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Sample records for leukemia virus replication

  1. Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence

    PubMed Central

    Carpentier, Alexandre; Barez, Pierre-Yves; Hamaidia, Malik; Gazon, Hélène; de Brogniez, Alix; Perike, Srikanth; Gillet, Nicolas; Willems, Luc

    2015-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans. PMID:26198240

  2. Molecular cloning of osteoma-inducing replication-competent murine leukemia viruses from the RFB osteoma virus stock.

    PubMed Central

    Pedersen, L; Behnisch, W; Schmidt, J; Luz, A; Pedersen, F S; Erfle, V; Strauss, P G

    1992-01-01

    We report the molecular cloning of two replication-competent osteoma-inducing murine leukemia viruses from the RFB osteoma virus stock (M. P. Finkel, C. A. Reilly, Jr., B. O. Biskis, and I. L. Greco, p. 353-366, in C. H. G. Price and F. G. M. Ross, ed., Bone--Certain Aspects of Neoplasia, 1973). Like the original RFB osteoma virus stock, viruses derived from the molecular RFB clones induced multiple osteomas in mice of the CBA/Ca strain. The cloned RFB viruses were indistinguishable by restriction enzyme analysis and by nucleotide sequence analysis of their long-terminal-repeat regions and showed close relatedness to the Akv murine leukemia virus. Images PMID:1326664

  3. Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis.

    PubMed

    de Brogniez, Alix; Mast, Jan; Willems, Luc

    2016-03-24

    Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis.

  4. Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications.

    PubMed Central

    Parthasarathi, S; Varela-Echavarría, A; Ron, Y; Preston, B D; Dougherty, J P

    1995-01-01

    Retroviruses evolve at rapid rates, which is presumably advantageous for responding to selective pressures. Understanding the basic mutational processes involved during retroviral replication is important for comprehending the ability of retroviruses to escape immunosurveillance and antiviral drug treatment. Moreover, since retroviral vectors are important vehicles for somatic cell gene therapy, knowledge of the mechanism of retroviral variation is critical for anticipating untoward mutational events occurring during retrovirus-medicated gene transfer. The focus of this report is to examine the spectrum of genomic rearrangements arising during a single cycle of Moloney murine leukemia virus (MoMLV) vector virus replication. An MoMLV vector containing the herpes simplex virus thymidine kinase (tk) gene was constructed. MoMLV vector virus was produced in packaging lines, and target cells were infected. From a total of 224 mutant proviruses analyzed, 114 had gross rearrangements readily detectable by Southern blotting. The remaining proviruses were of parental size. PCR and DNA sequence analysis of 73 of the grossly rearranged mutant proviruses indicated they resulted from deletions, combined with insertions, duplications, and complex mutations that were a result of multiple genomic alterations in the same provirus. Complex hypermutations distinct from those previously described for spleen necrosis virus and human immunodeficiency virus were detected. There was a correlation between the mutation breakpoints and single-stranded regions in the predicted viral RNA secondary structure. The results also confirmed that the tk gene is inactivated at an average rate of about 8.8% per cycle of retroviral replication, which corresponds to a rate of mutation of 3%/kbp. PMID:7494312

  5. [Efficacy of siRNA on feline leukemia virus replication in vitro].

    PubMed

    Lehmann, Melanie; Weber, Karin; Rauch, Gisep; Hofmann-Lehmann, Regina; Hosie, Margaret J; Meli, Marina L; Hartmann, Katrin

