of each virus. The following figure depicts the microfluidic process. For the abovementioned microfluidic process to work, a method for detecting amplification of target viral nucleic acids that does not interfere with the multiplexed biochemical reaction is required for downstream sorting and analysis. In this report, the successful development of a multiplexed PCR assay using SYBR Green I

  5. Microbiological diagnostics of viral hepatitis

    OpenAIRE

    HASDEMİR, Ufuk

    2016-01-01

    Viral hepatitis is an infection that primarily affects the liverbut may also have systemic clinical manifestations. The vastmajority of viral hepatitis are caused by one of five hepatotropicviruses: hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis D (delta) virus (HDV), andhepatitis E virus (HEV) (Table I) [1]. HBV, HCV, and HDValso cause chronic hepatitis, whereas HAV does not. HEVcauses acute hepatitis in normal hosts but can cause protractedand chronic he...

  6. Microbiological diagnostics of viral hepatitis

    OpenAIRE

    HASDEMİR, Ufuk

    2016-01-01

    Viral hepatitis is an infection that primarily affects the liverbut may also have systemic clinical manifestations. The vastmajority of viral hepatitis are caused by one of five hepatotropicviruses: hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), hepatitis D (delta) virus (HDV), andhepatitis E virus (HEV) (Table I) [1]. HBV, HCV, and HDValso cause chronic hepatitis, whereas HAV does not. HEVcauses acute hepatitis in normal hosts but can cause protractedand chronic he...

  7. Ultrashort pulsed laser treatment inactivates viruses by inhibiting viral replication and transcription in the host nucleus.

    Science.gov (United States)

    Tsen, Shaw-Wei D; Chapa, Travis; Beatty, Wandy; Xu, Baogang; Tsen, Kong-Thon; Achilefu, Samuel

    2014-10-01

    Ultrashort pulsed laser irradiation is a new method for virus reduction in pharmaceuticals and blood products. Current evidence suggests that ultrashort pulsed laser irradiation inactivates viruses through an impulsive stimulated Raman scattering process, resulting in aggregation of viral capsid proteins. However, the specific functional defect(s) in viruses inactivated in this manner have not been demonstrated. This information is critical for the optimization and the extension of this treatment platform to other applications. Toward this goal, we investigated whether viral internalization, replication, or gene expression in cells were altered by ultrashort pulsed laser irradiation. Murine Cytomegalovirus (MCMV), an enveloped DNA virus, was used as a model virus. Using electron and fluorescence microscopy, we found that laser-treated MCMV virions successfully internalized in cells, as evidenced by the detection of intracellular virions, which was confirmed by the detection of intracellular viral DNA via PCR. Although the viral DNA itself remained polymerase-amplifiable after laser treatment, no viral replication or gene expression was observed in cells infected with laser-treated virus. These results, along with evidence from previous studies, support a model whereby the laser treatment stabilizes the capsid, which inhibits capsid uncoating within cells. By targeting the mechanical properties of viral capsids, ultrashort pulsed laser treatment represents a unique potential strategy to overcome viral mutational escape, with implications for combatting emerging or drug-resistant pathogens.

  8. Viral control of mitochondrial apoptosis.

    Directory of Open Access Journals (Sweden)

    Lorenzo Galluzzi

    2008-05-01

    Full Text Available Throughout the process of pathogen-host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against infection, disabling host cell apoptosis might represent an almost obligate step in the viral life cycle. Conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system, or to induce the breakdown of infected cells, thereby favoring viral dissemination. Several viral polypeptides are homologs of host-derived apoptosis-regulatory proteins, such as members of the Bcl-2 family. Moreover, viral factors with no homology to host proteins specifically target key components of the apoptotic machinery. Here, we summarize the current knowledge on the viral modulation of mitochondrial apoptosis, by focusing in particular on the mechanisms by which viral proteins control the host cell death apparatus.

  9. Viral RNAs are unusually compact.

    Directory of Open Access Journals (Sweden)

    Ajaykumar Gopal

    Full Text Available A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly.

  10. Anti-viral RNA silencing: do we look like plants ?

    Directory of Open Access Journals (Sweden)

    Lecellier Charles-Henri

    2006-01-01

    Full Text Available Abstract The anti-viral function of RNA silencing was first discovered in plants as a natural manifestation of the artificial 'co-suppression', which refers to the extinction of endogenous gene induced by homologous transgene. Because silencing components are conserved among most, if not all, eukaryotes, the question rapidly arose as to determine whether this process fulfils anti-viral functions in animals, such as insects and mammals. It appears that, whereas the anti-viral process seems to be similarly conserved from plants to insects, even in worms, RNA silencing does influence the replication of mammalian viruses but in a particular mode: micro(miRNAs, endogenous small RNAs naturally implicated in translational control, rather than virus-derived small interfering (siRNAs like in other organisms, are involved. In fact, these recent studies even suggest that RNA silencing may be beneficial for viral replication. Accordingly, several large DNA mammalian viruses have been shown to encode their own miRNAs. Here, we summarize the seminal studies that have implicated RNA silencing in viral infection and compare the different eukaryotic responses.

  11. Use of computer algorithms to reduce viral quasispecies sequence space.

    Science.gov (United States)

    Epperson, E S; Tyrer, H W

    1995-01-01

    A virus may express multiple simple mutations producing a set of viral subspecies called quasispecies: the quasispecies are considered the same species as the original virus. We are interested in reducing the point mutation space to enumerate that sequence space. We form a point mutation by applying a single bit mutation to a strand of viral DNA. How many differing viruses are possible if we allow any of the base pairs to change along the strand? For a strand of arbitrary length n we see that there is a possible sequence space of 4n-1.4 = 4n combinations. We can further remove identical sequences due to redundant amino acid codon encoding. This requires the use of a computer, but this time the complexity is a product: the number of possible amino acids times the number of codons. This substantial reduction from an exponential complexity O(4n) to a product O(n.amino-acid-number) gives us the complete list of mutant viral entities which are one mutation away from the original. Further reduction is possible, but requires biological insight regarding the viability of the mutation. By recognizing the possible sequence space, prediction can be made toward identifying future viral strains of HIV and influenza (to name two important viral particles), and perhaps develop a predictive intervention.

  12. VirSorter: mining viral signal from microbial genomic data

    Directory of Open Access Journals (Sweden)

    Simon Roux

    2015-05-01

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

  13. Hepatitis B virus: pathogenesis, viral intermediates, and viral replication.

    Science.gov (United States)

    Lee, Jia-Yee; Locarnini, Stephen

    2004-05-01

    Although HBV has the potential to generate an almost limitless spectrum of quasispecies during chronic infection, the viability of the majority of these quasispecies is almost certainly impaired due to constraints imposed by the remarkably compact organization of the HBV genome. On the other hand, single mutations may affect more than one gene and result in complex and unpredictable effects on viral phenotype. Better understanding of the constraints imposed by gene overlap and of genotype-phenotype relationships should help in the development of improved antiviral strategies and management approaches. Although the probability of developing viral resistance is directly proportional to the intensity of selection pressure and the diversity of quasispecies, potent inhibition of HBV replication should be able to prevent development of drug resistance because mutagenesis is replication dependent. If viral replication can be suppressed for a sufficient length of time, viral load should decline to a point where the continued production of quasispecies with the potential to resist new drug treatments no longer occurs. Clinical application of this concept will require optimization of combination therapies analogous to highly active antiretroviral therapy (HAART) for HIV infection. Total cure of hepatitis B will require elimination of the intranuclear pool of viral minichromosomes, which will probably only be achieved by normal cell turnover, reactivation of host immunity, or elucidation of the antiviral mechanisms operating during cytokine clearance in acute hepatitis B (see Fig. 1).

  14. Tv-commercial verliest het van virals

    OpenAIRE

    Gisbergen, M.S. van; Ketelaar, P.E.; Baudoin, P,

    2007-01-01

    Viral commercials werken goed. De meeste jongeren bekijken viral commercials en beoordelen ze positiever dan tv-commercials. Doordat viral commercials volgens consumenten voor vermaak zorgen, ontwijken zij ze minder dan tv-commercials. Ook onthouden jongeren de in viral commercials geadverteerde producten en merken goed. Dit blijkt uit onderzoek van de Radboud Universiteit in samenwerking met De Vos & Jansen onder 412 jongeren.

  15. [Neuropsychiatric sequelae of viral meningitis in adults].

    Science.gov (United States)

    Damsgaard, Jesper; Hjerrild, Simon; Renvillard, Signe Groth; Leutscher, Peter Derek Christian

    2011-10-10

    Viral meningitis is considered to be a benign illness with only mild symptoms. In contrast to viral encephalitis and bacterial meningitis, the prognosis is usually good. However, retrospective studies have demonstrated that patients suffering from viral meningitis may experience cognitive impairment following the acute course of infection. Larger controlled studies are needed to elucidate the potential neuropsychiatric adverse outcome of viral meningitis.

  16. IMMUNE INHIBITION OF VIRUS RELEASE FROM HUMAN AND NONHUMAN CELLS BY ANTIBODY TO VIRAL AND HOST-CELL DETERMINANTS

    NARCIS (Netherlands)

    SHARIFF, DM; DESPERBASQUES, M; BILLSTROM, M; GEERLIGS, HJ; WELLING, GW; WELLINGWESTER, S; BUCHAN, A; SKINNER, GRB

    1991-01-01

    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and su

  17. IMMUNE INHIBITION OF VIRUS RELEASE FROM HUMAN AND NONHUMAN CELLS BY ANTIBODY TO VIRAL AND HOST-CELL DETERMINANTS

    NARCIS (Netherlands)

    SHARIFF, DM; DESPERBASQUES, M; BILLSTROM, M; GEERLIGS, HJ; WELLING, GW; WELLINGWESTER, S; BUCHAN, A; SKINNER, GRB

    1991-01-01

    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and

  18. DNA Topology and the Initiation of Virus DNA Packaging.

    Directory of Open Access Journals (Sweden)

    Choon Seok Oh

    Full Text Available 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.

  19. DNA Topology and the Initiation of Virus DNA Packaging.

    Science.gov (United States)

    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.

  20. A thermodynamic theory of codon bias in viral genes.

    Science.gov (United States)

    Rowe, G W; Trainor, L E

    1983-03-21

    The relationship between degeneracy in the genetic code and the occurrence of a strong codon bias is examined, with particular reference to a group of viral genomes. The present paper shows how codon bias may have been imposed by thermodynamic considerations at the time the primitive DNA first formed in the primordial soup. Using a four-state Ising-like model with stacking interactions between successive base pairs, we show how primeval periodic DNA polymers could have arisen the remnants of which are still observed in codon biases today.

  1. Surveillance of viral contamination of invasive medical instruments in dentistry

    Institute of Scientific and Technical Information of China (English)

    ZHOU Lin-fu; ZHU Hai-hong; LIN Jun; HU Min-jun; CHEN Feng; CHEN Zhi

    2006-01-01

    Objective: To investigate the viral contamination of invasive medical instruments in dentistry and to provide health administrative institutions with surveillance data. Methods: Sterilized samples were randomly collected from the department of dentistry to detect HBV-DNA, HCV-RNA, HIV-RNA and HBsAg. Results: Of the invasive medical instruments that were sterilized with 2% glutaraldehyde, one of the samples was positive for HBV-DNA, and another sample was positive for HBsAg.Conclusion: Though massive virus contamination of invasive medical instruments in dentistry has been reduced to a low level, the occurrence of contamination still remains.

  2. Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.

    Directory of Open Access Journals (Sweden)

    Aviad Levin

    Full Text Available BACKGROUND: The human immunodeficiency virus type 1 (HIV-1 integrase protein (IN, catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3'-end processing in which IN removes a pGT dinucleotide from the 3' end of each viral long terminal repeat (LTR. Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication. METHODOLOGY/PRINCIPAL FINDINGS: Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR. CONCLUSIONS/SIGNIFICANCE: The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

  3. Cell-Associated Viral Burden Provides Evidence of Ongoing Viral Replication in Aviremic HIV-2-Infected Patients▿

    Science.gov (United States)

    Soares, Rui S.; Tendeiro, Rita; Foxall, Russell B.; Baptista, António P.; Cavaleiro, Rita; Gomes, Perpétua; Camacho, Ricardo; Valadas, Emília; Doroana, Manuela; Lucas, Margarida; Antunes, Francisco; Victorino, Rui M. M.; Sousa, Ana E.