    2015-01-01

    Feline leukemia virus (FeLV) can lead to severe clinical signs in cats. Until now, there is no effective therapy for FeLV-infected cats. RNA interference-based antiviral therapy is a new concept. Specific small interfering RNA (siRNA) are designed complementary to the mRNA of a target region, and thus inhibit replication. Several studies have proven efficacy of siRNAs in inhibiting virus replication. The aim of this study was to evaluate the inhibitory potential of siRNAs against FeLV replication in vitro. siRNAs against the FeLV env gene and the host cell surface receptor (feTHTR1) which is used by FeLV-A for entry as well as siRNA that were not complementary to the FeLV or cat genome, were tested. Crandell feline kidney cells (CrFK cells) were transfected with FeLV-A/Glasgow-1. On day 13, infected cells were transfected with siRNAs. As control, cells were mock-transfected or treated with azidothymidine (AZT) (5 μg/ml). Culture supernatants were analyzed for FeLV RNA using quantitative real-time RT-PCR and for FeLV p27 by ELISA every 24 hours for five days. All siRNAs significantly reduced viral RNA and p27 production, starting after 48 hours. The fact that non-complementary siRNAs also inhibited virus replication may lead to the conclusion that unspecific mechanisms rather than specific binding lead to inhibition.

  6. Effect of internal genomic sequences of the Moloney murine leukemia virus on replication

    SciTech Connect

    Fomin, I.K.; Lobanova, A.B.; Voitenok, N.N.

    1995-11-01

    Construction and use of retrovirus vectors derived from the Moloney murine leukemia virus (MoMuLV) are described. These vectors, designated minimal vectors, contain the left and right long terminal repeats (LTRs), a binding site for proline tRNA, a polypurine tract (PPT), and a dominant marker for selective introduction of vectors into a packaging cell line, but lack the internal sequences of the virus genome. The experiments showed that the minimal vectors can be replicated and that their titer was approximately 1500-fold lower than that of wild-type vectors. The minimal vectors were shown to contain all the cis-acting sequences necessary for correct reverse transcription. One infectious virion, like wild-type viruses, produced only one provirus. Unlike the avian reticuloendotheliosis virus (REV), {Psi}{sup +} and {Psi}{sup {minus}} genomes of MoMuLV did not compete for virion proteins in the {Psi}2 packaging cell line. When an insert was introduced into a central part of the LTR U5 region, the titer of the minimal vector remained the same, while the titer of the wild-type vector decreased approximately 40-fold. 28 refs., 2 figs., 2 tabs.

  7. Characterization of producer cell-dependent restriction of murine leukemia virus replication.

    PubMed

    Serhan, Fatima; Jourdan, Nathalie; Saleun, Sylvie; Moullier, Philippe; Duisit, Ghislaine

    2002-07-01

    We previously reported that the human bronchocarcinoma cell line A549 produces poorly infectious gibbon ape leukemia virus-pseudotyped Moloney murine leukemia virus (MLV). In contrast, similar amounts of virions recovered from human fibrosarcoma HT1080 cells result in 10-fold-higher transduction rates (G. Duisit, A. Salvetti, P. Moullier, and F. Cosset, Hum. Gene Ther. 10:189-200, 1999). We have now extended this initial observation to other type-C envelope (Env) pseudotypes and analyzed the mechanism involved. Structural and morphological analysis showed that viral particles recovered from A549 (A549-MLV) and HT1080 (HT1080-MLV) cells were normal and indistinguishable from each other. They expressed equivalent levels of mature Env proteins and bound similarly to the target cells. Furthermore, incoming particles reached the cytosol and directed the synthesis of linear viral DNA equally efficiently. However, almost no detectable circular DNAs could be detected in A549-MLV-infected cells, indicating that the block of infection resulted from defective nuclear translocation of the preintegration complex. Interestingly, pseudotyping of A549-MLV with vesicular stomatitis virus glycoprotein G restored the amount of circular DNA forms as well as the transduction rates to HT1080-MLV levels, suggesting that the postentry blockage could be overcome by endocytic delivery of the core particles downstream of the restriction point. Thus, in contrast to the previously described target cell-dependent Fv-1 (or Fv1-like) restriction in mammalian cells (P. Pryciak and H. E. Varmus, J. Virol. 66:5959-5966, 1992; G. Towers, M. Bock, S. Martin, Y. Takeuchi, J. P. Stoye, and O. Danos, Proc. Natl. Acad. Sci. USA 97:12295-12299, 2000), we report here a new restriction of MLV replication that relies only on the producer cell type.