    2011-01-01

    Viremia is significantly lower in HIV-2 than in HIV-1 infection, irrespective of disease stage. Nevertheless, the comparable proviral DNA burdens observed for these two infections indicate similar numbers of infected cells. Here we investigated this apparent paradox by assessing cell-associated viral replication. We found that untreated HIV-1-positive (HIV-1+) and HIV-2+ individuals, matched for CD4 T cell depletion, exhibited similar gag mRNA levels, indicating that significant viral transcription is occurring in untreated HIV-2+ patients, despite the reduced viremia (undetectable to 2.6 × 104 RNA copies/ml). However, tat mRNA transcripts were observed at significantly lower levels in HIV-2+ patients, suggesting that the rate of de novo infection is decreased in these patients. Our data also reveal a direct relationship of gag and tat transcripts with CD4 and CD8 T cell activation, respectively. Antiretroviral therapy (ART)-treated HIV-2+ patients showed persistent viral replication, irrespective of plasma viremia, possibly contributing to the emergence of drug resistance mutations, persistent hyperimmune activation, and poor CD4 T cell recovery that we observed with these individuals. In conclusion, we provide here evidence of significant ongoing viral replication in HIV-2+ patients, further emphasizing the dichotomy between amount of plasma virus and cell-associated viral burden and stressing the need for antiretroviral trials and the definition of therapeutic guidelines for HIV-2 infection. PMID:21159859

  4. CRISPR/Cas9-mediated viral interference in plants

    KAUST Repository

    Ali, Zahir

    2015-11-11

    Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

  5. [Pathology and viral metagenomics, a recent history].

    Science.gov (United States)

    Bernardo, Pauline; Albina, Emmanuel; Eloit, Marc; Roumagnac, Philippe

    2013-05-01

    Human, animal and plant viral diseases have greatly benefited from recent metagenomics developments. Viral metagenomics is a culture-independent approach used to investigate the complete viral genetic populations of a sample. During the last decade, metagenomics concepts and techniques that were first used by ecologists progressively spread into the scientific field of viral pathology. The sample, which was first for ecologists a fraction of ecosystem, became for pathologists an organism that hosts millions of microbes and viruses. This new approach, providing without a priori high resolution qualitative and quantitative data on the viral diversity, is now revolutionizing the way pathologists decipher viral diseases. This review describes the very last improvements of the high throughput next generation sequencing methods and discusses the applications of viral metagenomics in viral pathology, including discovery of novel viruses, viral surveillance and diagnostic, large-scale molecular epidemiology, and viral evolution.

  6. The role of unintegrated DNA in HIV infection

    Directory of Open Access Journals (Sweden)

    Wainberg Mark A

    2011-07-01

    Full Text Available Abstract Integration of the reverse transcribed viral genome into host chromatin is the hallmark of retroviral replication. Yet, during natural HIV infection, various unintegrated viral DNA forms exist in abundance. Though linear viral cDNA is the precursor to an integrated provirus, increasing evidence suggests that transcription and translation of unintegrated DNAs prior to integration may aid productive infection through the expression of early viral genes. Additionally, unintegrated DNA has the capacity to result in preintegration latency, or to be rescued and yield productive infection and so unintegrated DNA, in some circumstances, may be considered to be a viral reservoir. Recently, there has been interest in further defining the role and function of unintegrated viral DNAs, in part because the use of anti-HIV integrase inhibitors leads to an abundance of unintegrated DNA, but also because of the potential use of non-integrating lentiviral vectors in gene therapy and vaccines. There is now increased understanding that unintegrated viral DNA can either arise from, or be degraded through, interactions with host DNA repair enzymes that may represent a form of host antiviral defence. This review focuses on the role of unintegrated DNA in HIV infection and additionally considers the potential implications for antiviral therapy.

  7. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  8. The bacteriophage DNA packaging machine.

    Science.gov (United States)

    Feiss, Michael; Rao, Venigalla B

    2012-01-01

    Large dsDNA bacteriophages and herpesviruses encode a powerful ATP-driven DNA-translocating machine that encapsidates a viral genome into a preformed capsid shell or prohead. The key components of the packaging machine are the packaging enzyme (terminase, motor) and the portal protein that forms the unique DNA entrance vertex of prohead. The terminase complex, comprised of a recognition subunit (small terminase) and an endonuclease/translocase subunit (large terminase), cuts viral genome concatemers. The terminase-viral DNA complex docks on the portal vertex, assembling a motor complex containing five large terminase subunits. The pentameric motor processively translocates DNA until the head shell is full with one viral genome. The motor cuts the DNA again and dissociates from the full head, allowing head-finishing proteins to assemble on the portal, sealing the portal, and constructing a platform for tail attachment. A body of evidence from molecular genetics and biochemical, structural, and biophysical approaches suggests that ATP hydrolysis-driven conformational changes in the packaging motor (large terminase) power DNA motion. Various parts of the motor subunit, such as the ATPase, arginine finger, transmission domain, hinge, and DNA groove, work in concert to translocate about 2 bp of DNA per ATP hydrolyzed. Powerful single-molecule approaches are providing precise delineation of steps during each translocation event in a motor that has a speed as high as a millisecond/step. The phage packaging machine has emerged as an excellent model for understanding the molecular machines, given the mechanistic parallels between terminases, helicases, and numerous motor proteins.

  9. Studies of the silencing of Baculovirus DNA binding protein

    OpenAIRE

    Quadt, I.; Lent, van, J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P1...

  10. The impact of cidofovir treatment on viral loads in adult recurrent respiratory papillomatosis.

    Science.gov (United States)

    Mikolajczak, S; Quante, G; Weissenborn, S; Wafaisade, A; Wieland, U; Lüers, J C; Klussmann, J P; Beutner, D

    2012-12-01

    Cidofovir is an antiviral agent used in the therapy of recurrent respiratory papillomatosis (RRP). In this study, we hypothesized that cidofovir is effective in decreasing the viral load of human papillomavirus (HPV). We established a type specific real-time PCR and measured HPV DNA loads. The course of viral load of HPV types 6 and 11 after repeated applications of cidofovir intralesionally was compared to the clinical outcome using a modified Derkay score. In 6 of the 8 (75 %) patients, we detected HPV 6. In 2 (25 %) patients, we detected HPV 11. In all of the patients, the viral load and the modified Derkay score decreased significantly during the treatment. We conclude that viral load of HPV can be monitored using the technique described here. Cidofovir in combination with surgical debulking reduces the viral load in patients with RRP. Relapses of the symptoms cannot be avoided but might be delayed.

  11. Beyond viral suppression of HIV

    DEFF Research Database (Denmark)

    Lazarus, Jeffrey V.; Safreed-Harmon, Kelly; Barton, Simon E

    2016-01-01

    BACKGROUND: In 2016, the World Health Organization (WHO) adopted a new Global Health Sector Strategy on HIV for 2016-2021. It establishes 15 ambitious targets, including the '90-90-90' target calling on health systems to reduce under-diagnosis of HIV, treat a greater number of those diagnosed......, and ensure that those being treated achieve viral suppression. DISCUSSION: The WHO strategy calls for person-centered chronic care for people living with HIV (PLHIV), implicitly acknowledging that viral suppression is not the ultimate goal of treatment. However, it stops short of providing an explicit target...... for health-related quality of life. It thus fails to take into account the needs of PLHIV who have achieved viral suppression but still must contend with other intense challenges such as serious non-communicable diseases, depression, anxiety, financial stress, and experiences of or apprehension about HIV...

  12. Integrin Activation and Viral Infection

    Institute of Scientific and Technical Information of China (English)

    Shan-dian GAO; Jun-zheng DU; Jian-hua ZHOU; Hui-yun CHANG; Qing-ge XIE

    2008-01-01

    Integrins are members of a ubiquitous membrane receptor family which includes 18 different α subunits and 8 β subunits forming more than 20 α/β heterodimers. Integrins play key functions in vascular endothelial cell and tumour cell adhesion, lymphocyte trafficking, tumor growth and viral infection. Current understanding of the molecular basis of integrins as viral receptors has been achieved through many decades of study into the biology of transmembrane glycoproteins and their interactions with several viruses. This review provides a summary of the current knowledge on the molecular bases of interactions between viruses and integrins, which are of potential practical significance. Inhibition of virus-integrin interactions at the points of virus attachment or entry will provide a novel approach for the therapeutic treatment of viral diseases.

  13. Enfermedades virales emergentes y reemergentes

    Directory of Open Access Journals (Sweden)

    Jorge Eliécer Ossa Londoño

    1996-03-01

    Full Text Available Los virus no son una excepción al principio de que toda forma de vida de hoyes el producto de la evolución de información gen ética preexistente. Tradicionalmente se ha reconocido que ta expresión clínica de las enfermedades virales cambia con el tiempo; molecularmente se ha demostrado que esos cambios fenotípicos son el producto de variaciones en el genoma viral. La tasa de cambio
    gen ético y fenotípico no es la misma en todos los agentes virales y ello está determinado, principalmente, por factores intrínsecos del virus, como la naturaleza de su ácido nucleico, y por la longevidad
    y tasa reproductiva del huésped.

  14. Acute bacterial and viral meningitis.

    Science.gov (United States)

    Bartt, Russell

    2012-12-01

    Most cases of acute meningitis are infectious and result from a potentially wide range of bacterial and viral pathogens. The organized approach to the patient with suspected meningitis enables the prompt administration of antibiotics, possibly corticosteroids, and diagnostic testing with neuroimaging and spinal fluid analysis. Acute meningitis is infectious in most cases and caused by a potentially wide range of bacterial and viral pathogens. Shifts in the epidemiology of bacterial pathogens have been influenced by changes in vaccines and their implementation. Seasonal and environmental changes influence the likely viral and rickettsial pathogens. The organized approach to the patient with suspected meningitis enables the prompt administration of antibiotics, possibly corticosteroids, and diagnostic testing with neuroimaging and spinal fluid analysis. Pertinent testing and treatment can vary with the clinical presentation, season, and possible exposures. This article reviews the epidemiology, clinical presentation, diagnosis, and treatment of acute meningitis.

  15. Cutaneous manifestations of viral hepatitis.

    Science.gov (United States)

    Akhter, Ahmed; Said, Adnan

    2015-02-01

    There are several extrahepatic cutaneous manifestations associated with hepatitis B and hepatitis C virus infection. Serum sickness and polyarteritis nodosa are predominantly associated with hepatitis B infection, whereas mixed cryoglobulinemia associated vasculitis and porphyria cutanea tarda are more frequently seen in hepatitis C infection. The clinico-pathogenic associations of these skin conditions are not completely defined but appear to involve activation of the host immune system including the complement system. Management of the aforementioned cutaneous manifestations of viral hepatitis is often similar to that done in cases without viral hepatitis, with control of immune activation being a key strategy. In cases associated with hepatitis B and C, control of viral replication with specific antiviral therapy is also important and associated with improvement in most of the associated clinical manifestations.

  16. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies.

    Directory of Open Access Journals (Sweden)

    Susanne Eriksson

    2013-02-01

    Full Text Available HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART. The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4(+ T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4(+ T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4(+ T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy

  17. Viral infections and cell cycle G2/M regulation

    Institute of Scientific and Technical Information of China (English)

    Richard Y.ZHAO; Robert T.ELDER

    2005-01-01

    Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15(Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well-characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

  18. Going Viral with Fluorescent Proteins.

    Science.gov (United States)

    Costantini, Lindsey M; Snapp, Erik L

    2015-10-01

    Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

  19. [The vaccines based on the replicon of the venezuelan equine encephalomyelitis virus against viral hemorrhagic fevers].

    Science.gov (United States)

    Petrov, A A; Plekhanova, T M; Sidorova, O N; Borisevich, S V; Makhlay, A A

    2015-01-01

    The status of the various recombinant DNA and RNA-derived candidate vaccines, as well as the Venezuelan equine encephalomyelitis virus (VEEV) replicon vaccine system against extremely hazardous viral hemorrhagic fevers, were reviewed. The VEEV-based replication-incompetent vectors offer attractive features in terms of safety, high expression levels of the heterologous viral antigen, tropism to dendritic cells, robust immune responses, protection efficacy, low potential for pre-existing anti-vector immunity and possibility of engineering multivalent vaccines were tested. These features of the VEEV replicon system hold much promise for the development of new generation vaccine candidates against viral hemorrhagic fevers.

  20. Physical status and viral load in women with positive human papillomavirus (HPV) infection in uterine cervix

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Byoung Gie; Lee, Eui Don; Zin, Yong Jae [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1998-01-01

    This study was performed to determine the frequency of viral integration and viral load in women with positive HPV type 16 infection, and showing normal findings, CIN, and cervical cancer. Total 75 (normal, 15; CIN I, 20; CIN III, 20; cervical cancer, 20) cervical swab specimens were used. HPV detection, typing, and viral load was determined by PCR method. Seventy of 75 (93.3%) of cervical swab specimens showed same results with hybrid capture assay and PCR method for detecting HPV DNA. HPV type 16 DNA was identified more frequently with progression from normal to cervical cancer (normal, 13 %; CIN I, 15%; CIN III, 40 %; cervical cancer, 55 %). The frequency of HPV type 16 DNA integration also increased with grade of the lesion (normal, 0 %; CIN I, 33 %; CIN III, 87 %; cervical cancer, 91 %) suggesting most of HPV type 16 present as integration forms in the cells. In addition, high-level of HPV 16 viral load also was found more frequently in CIN III and cervical cancer (normal, 0 %; CIN I, 0 %; CIN III, 87 %; cervical cancer, 100 %). These results suggest that viral integration and high-level of viral load may play an important role in cervical carcinogenesis. (author). 13 refs., 5 figs.