  8. Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis

    PubMed Central

    de Brogniez, Alix; Mast, Jan; Willems, Luc

    2016-01-01

    Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis. PMID:27023592

  9. Inhibition of Feline leukemia virus replication by the integrase inhibitor Raltegravir.

    PubMed

    Cattori, Valentino; Weibel, Beatrice; Lutz, Hans

    2011-08-26

    The oncogenic gammaretrovirus Feline leukemia virus (FeLV) has been the leading cause of death among domestic cats until the introduction of efficient diagnostics and vaccines in the late 1980s. So far, no efficient treatment for viremic animals is available. Hence, use of the FeLV model to evaluate antiretroviral therapies applied to HIV is a timely task. The efficacy of the integrase inhibitor Raltegravir, which is widely used for the treatment of HIV in humans, has been assessed in vitro for the FeLV-A/Glasgow-1 strain. EC(50) values for FeLV-A inhibition in feline cell lines are in the range of that observed for HIV and xenotropic murine leukemia virus-related gammaretrovirus. Therefore, Raltegravir may be a potential therapeutical agent for felids with progressive FeLV infection.

  10. Highly efficient tumor transduction and antitumor efficacy in experimental human malignant mesothelioma using replicating gibbon ape leukemia virus.

    PubMed

    Kubo, S; Takagi-Kimura, M; Logg, C R; Kasahara, N

    2013-12-01

    Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), in human malignant mesothelioma cells. In vitro, both RRVs expressing the green fluorescent protein gene efficiently replicated in most mesothelioma cell lines tested, but not in normal mesothelial cells. Notably, in ACC-MESO-1 mesothelioma cells that were not permissive for AMLV-RRV, the GALV-RRV could spread efficiently in culture and in mice with subcutaneous xenografts by in vivo fluorescence imaging. Next, GALV-RRV expressing the cytosine deaminase prodrug activator gene showed efficient killing of ACC-MESO-1 cells in a prodrug 5-fluorocytosine dose-dependent manner, compared with AMLV-RRV. GALV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous ACC-MESO-1 tumor growth in nude mice. Quantitative reverse transcription PCR demonstrated that ACC-MESO-1 cells express higher PiT-1 (GALV receptor) and lower PiT-2 (AMLV receptor) compared with normal mesothelial cells and other mesothelioma cells, presumably accounting for the distinctive finding that GALV-RRV replicates much more robustly than AMLV-RRV in these cells. These data indicate the potential utility of GALV-RRV-mediated prodrug activator gene therapy in the treatment of mesothelioma.

  11. Overexpression of feline tripartite motif-containing 25 interferes with the late stage of feline leukemia virus replication.

    PubMed

    Koba, Ryota; Oguma, Keisuke; Sentsui, Hiroshi

    2015-06-02

    Tripartite motif-containing 25 (TRIM25) regulates various cellular processes through E3 ubiquitin ligase activity. Previous studies have revealed that the expression of TRIM25 is induced by type I interferon and that TRIM25 is involved in the host cellular innate immune response against retroviral infection. Although retroviral infection is prevalent in domestic cats, the roles of feline TRIM25 in the immune response against these viral infections are poorly understood. Because feline TRIM25 is expected to modulate the infection of feline leukemia virus (FeLV), we investigated its effects on early- and late-stage FeLV replication. This study revealed that ectopic expression of feline TRIM25 in HEK293T cells reduced viral protein levels leading to the inhibition of FeLV release. Our findings show that feline TRIM25 has a potent antiviral activity and implicate an antiviral mechanism whereby feline TRIM25 interferes with late-stage FeLV replication.