  1. Viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis.

    Directory of Open Access Journals (Sweden)

    Ashish V Chintakuntlawar

    2010-04-01

    Full Text Available Human adenovirus (HAdV infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9 signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/- mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD. These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.

  2. Mechanism of membranous tunnelling nanotube formation in viral genome delivery.

    Directory of Open Access Journals (Sweden)

    Bibiana Peralta

    2013-09-01

    Full Text Available In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.

  3. Virally vectored vaccine delivery: medical needs, mechanisms, advantages and challenges.

    Science.gov (United States)

    Pinschewer, Daniel D

    2017-08-14

    Vaccines represent one of the most successful chapters in the history of medicine. Over the past decades, the advent of recombinant cDNA technology has enabled the biomedical community to genetically engineer viruses for vaccine delivery purposes. As a starting point, this review evaluates the unmet medical needs, which drive scientists and industry to exploit such fundamentally new technology for human vaccination. The author discusses the molecular functioning, production and safety profile of replication-competent and -deficient viral vector systems, representing two fundamentally distinct classes of "genetic vaccines". Building upon this knowledge, he dissects the immunological mechanisms rendering immune responses to viral vectors qualitatively and quantitatively distinct from those elicited by non-live vaccination approaches. These mechanisms comprise (1) the vectors' innate immune recognition by the host cell, (2) potent priming of CD8+ cytotoxic T cells as a result of dendritic cell targeting and endogenous protein synthesis, (3) conformational antigen display for protective antibody induction as well as (4) prolonged availability of substantial quantities of antigen. Deduced from these features, preferential indications for virally vectored vaccines are discussed, taking into consideration specific medical needs as well as risk-benefit assessments of replicating vector systems. The limitations and challenges in virally vectored vaccination must also be given careful consideration. Pre-existing and vaccination-induced anti-vector immunity can interfere with vaccine immunogenicity and prime-boost vaccination, respectively. Additionally, the requirement for eukaryotic production systems imposes technological as well as regulatory hurdles. Existing strategies to overcome these challenges are outlined. With the recent licensure of the first virally vectored vaccine this review seems timely to herald the introduction of virally vectored vaccines into daily

  4. Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B

    Institute of Scientific and Technical Information of China (English)

    Gerald C Kimbi; Anna Kramvis; Michael C Kew

    2005-01-01

    AIM: To examine the serum from black African patients with acute hepatitis B to ascertain if integrants of viral DNA can be detected in fragments of cellular DNA leaking from damaged hepatocytes into the circulation.METHODS: DNA was extracted from the sera of five patients with uncomplicated acute hepatitis B and one with fulminant disease. Two subgenomic PCRs designed to amplify the complete genome of HBV were used and the resulting amplicons were cloned and sequenced.RESULTS: HBV and chromosomal DNA were amplified from the sera of all the patients. In one patient with uncomplicated disease, HBV DNA was integrated into host chromosome 7 q11.23 in the WBSCR1 gene. The viral DNA comprised 200 nucleotides covering the S and X genes in opposite orientation, with a 1 169 nudeotide deletion. The right virus/host junction was situated at nucleotide 1774 in the cohesive overlap region of the viral genome, at a preferred topoisomerase I cleavage motif. The chromosomal DNA was not rearranged.The patient made a full recovery and seroconverted to anti-HBs- and anti-HBe-positivity. Neither HBV nor chromosomal DNA could be amplified from his serum at that time.CONCLUSION: Integration of viral DNA into chromosomal DNA may occur rarely during acute hepatitis B and, with clonal propagation of the integrant, might play a role in hepatocarcinogenesis.

  5. Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes

    Science.gov (United States)

    Dembowski, Jill A.

    2017-01-01

    Herpes simplex virus type 1 (HSV-1) infects over half the human population. Much of the infectious cycle occurs in the nucleus of cells where the virus has evolved mechanisms to manipulate host processes for the production of virus. The genome of HSV-1 is coordinately expressed, maintained, and replicated such that progeny virions are produced within 4–6 hours post infection. In this study, we selectively purify HSV-1 replication forks and associated proteins from virus-infected cells and identify select viral and cellular replication, repair, and transcription factors that associate with viral replication forks. Pulse chase analyses and imaging studies reveal temporal and spatial dynamics between viral replication forks and associated proteins and demonstrate that several DNA repair complexes and key transcription factors are recruited to or near replication forks. Consistent with these observations we show that the initiation of viral DNA replication is sufficient to license late gene transcription. These data provide insight into mechanisms that couple HSV-1 DNA replication with transcription and repair for the coordinated expression and maintenance of the viral genome. PMID:28095497

  6. CK2 phosphorylation inactivates DNA binding by the papillomavirus E1 and E2 proteins.

    Science.gov (United States)

    Schuck, Stephen; Ruse, Cristian; Stenlund, Arne

    2013-07-01

    Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity. Phosphorylation at multiple sites in the N-terminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1.

  7. Ventilator and viral induced inflammation

    NARCIS (Netherlands)

    Hennus, M.P.

    2013-01-01

    This thesis expands current knowledge on ventilator induced lung injury and provides insights on the immunological effects of mechanical ventilation during viral respiratory infections. The experimental studies in the first part of this thesis improve our understanding of how mechanical ventilation

  8. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  9. Mast cells in viral infections

    Directory of Open Access Journals (Sweden)

    Piotr Witczak

    2012-04-01

    Full Text Available  There are some premises suggesting that mast cells are involved in the mechanisms of anti-virus defense and in viral disease pathomechanisms. Mast cells are particularly numerous at the portals of infections and thus may have immediate and easy contact with the external environment and invading pathogens. These cells express receptors responsible for recognition of virus-derived PAMP molecules, mainly Toll-like receptors (TLR3, TLR7/8 and TLR9, but also RIG-I-like and NOD-like molecules. Furthermore, mast cells generate various mediators, cytokines and chemokines which modulate the intensity of inflammation and regulate the course of innate and adaptive anti-viral immunity. Indirect evidence for the role of mast cells in viral infections is also provided by clinical observations and results of animal studies. Currently, more and more data indicate that mast cells can be infected by some viruses (dengue virus, adenoviruses, hantaviruses, cytomegaloviruses, reoviruses, HIV-1 virus. It is also demonstrated that mast cells can release pre formed mediators as well as synthesize de novo eicosanoids in response to stimulation by viruses. Several data indicate that virus-stimulated mast cells secrete cytokines and chemokines, including interferons as well as chemokines with a key role in NK and Tc lymphocyte influx. Moreover, some information indicates that mast cell stimulation via TLR3, TLR7/8 and TLR9 can affect their adhesion to extracellular matrix proteins and chemotaxis, and influence expression of some membrane molecules. Critical analysis of current data leads to the conclusion that it is not yet possible to make definitive statements about the role of mast cells in innate and acquired defense mechanisms developing in the course of viral infection and/or pathomechanisms of viral diseases.

  10. Autistic disorder and viral infections.

    Science.gov (United States)

    Libbey, Jane E; Sweeten, Thayne L; McMahon, William M; Fujinami, Robert S

    2005-02-01

    Autistic disorder (autism) is a behaviorally defined developmental disorder with a wide range of behaviors. Although the etiology of autism is unknown, data suggest that autism results from multiple etiologies with both genetic and environmental contributions, which may explain the spectrum of behaviors seen in this disorder. One proposed etiology for autism is viral infection very early in development. The mechanism, by which viral infection may lead to autism, be it through direct infection of the central nervous system (CNS), through infection elsewhere in the body acting as a trigger for disease in the CNS, through alteration of the immune response of the mother or offspring, or through a combination of these, is not yet known. Animal models in which early viral infection results in behavioral changes later in life include the influenza virus model in pregnant mice and the Borna disease virus model in newborn Lewis rats. Many studies over the years have presented evidence both for and against the association of autism with various viral infections. The best association to date has been made between congenital rubella and autism; however, members of the herpes virus family may also have a role in autism. Recently, controversy has arisen as to the involvement of measles virus and/or the measles, mumps, rubella (MMR) vaccine in the development of autism. Biological assays lend support to the association between measles virus or MMR and autism whereas epidemiologic studies show no association between MMR and autism. Further research is needed to clarify both the mechanisms whereby viral infection early in development may lead to autism and the possible involvement of the MMR vaccine in the development of autism.

  11. Illuminating structural proteins in viral "dark matter" with metaproteomics.

    Science.gov (United States)

    Brum, Jennifer R; Ignacio-Espinoza, J Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M; Roux, Simon; VerBerkmoes, Nathan C; Rich, Virginia I; Sullivan, Matthew B

    2016-03-01

    Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter.

  12. Aptamers in Diagnostics and Treatment of Viral Infections

    Directory of Open Access Journals (Sweden)

    Tomasz Wandtke

    2015-02-01

    Full Text Available Aptamers are in vitro selected DNA or RNA molecules that are capable of binding a wide range of nucleic and non-nucleic acid molecules with high affinity and specificity. They have been conducted through the process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment. It serves to reach specificity and considerable affinity to target molecules, including those of viral origin, both proteins and nucleic acids. Properties of aptamers allow detecting virus infected cells or viruses themselves and make them competitive to monoclonal antibodies. Specific aptamers can be used to interfere in each stage of the viral replication cycle and also inhibit its penetration into cells. Many current studies have reported possible application of aptamers as a treatment or diagnostic tool in viral infections, e.g., HIV (Human Immunodeficiency Virus, HBV (Hepatitis B Virus, HCV (Hepatitis C Virus, SARS (Severe Acute Respiratory Syndrome, H5N1 avian influenza and recently spread Ebola. This review presents current developments of using aptamers in the diagnostics and treatment of viral diseases.

  13. Classification of viral zoonosis through receptor pattern analysis

    Directory of Open Access Journals (Sweden)

    Son Hyeon

    2011-04-01

    Full Text Available Abstract Background Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses. Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell. Results We analysed host-cell receptor sequences for their hydrophobicity/hydrophilicity characteristics. We then analysed these properties for similarities among receptors of different species and used a statistical discriminant analysis to predict the likelihood of transmission among species. Conclusions This study is an attempt to predict zoonosis through simple computational analysis of receptor sequence differences. Our method may be useful in predicting the zoonotic potential of newly discovered viral strains.

  14. Classification of viral zoonosis through receptor pattern analysis.

    Science.gov (United States)

    Bae, Se-Eun; Son, Hyeon Seok

    2011-04-13

    Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses). Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell. We analysed host-cell receptor sequences for their hydrophobicity/hydrophilicity characteristics. We then analysed these properties for similarities among receptors of different species and used a statistical discriminant analysis to predict the likelihood of transmission among species. This study is an attempt to predict zoonosis through simple computational analysis of receptor sequence differences. Our method may be useful in predicting the zoonotic potential of newly discovered viral strains.

  15. Structural and Functional Insights into Foamy Viral Integrase

    Directory of Open Access Journals (Sweden)

    Cha-Gyun Shin

    2013-07-01

    Full Text Available Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN and orchestrated by 3'-end processing and the strand transfer reaction. In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN have been described well in several reports. Recent studies on the three‑dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.

  16. Evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life

    DEFF Research Database (Denmark)

    Prangishvili, D.; Garrett, R. A.; Koonin, E.

    2006-01-01

    of bacteriophages. The proteins encoded by the genes belonging to this pool include predicted transcription regulators, ATPases implicated in viral DNA replication and packaging, enzymes of DNA precursor metabolism, RNA modification enzymes, and glycosylases. In addition, each of the crenarchaeal viruses encodes...... several proteins with prokaryotic but not viral homologs, some of which, predictably, seem to have been scavenged from the crenarchaeal hosts, but others might have been acquired from bacteria. We conclude that crenarchaeal viruses are, in general, evolutionarily unrelated to other known viruses and......, probably, evolved via independent accretion of genes derived from the hosts and, through more complex routes of horizontal gene transfer, from other prokaryotes....

  17. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    OpenAIRE

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G.

    1997-01-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene...

  18. Non-random patterns in viral diversity

    DEFF Research Database (Denmark)

    Anthony, Simon J.; Islam, Ariful; Johnson, Christine

    2015-01-01

    It is currently unclear whether changes in viral communities will ever be predictable. Here we investigate whether viral communities in wildlife are inherently structured (inferring predictability) by looking at whether communities are assembled through deterministic (often predictable) or stocha...

  19. Endemic Poultry Viral Diseases 2016 Research Update

    Science.gov (United States)

    Viral infections of the avian gastrointestinal tract negatively impact poultry production; however, determining the complex etiologies of the viral enteric diseases in poultry has been difficult. Project scientists are continuing to investigate the species specificity, molecular phylogenetics, and p...

  20. Exploring the viral world through metagenomics.

    Science.gov (United States)

    Rosario, Karyna; Breitbart, Mya

    2011-10-01

    Viral metagenomics, or shotgun sequencing of purified viral particles, has revolutionized the field of environmental virology by allowing the exploration of viral communities in a variety of sample types throughout the biosphere. The introduction of viral metagenomics has demonstrated that dominant viruses in environmental communities are not well-represented by the cultured viruses in existing sequence databases. Viral metagenomic studies have provided insights into viral ecology by elucidating the genetic potential, community structure, and biogeography of environmental viruses. In addition, viral metagenomics has expanded current knowledge of virus-host interactions by uncovering genes that may allow viruses to manipulate their hosts in unexpected ways. The intrinsic potential for virus discovery through viral metagenomics can help advance a wide array of disciplines including evolutionary biology, pathogen surveillance, and biotechnology.

  1. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication.

    Science.gov (United States)

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the

  2. Viral load of human bocavirus-1 in stools from children with viral diarrhoea in Paraguay.

    Science.gov (United States)

    Proenca-Modena, J L; Martinez, M; Amarilla, A A; Espínola, E E; Galeano, M E; Fariña, N; Russomando, G; Aquino, V H; Parra, G I; Arruda, E

    2013-12-01

    Since their discovery, four species of human bocavirus (HBoV) have been described in patients with respiratory and gastrointestinal diseases. However, a clear causal association between HBoV-1 and gastroenteritis has not been demonstrated. In this study, we describe the detection and quantification of HBoV-1 in stools from children with acute non-bacterial gastroenteritis using quantitative polymerase chain reaction. HBoV-1 genome was detected in 10.6% of stools with frequent association with rotavirus and norovirus. The median of HBoV-1 viral load was 1.88 × 104 genome/ml, lower than previously shown in secretions of patients with respiratory infections, without any obvious association between high viral load and presence of HBoV as single agent. Thus, although HBoV-1 was frequently detected in these patients, there is no clear causal association of this agent with diarrhoea. Indeed, HBoV-1 DNA in stools of patients with gastroenteritis without respiratory symptoms may be a remnant of previous infections or associated with prolonged shedding of virus in the respiratory or digestive tracts.

  3. DNA/chitosan electrostatic complex.

    Science.gov (United States)

    Bravo-Anaya, Lourdes Mónica; Soltero, J F Armando; Rinaudo, Marguerite

    2016-07-01

    Up to now, chitosan and DNA have been investigated for gene delivery due to chitosan advantages. It is recognized that chitosan is a biocompatible and biodegradable non-viral vector that does not produce immunological reactions, contrary to viral vectors. Chitosan has also been used and studied for its ability to protect DNA against nuclease degradation and to transfect DNA into several kinds of cells. In this work, high molecular weight DNA is compacted with chitosan. DNA-chitosan complex stoichiometry, net charge, dimensions, conformation and thermal stability are determined and discussed. The influence of external salt and chitosan molecular weight on the stoichiometry is also discussed. The isoelectric point of the complexes was found to be directly related to the protonation degree of chitosan. It is clearly demonstrated that the net charge of DNA-chitosan complex can be expressed in terms of the ratio [NH3(+)]/[P(-)], showing that the electrostatic interactions between DNA and chitosan are the main phenomena taking place in the solution. Compaction of DNA long chain complexed with low molar mass chitosan gives nanoparticles with an average radius around 150nm. Stable nanoparticles are obtained for a partial neutralization of phosphate ionic sites (i.e.: [NH3(+)]/[P(-)] fraction between 0.35 and 0.80).

  4. Drug Use and Viral Infections (HIV, Hepatitis)

    Science.gov (United States)

    ... Genetics Global Health Health Consequences of Drug Misuse Hepatitis (Viral) HIV/AIDS Mental Health Military Opioid Overdose Reversal ... Publications » DrugFacts » Drug Use and Viral Infections (HIV, Hepatitis) Drug Use and Viral Infections (HIV, Hepatitis) Email Facebook Twitter Revised March ...

  5. Viral commercials: the consumer as marketeer

    NARCIS (Netherlands)

    Ketelaar, P.E.; Lucassen, P.; Kregting, G.H.J.

    2010-01-01

    Research into the reasons why consumers pass along viral commercials: their motives, the content characteristics of viral commercials and the medium context in which viral commercials appear. Based on the uses and gratifications perspective this study has determined which motives of consumers,

  6. Viral ecology of a shallow eutrophic lake

    NARCIS (Netherlands)

    Tijdens, M.

    2007-01-01

    This thesis aims to give an insight into the ecology of the viral community in a shallow eutrophic lake. To achieve this, the population dynamics, diversity and control of the viral community in Lake Loosdrecht were studied, as well as the impact of the viral community on plankton mortality and comm

  7. Assembly of viral genomes from metagenomes

    NARCIS (Netherlands)

    S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

    2014-01-01

    textabstractViral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow r

  8. DNA vaccine for cancer immunotherapy.

    Science.gov (United States)

    Yang, Benjamin; Jeang, Jessica; Yang, Andrew; Wu, T C; Hung, Chien-Fu

    2014-01-01

    DNA vaccination has emerged as an attractive immunotherapeutic approach against cancer due to its simplicity, stability, and safety. Results from numerous clinical trials have demonstrated that DNA vaccines are well tolerated by patients and do not trigger major adverse effects. DNA vaccines are also very cost effective and can be administered repeatedly for long-term protection. Despite all the practical advantages, DNA vaccines face challenges in inducing potent antigen specific cellular immune responses as a result of immune tolerance against endogenous self-antigens in tumors. Strategies to enhance immunogenicity of DNA vaccines against self-antigens have been investigated including encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. This review will focus on discussing strategies that circumvent immune tolerance and provide updates on findings from recent clinical trials.

  9. Non-viral vectors for gene-based therapy.

    Science.gov (United States)

    Yin, Hao; Kanasty, Rosemary L; Eltoukhy, Ahmed A; Vegas, Arturo J; Dorkin, J Robert; Anderson, Daniel G

    2014-08-01

    Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions. This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides. Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors. In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials. The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches.

  10. Hepatoepigenetic Alterations in Viral and Nonviral-Induced Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Mankgopo M. Kgatle

    2016-01-01

    Full Text Available Hepatocellular carcinoma (HCC is a major public health concern and one of the leading causes of tumour-related deaths worldwide. Extensive evidence endorses that HCC is a multifactorial disease characterised by hepatic cirrhosis mostly associated with chronic inflammation and hepatitis B/C viral infections. Interaction of viral products with the host cell machinery may lead to increased frequency of genetic and epigenetic aberrations that cause harmful alterations in gene transcription. This may provide a progressive selective advantage for neoplastic transformation of hepatocytes associated with phenotypic heterogeneity of intratumour HCC cells, thus posing even more challenges in HCC treatment development. Epigenetic aberrations involving DNA methylation, histone modifications, and noncoding miRNA dysregulation have been shown to be intimately linked with and play a critical role in tumour initiation, progression, and metastases. The current review focuses on the aberrant hepatoepigenetics events that play important roles in hepatocarcinogenesis and their utilities in the development of HCC therapy.

  11. Virally encoded 7TM receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Waldhoer, M; Lüttichau, H R

    2001-01-01

    A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion...... in various parts of the viral life cyclus. Most of the receptors encoded by human pathogenic virus are still orphan receptors, i.e. the endogenous ligand is unknown. In the few cases where it has been possible to characterize these receptors pharmacologically, they have been found to bind a broad spectrum...... expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets....

  12. [Recent acquisitions on viral hepatitis].

    Science.gov (United States)

    Resti, M; Tucci, F; Vierucci, A

    1990-01-01

    In the last years the research on viral hepatitis let to better understand the biological, molecular, immunological and epidemiologic characteristics of the viruses that are responsible for hepatitis. The first studied virus was hepatitis B virus (HBv). The scientific attention is still, today, focused on that virus since new markers of infectivity and biological importance in early diagnosis and in disease evolution have been found. The most important result in the last years in the field of viral hepatitis has been, however, the identification of agents responsible for Non-A-Non-B hepatitis. Its epidemiology and clinical importance are discussed in the present paper. Virus C is the most important parenteral agent of NANB hepatitis. Its epidemiology in at risk populations and its role in post-transfusional and cryptogenetic hepatitis are here discussed. The research of new markers of HCV infection is today considered a main goal since the role of the only marker now available is still under discussion.

  13. Viral diseases and human evolution

    Directory of Open Access Journals (Sweden)

    Leal Élcio de Souza

    2000-01-01

    Full Text Available The interaction of man with viral agents was possibly a key factor shaping human evolution, culture and civilization from its outset. Evidence of the effect of disease, since the early stages of human speciation, through pre-historical times to the present suggest that the types of viruses associated with man changed in time. As human populations progressed technologically, they grew in numbers and density. As a consequence different viruses found suitable conditions to thrive and establish long-lasting associations with man. Although not all viral agents cause disease and some may in fact be considered beneficial, the present situation of overpopulation, poverty and ecological inbalance may have devastating effets on human progress. Recently emerged diseases causing massive pandemics (eg., HIV-1 and HCV, dengue, etc. are becoming formidable challenges, which may have a direct impact on the fate of our species.

  14. RHEUMATIC MANIFESTATIONS IN VIRAL HEPATITIS

    Directory of Open Access Journals (Sweden)

    L P Anan'eva

    2008-01-01

    Full Text Available Autoimmune reactions are of primary importance in the development of extrahepatic manifestations of viral hepatitis, among which there are rheumatic symptoms and syndromes. The incidence of clinically significant extrahepatic manifestations is shown to be relatively low, but they may be in the foreground in the clinical picture of the disease and are noted for severity. It is concluded that due to the high prevalence of hepatitis and the systemic pattern of their chronic forms, patients with extrahepatic manifestations of viral hepatitis may be encountered in the practice of a therapist and a rheumatologist. The onset of the infection caused by hepatitis viruses may be accompanied by articular lesion therefore the rheumatologist may be the first physician such a patient may resort to.

  15. RHEUMATIC MANIFESTATIONS IN VIRAL HEPATITIS

    Directory of Open Access Journals (Sweden)

    L P Anan'eva

    2008-12-01

    Full Text Available Autoimmune reactions are of primary importance in the development of extrahepatic manifestations of viral hepatitis, among which there are rheumatic symptoms and syndromes. The incidence of clinically significant extrahepatic manifestations is shown to be relatively low, but they may be in the foreground in the clinical picture of the disease and are noted for severity. It is concluded that due to the high prevalence of hepatitis and the systemic pattern of their chronic forms, patients with extrahepatic manifestations of viral hepatitis may be encountered in the practice of a therapist and a rheumatologist. The onset of the infection caused by hepatitis viruses may be accompanied by articular lesion therefore the rheumatologist may be the first physician such a patient may resort to.

  16. DNA-templated assembly of viral protein hydrogel

    Science.gov (United States)

    Xu, Xin; Tao, Ailin; Xu, Yun

    2014-11-01

    Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs.Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02414a

  17. Viral diseases and human evolution

    OpenAIRE

    2000-01-01

    The interaction of man with viral agents was possibly a key factor shaping human evolution, culture and civilization from its outset. Evidence of the effect of disease, since the early stages of human speciation, through pre-historical times to the present suggest that the types of viruses associated with man changed in time. As human populations progressed technologically, they grew in numbers and density. As a consequence different viruses found suitable conditions to thrive and establish l...

  18. Treatment of acute viral bronchiolitis.

    Science.gov (United States)

    Eber, Ernst

    2011-01-01

    Acute viral bronchiolitis represents the most common lower respiratory tract infection in infants and young children and is associated with substantial morbidity and mortality. Respiratory syncytial virus is the most frequently identified virus, but many other viruses may also cause acute bronchiolitis. There is no common definition of acute viral bronchiolitis used internationally, and this may explain part of the confusion in the literature. Most children with bronchiolitis have a self limiting mild disease and can be safely managed at home with careful attention to feeding and respiratory status. Criteria for referral and admission vary between hospitals as do clinical practice in the management of acute viral bronchiolitis, and there is confusion and lack of evidence over the best treatment for this condition. Supportive care, including administration of oxygen and fluids, is the cornerstone of current treatment. The majority of infants and children with bronchiolitis do not require specific measures. Bronchodilators should not be routinely used in the management of acute viral bronchiolitis, but may be effective in some patients. Most of the commonly used management modalities have not been shown to have a clear beneficial effect on the course of the disease. For example, inhaled and systemic corticosteroids, leukotriene receptor antagonists, immunoglobulins and monoclonal antibodies, antibiotics, antiviral therapy, and chest physiotherapy should not be used routinely in the management of bronchiolitis. The potential effect of hypertonic saline on the course of the acute disease is promising, but further studies are required. In critically ill children with bronchiolitis, today there is little justification for the use of surfactant and heliox. Nasal continuous positive airway pressure may be beneficial in children with severe bronchiolitis but a large trial is needed to determine its value. Finally, very little is known on the effect of the various

  19. STUDY OF PERSISTENT VIRAL INFECTION IN AN ANIMAL MODEL OF VIRAL MYOCARDITIS BY PCR

    Institute of Scientific and Technical Information of China (English)

    马睿; 陈曙霞; 刘晶星

    2000-01-01

    ffeStnn6 Objectif Etudier ie r6le de l'infection virale persistante dans ie pethog4de de la myOCardite virale.ANt~ L' ARN viral dens ie my~rde et ie mug et l' alteration potholedque du m~rde ent ate ewilnd per la techniquede PCR adns un mangle de myrmrdite virale chez ies ~ris. Rhaltats L 'ARN viral a ate detects an 3'jour dens ie mug etie myrmrde. An 8'jour, I 'ARN viral an niveau du mug a ate pertiellement dewnu then f lorsque l' alteration pethologiquedu myocarde a atteint un maximum. he 12'jour, L' ARN ...