  12. Inefficient viral replication of bovine leukemia virus induced by spontaneous deletion mutation in the G4 gene.

    PubMed

    Murakami, Hironobu; Uchiyama, Jumpei; Nikaido, Sae; Sato, Reiichiro; Sakaguchi, Masahiro; Tsukamoto, Kenji

    2016-10-01

    Enzootic bovine leucosis is caused by bovine leukemia virus (BLV) infection, which is highly prevalent in several regions of the world and significantly impacts the livestock industry. In BLV infection, the proviral load in the blood reflects disease progression. Although the BLV genome is highly conserved among retroviruses, genetic variation has been reported. However, the relationship between proviral load and genetic variation is poorly understood. In this study, we investigated the changes in proviral load in BLV-infected cattle in Japan and then identified and analysed a BLV strain pvAF967 that had a static proviral load. First, examining the proviral load in the aleukaemic cattle in 2014 and 2015, cow AF967 showed a static proviral load, while the other cows showed significant increases in proviral load. Sequencing the provirus in cow AF967 showed a deletion of 12 nt located in the G4 gene. An in vitro assay system using BLV molecular clone was set up to evaluate viral replication and production. In this in vitro assay, the deletion mutation in the G4 gene resulted in a significant decrease in viral replication and production. In addition, we showed that the deletion mutation did not affect the viral transcriptional activity of Tax protein, which is also important for virus replication. The emergence of strain pvAF967 that showed a static proviral load, combined with other retrovirus evolutionary traits, suggests that some BLV strains may have evolved to be symbiotic with cattle.

  13. Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo

    PubMed Central

    Hamaidia, Malik; de Brogniez, Alix; Gutiérrez, Gerónimo; Renotte, Nathalie; Reichert, Michal; Trono, Karina; Willems, Luc

    2016-01-01

    Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expression of genes involved in cell signaling, cancer and immunity. We further demonstrate that BLV miRNAs are essential to induce B-cell tumors in an experimental model and to promote efficient viral replication in the natural host. PMID:27123579

  14. Four Moloney murine leukemia virus-infected rat cell clones producing replication-defective particles: protein and nucleic acid analyses.

    PubMed Central

    Yoshimura, F K; Yamamura, J M

    1981-01-01

    Four cloned rat cell lines (NX-1 to -4) infected with Moloney murine leukemia virus and defective in virus replication were found to be all different by viral protein and nucleic acid analyses. All four clones produced noninfectious particles and, except for NX-2, at about the same level as wild type. Compared with wild-type virions these defective particles contained larger amounts of gag precursor proteins and very little or no p30 or p15. Analysis of intracellular precursor proteins revealed that NX-2 to -4 synthesized normal Pr65gag, whereas NX-1 produced a slightly smaller precursor. Both NX-1 and NX-4 synthesized an intracellular polyprotein with a size similar to that of wild-type Pr180 gag-pol. Restriction endonuclease analysis of NX-1 to -4 cellular DNA showed that each clone contained a single integrated provirus which possessed large terminal repeat sequences at both the 5' and 3' ends. The proviruses of NX-1 to -3 appeared normal by restriction endonuclease analysis, but NX-4 provirus had a deletion of 1,700 base pairs comprising part of the polymerase region. The noninfectious particles produced by all four clones packaged Moloney viral RNAs and rat RNAs of two different sizes. Images PMID:6165841

  15. miR-28-3p is a cellular restriction factor that inhibits human T cell leukemia virus, type 1 (HTLV-1) replication and virus infection.

    PubMed

    Bai, Xue Tao; Nicot, Christophe

    2015-02-27

    Human T cell leukemia virus, type 1 (HTLV-1) replication and spread are controlled by different viral and cellular factors. Although several anti-HIV cellular microRNAs have been described, such a regulation for HTLV-1 has not been reported. In this study, we found that miR-28-3p inhibits HTLV-1 virus expression and its replication by targeting a specific site within the genomic gag/pol viral mRNA. Because miR-28-3p is highly expressed in resting T cells