  20. Pediatric Asthma and Viral Infection.

    Science.gov (United States)

    Garcia-Garcia, M Luz; Calvo Rey, Cristina; Del Rosal Rabes, Teresa

    2016-05-01

    Respiratory viral infections, particularly respiratory syncytial virus (RSV) and rhinovirus, are the most importance risk factors for the onset of wheezing in infants and small children. Bronchiolitis is the most common acute respiratory infection in children under 1year of age, and the most common cause of hospitalization in this age group. RSV accounts for approximately 70% of all these cases, followed by rhinovirus, adenovirus, metapneumovirus and bocavirus. The association between bronchiolitis caused by RSV and the development of recurrent wheezing and/or asthma was first described more than 40years ago, but it is still unclear whether bronchiolitis causes chronic respiratory symptoms, or if it is a marker for children with a genetic predisposition for developing asthma in the medium or long term. In any case, sufficient evidence is available to corroborate the existence of this association, which is particularly strong when the causative agent of bronchiolitis is rhinovirus. The pathogenic role of respiratory viruses as triggers for exacerbations in asthmatic patients has not been fully characterized. However, it is clear that respiratory viruses, and in particular rhinovirus, are the most common causes of exacerbation in children, and some type of respiratory virus has been identified in over 90% of children hospitalized for an episode of wheezing. Changes in the immune response to viral infections in genetically predisposed individuals are very likely to be the main factors involved in the association between viral infection and asthma.

  1. Problems in diagnosing viral hepatitis.

    Science.gov (United States)

    Bonino, F; Colloredo Mels, G; Bellati, G; Ideo, G; Oliveri, F; Colombatto, P; Brunetto, M R

    1993-01-01

    The most reliable method of making a specific aetiological diagnosis of chronic viral hepatitis would be to identify virus specific cytotoxic T lymphocytes responsible for the killing of virus infected hepatocytes in each patient's liver. Unfortunately, this can not be proposed for routine diagnosis and surrogate tests are required. The detection of virus markers, and even of the virus itself, does not imply that liver damage is caused by virus infection. Indirect markers of the host's antiviral immunoresponse have to be used to confirm more specifically the diagnosis of viral hepatitis. IgM antibodies against viral antigens implicated in the elimination of the virus seem to be suitable alternative candidates. Significant changes in the serum values of viraemia and aminotransferases occur within a few days, while a significant variation in liver histology takes much longer. Only the kinetics of the highly variable parameters can be used for an appropriate study of the relationship between viraemia, antiviral immunoresponse, and liver cell necrosis. Quantitative and dynamic analyses of hepatitis virus markers seem the most suitable and reliable methods of monitoring the patients eligible for antiviral treatment and identifying the most appropriate time to start this. PMID:8314490

  2. Recycling Endosomes and Viral Infection

    Directory of Open Access Journals (Sweden)

    Sílvia Vale-Costa

    2016-03-01

    Full Text Available Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral–host interactions occurring on the endocytic recycling compartment (ERC, and mediated by its regulatory Ras-related in brain (Rab GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.

  3. Comparative metagenomics of viral assemblages inhabiting four phyla of marine invertebrates

    Directory of Open Access Journals (Sweden)

    Brent Michael Gudenkauf

    2016-03-01

    Full Text Available Viruses are the most abundant biological entities on Earth, killing 10-20% of oceanic biomass each day. However, despite their ecological importance, viruses inhabiting many echinoderms, cnidarians, urochordates, and marine arthropods have not been investigated with significant breadth. We conducted a broad survey of the viral assemblages inhabiting these hosts through viral metagenomics and phylogenetic analysis. Results indicate that different invertebrate groups harbor distinct viral assemblages. Interestingly, however, no significant difference is observed between the viral assemblages of echinoderms and arthropods. These similarities and differences may be due to cellular, immunological, geographical, and ecological differences amongst host phyla, although mechanistic determination is beyond the purview of this work. Additionally, we present evidence of the detection of several viral families that have not yet been observed in these hosts. Finally, we confirm the result of previous investigation that method of library construction significantly biases metagenomic results by altering the representation of of ssDNA and dsDNA viral genomes.

  4. Viral-templated Palladium Nanocatalysts

    Science.gov (United States)

    Yang, Cuixian

    Despite recent progress on nanocatalysis, there exist several critical challenges in simple and readily controllable nanocatalyst synthesis including the unpredictable particle growth, deactivation of catalytic activity, cumbersome catalyst recovery and lack of in-situ reaction monitoring. In this dissertation, two novel approaches are presented for the fabrication of viral-templated palladium (Pd) nanocatalysts, and their catalytic activities for dichromate reduction reaction and Suzuki Coupling reaction were thoroughly studied. In the first approach, viral template based bottom-up assembly is employed for the Pd nanocatalyst synthesis in a chip-based format. Specifically, genetically displayed cysteine residues on each coat protein of Tobacco Mosaic Virus (TMV) templates provide precisely spaced thiol functionalities for readily controllable surface assembly and enhanced formation of catalytically active Pd nanoparticles. Catalysts with the chip-based format allow for simple separation and in-situ monitoring of the reaction extent. Thorough examination of synthesis-structure-activity relationship of Pd nanoparticles formed on surface-assembled viral templates shows that Pd nanoparticle size, catalyst loading density and catalytic activity of viral-templated Pd nanocatalysts can be readily controlled simply by tuning the synthesis conditions. The viral-templated Pd nanocatalysts with optimized synthesis conditions are shown to have higher catalytic activity per unit Pd mass than the commercial Pd/C catalysts. Furthermore, tunable and selective surface assembly of TMV biotemplates is exploited to control the loading density and location of Pd nanocatalysts on solid substrates via preferential electroless deposition. In addition, the catalytic activities of surface-assembled TMV-templated Pd nanocatalysts were also investigated for the ligand-free Suzuki Coupling reaction under mild reaction conditions. The chip-based format enables simple catalyst separation and

  5. Extracting viral RNAs from plant protoplasts.

    Science.gov (United States)

    Fabian, Marc R; Andrew White, K

    2007-08-01

    The analysis of viral RNA is a fundamental aspect of plant RNA virus research. Studies that focus on viral RNAs often involve virus infections of plant protoplasts (see UNITS 16D.1-16D.4). Protoplast offer the advantage of simultaneous initiation of infections, which allows for superior temporal and quantitative analyses of viral RNAs. The efficient isolation of intact viral RNA is key to any such investigations. This unit describes two basic protocols for extracting viral RNAs from plant protoplasts. An approach for preparing double-stranded viral RNA from total RNA pools is also provided. The viral RNA prepared by using these techniques can be used for further analyses such as primer extension, reverse transcription-PCR, and northern blotting.

  6. Influence of maintained hemodialysis on viral load in patients with end-stage renal disease with HBV infection

    Directory of Open Access Journals (Sweden)

    ZHANG Huifang

    2017-07-01

    Full Text Available In the patients with end-stage renal disease (ESRD with hepatitis B virus (HBV infection who underwent hemodialysis, the viral load of HBV DNA is relatively low and stable. For this phenomenon, some studies suggest that hemodialysis can reduce the HBV DNA load. The mechanism, which remains unclear, may be as follows: when HBV DNA enters the dialysate through the dialysis membrane, it was adsorbed onto the dialysis membrane; some virus particles were destroyed, and antiviral substances were produced in the course of hemodialysis. At present, there is no consensus on the mechanism responsible for the influence of maintained hemodialysis on the viral load of HBV DNA. This article reviews the factors involved in the influence of maintained hemodialysis on the viral load in ESRD patients with HBV infection and the recent progress.

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

    Science.gov (United States)

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

    2017-06-01

    Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of

  8. Chromatin structure of adenovirus DNA throughout infection

    OpenAIRE

    Giberson, Andrea N.; Davidson, Adam R.; Parks, Robin J.

    2011-01-01

    For more than half a century, researchers have studied the basic biology of Adenovirus (Ad), unraveling the subtle, yet profound, interactions between the virus and the host. These studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. In the infecting virion, the viral DNA is tightly condensed in a virally encoded protamine-like protein which must be remodeled within...

  9. Synthesis and Biological Evaluation of Brain-Specific Anti-RNA Viral Agents

    Science.gov (United States)

    1990-03-31

    genetic material either DNA or RNA and the type of nucleic acid give rise to the system of nomenclature for these entities. The viral DNA or RNA can then... anhydride (3) to produce the 5’-trigonellinate iodide of the protected sugar (4). Reduction of this quaternary salt in aqueous basic sodium dithionite...yielded the 5’-(1,4- dihydrotrigonellinate) of the ribavirin acetonide (5, AVS 5505). In the above synthesis, trigonelline anhydride was used to avoid

  10. Strategies and hurdles using DNA vaccines to fish.

    Science.gov (United States)

    Hølvold, Linn B; Myhr, Anne I; Dalmo, Roy A

    2014-01-01

    DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen - and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish.

  11. p53 Activation following Rift Valley fever virus infection contributes to cell death and viral production.

    Directory of Open Access Journals (Sweden)

    Dana Austin

    Full Text Available Rift Valley fever virus (RVFV is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production.

  12. Pattern Recognition Receptors and the Innate Immune Response to Viral Infection

    Directory of Open Access Journals (Sweden)

    Katherine A. Fitzgerald

    2011-06-01

    Full Text Available The innate immune response to viral pathogens is critical in order to mobilize protective immunity. Cells of the innate immune system detect viral infection largely through germline-encoded pattern recognition receptors (PRRs present either on the cell surface or within distinct intracellular compartments. These include the Toll-like receptors (TLRs, the retinoic acid-inducble gene I-like receptors (RLRs, the nucleotide oligomerization domain-like receptors (NLRs, also called NACHT, LRR and PYD domain proteins and cytosolic DNA sensors. While in certain cases viral proteins are the trigger of these receptors, the predominant viral activators are nucleic acids. The presence of viral sensing PRRs in multiple cellular compartments allows innate cells to recognize and quickly respond to a broad range of viruses, which replicate in different cellular compartments. Here, we review the role of PRRs and associated signaling pathways in detecting viral pathogens in order to evoke production of interferons and cytokines. By highlighting recent progress in these areas, we hope to convey a greater understanding of how viruses activate PRR signaling and how this interaction shapes the anti-viral immune response.

  13. Effects of interferon-α/β on HBV replication determined by viral load.

    Directory of Open Access Journals (Sweden)

    Yongjun Tian

    2011-07-01

    Full Text Available Interferons α and β (IFN-α/β are type I interferons produced by the host to control microbial infections. However, the use of IFN-α to treat hepatitis B virus (HBV patients generated sustained response to only a minority of patients. By using HBV transgenic mice as a model and by using hydrodynamic injection to introduce HBV DNA into the mouse liver, we studied the effect of IFN-α/β on HBV in vivo. Interestingly, our results indicated that IFN-α/β could have opposite effects on HBV: they suppressed HBV replication when viral load was high and enhanced HBV replication when viral load was low. IFN-α/β apparently suppressed HBV replication via transcriptional and post-transcriptional regulations. In contrast, IFN-α/β enhanced viral replication by inducing the transcription factor HNF3γ and activating STAT3, which together stimulated HBV gene expression and replication. Further studies revealed an important role of IFN-α/β in stimulating viral growth and prolonging viremia when viral load is low. This use of an innate immune response to enhance its replication and persistence may represent a novel strategy that HBV uses to enhance its growth and spread in the early stage of viral infection when the viral level is low.

  14. Pattern recognition receptors and the innate immune response to viral infection.

    Science.gov (United States)

    Thompson, Mikayla R; Kaminski, John J; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A

    2011-06-01

    The innate immune response to viral pathogens is critical in order to mobilize protective immunity. Cells of the innate immune system detect viral infection largely through germline-encoded pattern recognition receptors (PRRs) present either on the cell surface or within distinct intracellular compartments. These include the Toll-like receptors (TLRs), the retinoic acid-inducble gene I-like receptors (RLRs), the nucleotide oligomerization domain-like receptors (NLRs, also called NACHT, LRR and PYD domain proteins) and cytosolic DNA sensors. While in certain cases viral proteins are the trigger of these receptors, the predominant viral activators are nucleic acids. The presence of viral sensing PRRs in multiple cellular compartments allows innate cells to recognize and quickly respond to a broad range of viruses, which replicate in different cellular compartments. Here, we review the role of PRRs and associated signaling pathways in detecting viral pathogens in order to evoke production of interferons and cytokines. By highlighting recent progress in these areas, we hope to convey a greater understanding of how viruses activate PRR signaling and how this interaction shapes the anti-viral immune response.

  15. Divergent responses of viral and bacterial communities in the gut microbiome to dietary disturbances in mice

    Energy Technology Data Exchange (ETDEWEB)

    Howe, Adina; Ringus, Daina L.; Williams, Ryan J.; Choo, Zi-Ning; Greenwald, Stephanie M.; Owens, Sarah M.; Coleman, Maureen L.; Meyer, Folker; Chang, Eugene B.

    2015-10-16

    To improve our understanding of the stability of mammalian intestinal communities, we characterized the responses of both bacterial and viral communities in murine fecal samples to dietary changes between high- and low-fat (LF) diets. Targeted DNA extraction methods for bacteria, virus-like particles and induced prophages were used to generate bacterial and viral metagenomes as well as 16S ribosomal RNA amplicons. Gut microbiome communities from two cohorts of C57BL/6 mice were characterized in a 6-week diet perturbation study in response to high fiber, LF and high-refined sugar, milkfat (MF) diets. The resulting metagenomes from induced bacterial prophages and extracellular viruses showed significant overlap, supporting a largely temperate viral lifestyle within these gut microbiomes. The resistance of baseline communities to dietary disturbances was evaluated, and we observed contrasting responses of baseline LF and MF bacterial and viral communities. In contrast to baseline LF viral communities and bacterial communities in both diet treatments, baseline MF viral communities were sensitive to dietary disturbances as reflected in their non-recovery during the washout period. The contrasting responses of bacterial and viral communities suggest that these communities can respond to perturbations independently of each other and highlight the potentially unique role of viruses in gut health.

  16. Divergent responses of viral and bacterial communities in the gut microbiome to dietary disturbances in mice

    Science.gov (United States)

    Howe, Adina; Ringus, Daina L; Williams, Ryan J; Choo, Zi-Ning; Greenwald, Stephanie M; Owens, Sarah M; Coleman, Maureen L; Meyer, Folker; Chang, Eugene B

    2016-01-01

    To improve our understanding of the stability of mammalian intestinal communities, we characterized the responses of both bacterial and viral communities in murine fecal samples to dietary changes between high- and low-fat (LF) diets. Targeted DNA extraction methods for bacteria, virus-like particles and induced prophages were used to generate bacterial and viral metagenomes as well as 16S ribosomal RNA amplicons. Gut microbiome communities from two cohorts of C57BL/6 mice were characterized in a 6-week diet perturbation study in response to high fiber, LF and high-refined sugar, milkfat (MF) diets. The resulting metagenomes from induced bacterial prophages and extracellular viruses showed significant overlap, supporting a largely temperate viral lifestyle within these gut microbiomes. The resistance of baseline communities to dietary disturbances was evaluated, and we observed contrasting responses of baseline LF and MF bacterial and viral communities. In contrast to baseline LF viral communities and bacterial communities in both diet treatments, baseline MF viral communities were sensitive to dietary disturbances as reflected in their non-recovery during the washout period. The contrasting responses of bacterial and viral communities suggest that these communities can respond to perturbations independently of each other and highlight the potentially unique role of viruses in gut health. PMID:26473721

  17. Evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life

    DEFF Research Database (Denmark)

    Prangishvili, D.; Garrett, R. A.; Koonin, E.

    2006-01-01

    . In accord with this distinction, the sequenced genomes of euryarchaeal viruses encode many proteins homologous to bacteriophage capsid proteins. In contrast, initial analysis of the crenarchaeal viral genomes revealed no relationships with bacteriophages and, generally, very few proteins with detectable...... the proteins of crenarchaeal viruses and between viral proteins and those from cellular life forms and allowed functional predictions for some of these conserved genes. A small pool of genes is shared by overlapping subsets of crenarchaeal viruses, in a general analogy with the metagenome structure...... of bacteriophages. The proteins encoded by the genes belonging to this pool include predicted transcription regulators, ATPases implicated in viral DNA replication and packaging, enzymes of DNA precursor metabolism, RNA modification enzymes, and glycosylases. In addition, each of the crenarchaeal viruses encodes...

  18. Alternative autophagy, brefeldin A and viral trafficking pathways

    Science.gov (United States)

    Grose, Charles; Klionsky, Daniel J.

    2016-01-01

    ABSTRACT Two topics that have attracted recent attention in the field of autophagy concern the source of the membrane that is used to form the autophagosome during macroautophagy and the role of noncanonical autophagic pathways. The 2 topics may converge when considering the intersection of autophagy with viral infection. We suggest that noncanonical autophagy, which is sensitive to treatment with brefeldin A, may converge with the infectious cycles of certain DNA and RNA viruses that utilize membrane from the ER and cis-Golgi. PMID:27439673

  19. A core viral protein binds host nucleosomes to sequester immune danger signals.

    Science.gov (United States)

    Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C; Blumenthal, Daniel; Paris, Andrew J; Reyes, Emigdio D; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H; Worthen, G Scott; Black, Ben E; Garcia, Benjamin A; Weitzman, Matthew D

    2016-07-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

  20. Encefalitis virales en la infancia

    Directory of Open Access Journals (Sweden)

    Monserrat Téllez de Meneses

    2013-09-01

    Full Text Available La encefalitis viral es una enfermedad grave que implica el compromiso inflamatorio del parénquima cerebral. Las infecciones virales del SNC ocurren con frecuencia como complicación de infecciones virales sistémicas. Más de 100 virus están implicados como agentes causales, entre los cuales el virus Herpes simplex tipo I, es el agente causal más frecuente de encefalitis no epidémica en todos los grupos poblacionales del mundo; es el responsable de los casos más graves en todas las edades. Muchos de los virus para los cuales existe vacunas también pueden causar encefalitis como: sarampión, paperas, polio, rabia, rubéola, varicela. El virus produce una inflamación del tejido cerebral, la cual puede evolucionar a una destrucción de neuronas, provocar hemorragia y daño cerebral, dando lugar a encefalitis graves, como la encefalitis necrotizante o hemorrágica, con mucho peor pronóstico, produciendo secuelas graves, incluso la muerte. El cuadro clínico, incluye la presencia de cefalea, fiebre y alteración de la conciencia, de rápida progresión. El pronóstico de las encefalitis víricas es variable, algunos casos son leves, con recuperación completa, sin embargo existen casos graves que pueden ocasionar secuelas importantes a nivel cerebral. Es fundamental realizar un diagnóstico lo antes posible, a través de pruebas de laboratorio (bioquímica, PCR, cultivos y de neuroimagen (TAC, RM y ante todo, la instauración de un tratamiento precoz para evitar la evolución del proceso y sus posibles complicaciones. El pronóstico empeora si se retrasa la instauración del tratamiento.

  1. Evaluation of Viral Meningoencephalitis Cases

    Directory of Open Access Journals (Sweden)

    Handan Ilhan

    2012-08-01

    Full Text Available AIM: To evaluate retrospectively adult cases of viral encephalitis. METHOD: Fifteen patients described viral encephalitis hospitalized between the years 2006-2011 follow-up and treatment at the infectious diseases clinic were analyzed retrospectively. RESULTS: Most of the patients (%60 had applied in the spring. Fever (87%, confusion (73%, neck stiffness (73%, headache (73%, nausea-vomiting (33%, loss of consciousness (33%, amnesia (33%, agitation (20%, convulsion (%20, focal neurological signs (13%, Brudzinski-sign (13% were most frequently encountered findings. Electroencephalography test was applied to 13 of 14 patients, and pathological findings compatible with encephalitis have been found. Radiological imaging methods such as CT and MRI were performed in 9 of the 14 patients, and findings consistent with encephalitis were reported. All of initial cerebrospinal fluid (CSF samples were abnormal. The domination of the first examples was lymphocytes in 14 patients; only one patient had an increase in neutrophilic cells have been found. CSF protein level was high in nine patients, and low glucose level was detected in two patients. Herpes simplex virus polymerized chain reaction (PCR analyze was performed to fourteen patients CSF. Only two of them (14% were found positive. One of the patients sample selectively examined was found to be Parvovirus B19 (+, the other patient urine sample Jacobs-creutzfeld virus PCR was found to be positively. Empiric acyclovir therapy was given to all patients. Neuropsychiatric squeal developed at the one patient. CONCLUSION: The cases in the forefront of change in mental status viral meningoencephalitis should be considered and empirical treatment with acyclovir should be started. [TAF Prev Med Bull 2012; 11(4.000: 447-452

  2. Marked variability in the extent of protein disorder within and between viral families.

    Directory of Open Access Journals (Sweden)

    Ravindra Pushker

    Full Text Available Intrinsically disordered regions in eukaryotic proteomes contain key signaling and regulatory modules and mediate interactions with many proteins. Many viral proteomes encode disordered proteins and modulate host factors through the use of short linear motifs (SLiMs embedded within disordered regions. However, the degree of viral protein disorder across different viruses is not well understood, so we set out to establish the constraints acting on viruses, in terms of their use of disordered protein regions. We surveyed predicted disorder across 2,278 available viral genomes in 41 families, and correlated the extent of disorder with genome size and other factors. Protein disorder varies strikingly between viral families (from 2.9% to 23.1% of residues, and also within families. However, this substantial variation did not follow the established trend among their hosts, with increasing disorder seen across eubacterial, archaebacterial, protists, and multicellular eukaryotes. For example, among large mammalian viruses, poxviruses and herpesviruses showed markedly differing disorder (5.6% and 17.9%, respectively. Viral families with smaller genome sizes have more disorder within each of five main viral types (ssDNA, dsDNA, ssRNA+, dsRNA, retroviruses, except for negative single-stranded RNA viruses, where disorder increased with genome size. However, surveying over all viruses, which compares tiny and enormous viruses over a much bigger range of genome sizes, there is no strong association of genome size with protein disorder. We conclude that there is extensive variation in the disorder content of viral proteomes. While a proportion of this may relate to base composition, to extent of gene overlap, and to genome size within viral types, there remain important additional family and virus-specific effects. Differing disorder strategies are likely to impact on how different viruses modulate host factors, and on how rapidly viruses can evolve novel

  3. Murine cytomegalovirus protein pM92 is a conserved regulator of viral late gene expression.

    Science.gov (United States)

    Chapa, Travis J; Perng, Yi-Cheih; French, Anthony R; Yu, Dong

    2014-01-01

    In this study, we report that murine cytomegalovirus (MCMV) protein pM92 regulates viral late gene expression during virus infection. Previously, we have shown that MCMV protein pM79 and its human cytomegalovirus (HCMV) homologue pUL79 are required for late viral gene transcription. Identification of additional factors involved is critical to dissecting the mechanism of this regulation. We show here that pM92 accumulated abundantly at late times of infection in a DNA synthesis-dependent manner and localized to nuclear viral replication compartments. To investigate the role of pM92, we constructed a recombinant virus SMin92, in which pM92 expression was disrupted by an insertional/frameshift mutation. During infection, SMin92 accumulated representative viral immediate-early gene products, early gene products, and viral DNA sufficiently but had severe reduction in the accumulation of late gene products and was thus unable to produce infectious progeny. Coimmunoprecipitation and mass spectrometry analysis revealed an interaction between pM92 and pM79, as well as between their HCMV homologues pUL92 and pUL79. Importantly, we showed that the growth defect of pUL92-deficient HCMV could be rescued in trans by pM92. This study indicates that pM92 is an additional viral regulator of late gene expression, that these regulators (represented by pM92 and pM79) may need to complex with each other for their activity, and that pM92 and pUL92 share a conserved function in CMV infection. pM92 represents a potential new target for therapeutic intervention in CMV disease, and a gateway into studying a largely uncharted viral process that is critical to the viral life cycle.

  4. Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses.

    Science.gov (United States)

    Kim, Kyoung-Ho; Bae, Jin-Woo

    2011-11-01

    Investigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

  5. Viral diseases of marine invertebrates

    Science.gov (United States)

    Johnson, P. T.

    1984-03-01

    Approximately 40 viruses are known from marine sponges; turbellarian and monogenetic flatworms; cephalopod, bivalve, and gastropod mollusks; nereid polychaetes; and isopod and decapod crustaceans. Most of the viruses can be tentatively assigned to the Herpesviridae, Baculoviridae, Iridoviridae, Adenoviridae, Papovaviridae, Reoviridae, “Birnaviridae”, Bunyaviridae, Rhabdoviridae, and Picornaviridae. Viruslike particles found in oysters might be representatives of the Togaviridae and Retroviridae. Enveloped single-stranded RNA viruses from crustaceans have developmental and morphological characteristics intermediate between families, and some show evidence of relationships to the Paramyxoviridae as well as the Bunyaviridae or Rhabdoviridae. Certain small viruses of shrimp cannot be assigned, even tentatively, to a particular family. Some viruses cause disease in wild and captive hosts, others are associated with disease states but may not be primary instigators, and many occur in apparently normal animals. The frequency of viral disease in natural populations of marine invertebrates is unknown. Several viruses that cause disease in captive animals, with or without experimental intervention, have also been found in diseased wild hosts, including herpeslike viruses of crabs and oysters, iridovirus of octopus, and reolike and bunyalike viruses of crabs. Iridolike viruses have been implicated in massive mortalities of cultured oysters. Baculoviruses, and IHHN virus, which is of uncertain affinities, cause economically damaging diseases in cultured penaeid shrimp. Double or multiple viral infection is common in crabs. For example, a reolike virus and associated rhabdolike virus act synergistically to cause paralytic and fatal disease in Callinectes sapidus. Information on host range, most susceptible stage, and viral latency is available only for viruses of shrimp. One baculovirus attacks five species of New World penaeid shrimp. IHHN virus infects three species of

  6. [The ABC of viral hepatitis].

    Science.gov (United States)

    Van Bambeke, F

    2008-03-01

    Viral hepatitis has long been under-diagnosed. Hepatitis A is an acute disease, while patients infected by hepatitis B and hepatitis C viruses are likely to develop chronical infections and severe complications (cancer, cirrhosis). The current treatment of hepatitis B and C consists in alpha interferon (preferably under its pegylated form), in combination with ribavirin for hepatitis C. The frequent and severe adverse effects of interferon-based therapy constitute, however, a major limiting factor (reactions at the injection site, flu-like syndrome, neurological disorders, ...). For hepatitis B, two alternatives are available so far, namely lamivudine and adefovir (used as a prodrug with highe oral bioavailability).

  7. Authentic counterfeit in viral marketing

    OpenAIRE

    2016-01-01

    This master thesis deals with authentic fake, a phenomenon present in viral marketing. After a brief introduction into the issue and clarification of terms, the study aims to find out whether a seemingly authentic video recording of a catchy incident helps a marketing campaign to succeed, that is to say to achieve high sharing figures amongst internet users. In a theoretical part, the thesis elaborates information from technical books, publications and internet sources dealing with authentici...

  8. Mast cells in viral infections

    OpenAIRE

    Piotr Witczak; Ewa Brzezińska-Błaszczyk

    2012-01-01

     There are some premises suggesting that mast cells are involved in the mechanisms of anti-virus defense and in viral disease pathomechanisms. Mast cells are particularly numerous at the portals of infections and thus may have immediate and easy contact with the external environment and invading pathogens. These cells express receptors responsible for recognition of virus-derived PAMP molecules, mainly Toll-like receptors (TLR3, TLR7/8 and TLR9), but also RIG-I-like and NOD-like molecules. Fu...

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

    Directory of Open Access Journals (Sweden)

    Molly L. Bristol

    2016-06-01

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

  10. Construction and immune effect of DNA vaccine against bovine viral diarrhea-mucosal virus%牛病毒性腹泻/粘膜病病毒核酸疫苗的构建及其免疫效果研究

    Institute of Scientific and Technical Information of China (English)

    宫玉玲; 冉多良; 季新成; 刘建华

    2013-01-01

    为提高牛病毒性腹泻/粘膜病(BVD/MD)核酸疫苗的免疫效力,本实验应用PCR方法扩增BVD病毒(BVDV) E0基因,构建真核表达质粒pVAX1-E0,转染293T细胞,经RT-PCR和western blot分析显示,转染细胞能够瞬时表达E0蛋白.并分别将pVAX1、pVAX1-E0或将pVAX1-E0分别与一种表达细胞因子基因的重组质粒作为佐剂(pVAX1-IL-2、pVAX1-IL-4及pVAX1-IFN-γ)免疫小鼠,采用间接ELISA法检测免疫小鼠BVDV抗体效价,以MTT法检测免疫小鼠脾淋巴细胞的增殖活性.实验结果表明,与pVAX1-E0相比,接种pVAX1-E0/pVAX1-IL-2小鼠血清E0抗体水平及淋巴细胞增殖水平显著提高(p<0.01),表明细胞因子基因佐剂IL-2能够有效提高BVDV E0核酸疫苗免疫效果,可以刺激小鼠产生良好的免疫应答.%To improve the immune efficacy of DNA vaccine against the bovine viral diarrhea virus-mucosal disease virus (BVDV/MDV),BVDV E0 gene was amplified by PCR and inserted into pVAX1 eukaryotic expression vector to construct recombinant plasmid pVAX1-E0.In addition,the BVDV E0 gene transiently expressed in 293T cells transfected with pVAX1-E0 was verified by RT-PCR and western blot.Furthermore,the mice were immunized by co-inoculation of pVAV1-E0 with one of three recombinant plasmids expressing cytokine gene as adjuvant (pVAX1-IL-2,pVAX1-IL-4 and pVAX1-IFN-γ),and single inoculation with pVAX1 and pVAV1-E0,respectively.The results showed that the antibody titers against E0 and the ability to promote lymphocytes proliferation were significantly higher in mice group co-inoculated with pVAX1-E0 and pVAX1-IL-2 than that the mice in control groups (p<0.01),indicating the IL-2 was able to improve the immune effect of DNA vaccine.

  11. Cationic influenza virosomes as an adjuvanted delivery system for CTL induction by DNA vaccination

    NARCIS (Netherlands)

    Jamali, Abbas; Holtrop, Marijke; de Haan, Aalzen; Hashemi, Hamidreza; Shenagari, Mohammad; Memarnejadian, Arash; Roohvand, Farzin; Sabahi, Farzaneh; Kheiri, Masumeh Tavassoti; Huckriede, Anke

    2012-01-01

    DNA vaccines have emerged as an attractive approach to induce CTL responses against cancer and infectious agents in recent years. Although CTL induction by DNA vaccination would be a valuable strategy for controlling viral infections, increasing the potency of DNA vaccines is mandatory before DNA

  12. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    NARCIS (Netherlands)

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, r

  13. Commercialization of veterinary viral vaccines.

    Science.gov (United States)

    Flore, P H

    2004-12-01

    If vaccines are to reliably prevent disease, they must be developed, produced and quality-controlled according to very strict regulations and procedures. Veterinary viral vaccine registrations are governed by different rules in different countries, but these rules all emphasize that the quality of the raw materials--the cells, eggs, animals or plants that are used in production--need to be carefully controlled. The veterinary vaccine business is also very cost-conscious. Emphasis over the last 5-10 years has therefore been to develop culture systems that minimize labor and sterility problems and thus provide for reliable and cost-effective production. Implementing these often more complex systems in a production environment takes considerable effort, first in scale-up trials and further down the line in convincing production personnel to change their familiar system for something new and possibly untried. To complete scale-up trials successfully, it is absolutely necessary to understand the biochemistry of the cells and the influence of the virus on the cells under scale-up and later production conditions. Once a viral product can be produced on a large scale, it is imperative that the quality of the end-product is controlled in an intelligent way. One needs to know whether the end-product performs in the animal as was intended during its conception in the research and development department. The development of the appropriate tests to demonstrate this plays an important role in the successful development of a vaccine.

  14. Metagenomic Investigation of Viral Communities in Ballast Water.

    Science.gov (United States)

    Kim, Yiseul; Aw, Tiong Gim; Teal, Tracy K; Rose, Joan B

    2015-07-21

    Ballast water is one of the most important vectors for the transport of non-native species to new aquatic environments. Due to the development of new ballast water quality standards for viruses, this study aimed to determine the taxonomic diversity and composition of viral communities (viromes) in ballast and harbor waters using metagenomics approaches. Ballast waters from different sources within the North America Great Lakes and paired harbor waters were collected around the Port of Duluth-Superior. Bioinformatics analysis of over 550 million sequences showed that a majority of the viral sequences could not be assigned to any taxa associated with reference sequences, indicating the lack of knowledge on viruses in ballast and harbor waters. However, the assigned viruses were dominated by double-stranded DNA phages, and sequences associated with potentially emerging viral pathogens of fish and shrimp were detected with low amino acid similarity in both ballast and harbor waters. Annotation-independent comparisons showed that viromes were distinct among the Great Lakes, and the Great Lakes viromes were closely related to viromes of other cold natural freshwater systems but distant from viromes of marine and human designed/managed freshwater systems. These results represent the most detailed characterization to date of viruses in ballast water, demonstrating their diversity and the potential significance of the ship-mediated spread of viruses.

  15. Assembly of a marine viral metagenome after physical fractionation.

    Directory of Open Access Journals (Sweden)

    Jennifer R Brum

    Full Text Available Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne'ohe Bay, Hawai'i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.

  16. Viral communities associated with human pericardial fluids in idiopathic pericarditis.

    Directory of Open Access Journals (Sweden)

    Laura Fancello

    Full Text Available Pericarditis is a common human disease defined by inflammation of the pericardium. Currently, 40% to 85% of pericarditis cases have no identified etiology. Most of these cases are thought to be caused by an infection of undetected, unsuspected or unknown viruses. In this work, we used a culture- and sequence-independent approach to investigate the viral DNA communities present in human pericardial fluids. Seven viral metagenomes were generated from the pericardial fluid of patients affected by pericarditis of unknown etiology and one metagenome was generated from the pericardial fluid of a sudden infant death case. As a positive control we generated one metagenome from the pericardial fluid of a patient affected by pericarditis caused by herpesvirus type 3. Furthermore, we used as negative controls a total of 6 pericardial fluids from 6 different individuals affected by pericarditis of non-infectious origin: 5 of them were sequenced as a unique pool and the remaining one was sequenced separately. The results showed a significant presence of torque teno viruses especially in one patient, while herpesviruses and papillomaviruses were present in the positive control. Co-infections by different genotypes of the same viral type (torque teno viruses or different viruses (herpesviruses and papillomaviruses were observed. Sequences related to bacteriophages infecting Staphylococcus, Enterobacteria, Streptococcus, Burkholderia and Pseudomonas were also detected in three patients. This study detected torque teno viruses and papillomaviruses, for the first time, in human pericardial fluids.

  17. Sponging of Cellular Proteins by Viral RNAs

    OpenAIRE

    Charley, Phillida A.; Wilusz, Jeffrey

    2014-01-01

    Viral RNAs accumulate to high levels during infection and interact with a variety of cellular factors including miRNAs and RNA-binding proteins. Although many of these interactions exist to directly modulate replication, translation and decay of viral transcripts, evidence is emerging that abundant viral RNAs may in certain cases serve as a sponge to sequester host non coding RNAs and proteins. By effectively reducing the ability of cellular RNA binding proteins to regulate host cell gene exp...

  18. Viral Advertising on Facebook in Vietnam

    OpenAIRE

    Tran, Phuong

    2014-01-01

    The purpose of this thesis is to explore which factors affect the effectiveness of viral advertising on Facebook in Vietnam. The quantitative research method is applied in this research and the sample is Vietnamese Facebook users. After the data analysis stage using SPSS, it became clear that weak ties, perceptual affinity and emotions have an impact on the effectiveness of viral advertising. The results provide a pratical implication of how to make an Ad which can go viral on Facebook. Moreo...

  19. Pediatric knowledge about acute viral hepatitis

    OpenAIRE

    Franca,Rita; Silva,Luciana; Melo, Maria Clotildes; Cavalcante,Suzy; Lima, Bruno; Rocha, Anita; Gomes, Cristiana; Franca, Mônica

    2004-01-01

    p.227-235 Knowledge about hepatotropic viruses is crucial for pediatricians because of the high prevalence of viral hepatitis during childhood. The multiplicity of hepatotropic viruses, the spectrum of acute and chronic infections, and the sequels of viral hepatitis result in a need for physicians to better understand the clinical and epidemiological context of patients with viral hepatitis, as well as the importance of prevention measures for hepatitis. A descriptive cross-sectional study...

  20. Consumers’ attitude towards viral marketing in Pakistan

    OpenAIRE

    Kiani Irshad ZERNIGAH; Kamran SOHAIL

    2012-01-01

    The rapid advancement of technology has opened many costeffective avenues for marketers to promote their products. One of the emerging techniques of products promotion through the use of technology is viral marketing that is becoming a popular direct marketing tool for marketers across the world. Therefore, marketers should understand factors that result in increased acceptance of viral marketing by consumers. The present research was conducted to investigate consumers’ attitude towards viral...

  1. [Workshop on Molecular Epidemiology of Viral Diseases].

    Science.gov (United States)

    Gómez, B; Cabrera, L; Arias, C F

    1997-01-01

    A workshop on viral epidemiology was held on September 29, 1995 at the Medical School of the Universidad Nacional Autónoma de Mexico. The aim of this workshop was to promote interaction among scientists working in viral epidemiology. Eighteen scientists from ten institutions presented their experiences and work. General aspects of the epidemiology of meaningful viral diseases in the country were discussed, and lectures presented on the rota, polio, respiratory syncytial, dengue, papiloma, rabies, VIH and hepatitis viruses.

  2. Biotechnology and DNA vaccines for aquatic animals

    Science.gov (United States)

    Kurath, G.

    2008-01-01

    Biotechnology has been used extensively in the development of vaccines for aquaculture. Modern molecular methods such as polymerase chain reaction (PCR), cloning and microarray analysis have facilitated antigen discovery, construction of novel candidate vaccines, and assessments of vaccine efficacy, mode of action, and host response. This review focuses on DNA vaccines for finfish to illustrate biotechnology applications in this field. Although DNA vaccines for fish rhabdoviruses continue to show the highest efficacy, DNA vaccines for several other viral and bacterial fish pathogens have now been proven to provide significant protection against pathogen challenge. Studies of the fish rhabdovirus DNA vaccines have elucidated factors that affect DNA vaccine efficacy as well as the nature of the fish innate and adaptive immune responses to DNA vaccines. As tools for managing aquatic animal disease emergencies, DNA vaccines have advantages in speed, flexibility, and safety, and one fish DNA vaccine has been licensed.

  3. SOS processing of unique oxidative DNA damages in Escherichia coli.

    Science.gov (United States)

    Laspia, M F; Wallace, S S

    1989-05-05

    phi X174 replicative form (RF) I transfecting DNA containing thymine glycols (5,6-dihydroxy-5,6-dihydrothymine), urea glycosides or apurinic (AP) sites was used to study SOS processing of unique DNA damages in Escherichia coli. All three lesions can be found in DNA damaged by chemical oxidants or radiation and are representative of several common structural modifications of DNA bases. When phi X DNA containing thymine glycols was transfected into host cells that were ultraviolet-irradiated to induce the SOS response, a substantial increase in survival was observed compared to transfection into uninduced hosts. Studies with mutants demonstrated that both the activated form of RecA and UmuDC proteins were required for this reactivation. In contrast, no increase in survival was observed when DNA containing urea glycosides or AP sites was transfected into ultraviolet-induced hosts. These data suggest that SOS-induced reactivation does not reflect a generalized repair system for all replication-blocking, lethal lesions but rather that the efficiency of reactivation is damage dependent. Further, we found that a significant fraction of potentially lethal thymine glycols could be ultraviolet-reactivated in an umuC lexA recA-independent manner, suggesting the existence of an as yet uncharacterized damage-inducible SOS-independent mode of thymine glycol repair.

  4. Synthesis of Bacteriophage M13-Specific Proteins in a DNA-Dependent Cell-Free System II. In Vitro Synthesis of Biologically Active Gene 5 Protein

    Science.gov (United States)

    Konings, Ruud N. H.; Jansen, Josephine; Cuypers, Theo; Schoenmakers, John G. G.

    1973-01-01

    It is shown that gene 5 protein of bacteriophage M13 is one of the major proteins synthesized in vitro under the direction of M13 replicative-form DNA. By means of DNA-cellulose chromatography, this protein has been purified to homogeneity and its biological characteristics have been compared with those of its native counterpart. Like native gene 5 protein, the purified, in vitro-synthesized protein binds tightly and selectively to single-stranded, but not to double-stranded, DNAs. These results suggest that truly functional gene 5 protein is made in the cell-free system. Images PMID:4586780

  5. Exploring Text Virality in Social Networks

    CERN Document Server

    Guerini, Marco; Ozbal, Gozde

    2012-01-01

    This paper aims to shed some light on the concept of virality - especially in social networks - and to provide new insights on its structure. We argue that: (a) virality is a phenomenon strictly connected to the nature of the content being spread, rather than to the influencers who spread it, (b) virality is a phenomenon with many facets, i.e. under this generic term several different effects of persuasive communication are comprised and they only partially overlap. To give ground to our claims, we provide initial experiments in a machine learning framework to show how various aspects of virality can be independently predicted according to content features.

  6. Chronic viral hepatitis : diagnosis and therapeutics

    National Research Council Canada - National Science Library

    Wu, George Y; Koff, Raymond S

    2001-01-01

    .... The discussion of patient management includes contributions on developing novel therapeutics, supporting patients during therapy, alternative treatments, the use of drugs in chronic viral hepatitis...

  7. Viral Subversion of the Nuclear Pore Complex

    Directory of Open Access Journals (Sweden)

    Valerie Le Sage

    2013-08-01

    Full Text Available The nuclear pore complex (NPC acts as a selective barrier between the nucleus and the cytoplasm and is responsible for mediating communication by regulating the transport of RNA and proteins. Numerous viral pathogens have evolved different mechanisms to hijack the NPC in order to regulate trafficking of viral proteins, genomes and even capsids into and out of the nucleus thus promoting virus replication. The present review examines the different strategies and the specific nucleoporins utilized during viral infections as a means of promoting their life cycle and inhibiting host viral defenses.

  8. STUDY OF PERSISTENT VIRAL INFECTION IN AN ANIMALMODEL OF VIRAL MYOCARDITIS BY PCR

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To study the role of persistent viral infection in the mechanism of viral myocarditis. Methods A mice model of CVB3m viral myocarditis was made and the viral RNA in mice myocardium and whole blood sample was tested by using polymerase chain reaction ( PCR ) technique. The pathological changes in mice myocardium were determined. Results On day 3, the viral gene in whole blood and myocardium was found, which partly became negative on day 8, but the change of myocardial pathology became obvious. Although the blood specimens were tested negatively on day 12, the viral gene in mice myocardium remained positive within 120d. Conclusion This study indicates that persistent viral infection plays a role in the pathogenesis of viral myocarditis.

  9. Molecular architecture of the preinitiation complex in adenovirus DNA replication

    OpenAIRE

    Mysiak, Monika Elzbieta

    2004-01-01

    After infection of a host cell, adenovirus (Ad) aims for generation of progeny viruses, and thus it rapidly replicates its genomic DNA. The replication process starts with the assembly of the preinitiation complex (PIC) on the origin DNA. The PIC consists of three viral proteins, DNA polymerase (pol), precursor terminal protein (pTP), DNA binding protein (DBP) and two transcription factors of the host cell, Nuclear Factor I (NFI) and Octamer binding protein (Oct-1). Both transcription factors...

  10. Quantitative and qualitative differences in DNA complementary to avian myeloblastosis virus between normal and leukemic chicken cells.

    Science.gov (United States)

    Baluda, M A; Shoyab, M; Evans, R; Markham, P D; Ali, M

    1975-01-01

    Hybridization of avian myeloblastosis virus (AMV) RNA with DNA immobilized on filters or in liquid with a vast DNA excess was used to measure the viral specific DNA sequences in chicken cells. Newly synthesized viral DNA (v-DNA) appears within an hour after infection of chicken embryo fibroblasts (CEF) with avian oncornaviruses. A fraction of newly synthesized v-DNA becomes integrated into the cellular genome and the remainder gradually disappears. A covalent linkage between v-DNA and cellular DNA was demonstrated to exist in CEF and in leukemic myeloblasts by alkaline sucrose velocity sedimentation. Hybridization of AMV RNA in DNA excess has revealed that there are 2 clases of viral specific sequences within normal as well as in leukemic cells. The 2 types of sequences differ in their rate of hybridization. The amount of both types of DNA sequences is about 2 times higher in leukemic cells than in normal cells. Both the fast- and slowly reacting sequences in leukemic cells exhibit a higher Tm (2 degrees C) than the respective DNA sequences in normal cells. Furthermore, when nucleotide sequences in AMV RNA complementary to normal DNA are removed first by exhaustive hybridization with normal DNA, the residual RNA only hybridizes with leukemic DNA but not with normal DNA. These results suggest that leukemic cells contain viral specific DNA sequences which are absent in normal cells. Endogenous v-DNA has been shown to be integrated in cellular DNA region(s) with a reiteration frequency of approximately 1,200 copies per cell and each integration unit appears to have a size approximately equivalent to the 35S RNA subunit of the viral genome. Viral sequences acquired after infection appear to be integrated in the unique region of cell DNA, or in tandem with the endogenous viral sequences.

  11. Do microRNAs induced by Viral Hemorrhagic Septicemia virus in rainbow trout (Oncorhynchus mykiss) possess anti-viral activity?

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Lorenzen, Niels

    2013-01-01

    RNAs were also up regulated in the liver and muscle (vaccination site) of fish vaccinated with a DNA vaccine expressing the VHSV glycoprotein gene. Recent studies further indicate that the expression of these miRNAs is induced by interferons. In order to analyze if miRNA-462 and miRNA-731 have any anti...... processes. Some miRNAs have been shown to have direct anti-viral effects. We have previously observed and validated that the fish-specific miRNAs, miR-462 and miR-731, were among the most highly expressed miRNAs in rainbow trout liver following Viral hemorrhagic septicemia virus (VHSV) infection. These mi......-viral effects, we designed inhibitory synthetic oligonucleotides called antagomiRs or anti-miRNAs. These saline-formulated 2’-O-methylated Locked Nucleic Acid (LNA)-based antagomiRs were injected intraperitoneally into rainbow trout fingerlings followed by exposure of the fish to VHSV. Development of disease...

  12. Viral diseases of northern ungulates

    Directory of Open Access Journals (Sweden)

    K. Frölich

    2000-03-01

    Full Text Available This paper describes viral diseases reported in northern ungulates and those that are a potential threat to these species. The following diseases are discussed: bovine viral diarrhoea/mucosal disease (BVD/MD, alphaherpesvirus infections, malignant catarrhal fever (MCF, poxvirus infections, parainfluenza type 3 virus infection, Alvsborg disease, foot-and-mouth disease, epizootic haemorrhage disease of deer and bluetongue disease, rabies, respiratory syncytial virus infection, adenovirus infection, hog-cholera, Aujeszky's disease and equine herpesvirus infections. There are no significant differences in antibody prevalence to BVDV among deer in habitats with high, intermediate and low density of cattle. In addition, sequence analysis from the BVDV isolated from roe deer (Capreolus capreolus showed that this strain was unique within BVDV group I. Distinct BVDV strains might circulate in free-ranging roe deer populations in Germany and virus transmission may be independent of domestic livestock. Similar results have been obtained in a serological survey of alpha-herpesviruses in deer in Germany. Malignant catarrhal fever was studied in fallow deer (Cervus dama in Germany: the seroprevalence and positive PCR results detected in sheep originating from the same area as the antibody-positive deer might indicate that sheep are the main reservoir animals. Contagious ecthyma (CE is a common disease in domestic sheep and goats caused by the orf virus. CE has been diagnosed in Rocky Mountain bighorn sheep (Ovis canadensis, mountain goats (Oreamnos americanus, Dall sheep (Ovis dalli, chamois (Rupkapra rupi-capra, muskox {Ovibos moschatus and reindeer (Rangifer tarandus. Most parainfluenza type 3 virus infections are mild or clinically undetectable. Serological surveys in wildlife have been successfully conducted in many species. In 1985, a new disease was identified in Swedish moose (Alces alces, designated as Alvsborg disease. This wasting syndrome probably

  13. Metaviromics of Namib Desert Salt Pans: A Novel Lineage of Haloarchaeal Salterproviruses and a Rich Source of ssDNA Viruses

    OpenAIRE

    Adriaenssens, Evelien M.; Leonardo Joaquim van Zyl; Cowan, Don A.; Marla I. Trindade

    2016-01-01

    Viral communities of two different salt pans located in the Namib Desert, Hosabes and Eisfeld, were investigated using a combination of multiple displacement amplification of metaviromic DNA and deep sequencing, and provided comprehensive sequence data on both ssDNA and dsDNA viral community structures. Read and contig annotations through online pipelines showed that the salt pans harbored largely unknown viral communities. Through network analysis, we were able to assign a large portion of t...

  14. A genome-to-genome analysis of associations between human genetic variation, HIV-1 sequence diversity, and viral control.

    Science.gov (United States)

    Bartha, István; Carlson, Jonathan M; Brumme, Chanson J; McLaren, Paul J; Brumme, Zabrina L; John, Mina; Haas, David W; Martinez-Picado, Javier; Dalmau, Judith; López-Galíndez, Cecilio; Casado, Concepción; Rauch, Andri; Günthard, Huldrych F; Bernasconi, Enos; Vernazza, Pietro; Klimkait, Thomas; Yerly, Sabine; O'Brien, Stephen J; Listgarten, Jennifer; Pfeifer, Nico; Lippert, Christoph; Fusi, Nicolo; Kutalik, Zoltán; Allen, Todd M; Müller, Viktor; Harrigan, P Richard; Heckerman, David; Telenti, Amalio; Fellay, Jacques

    2013-10-29

    HIV-1 sequence diversity is affected by selection pressures arising from host genomic factors. Using paired human and viral data from 1071 individuals, we ran >3000 genome-wide scans, testing for associations between host DNA polymorphisms, HIV-1 sequence variation and plasma viral load (VL), while considering human and viral population structure. We observed significant human SNP associations to a total of 48 HIV-1 amino acid variants (pgenome-to-genome approach highlights sites of genomic conflict and is a strategy generally applicable to studies of host-pathogen interaction. DOI:http://dx.doi.org/10.7554/eLife.01123.001.

  15. Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing

    Directory of Open Access Journals (Sweden)

    Ali Idris

    2014-03-01

    Full Text Available Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes (genus, Begomovirus; family, Geminiviridae were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA. Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS. CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.

  16. Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing

    KAUST Repository

    Idris, Ali

    2014-03-12

    Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched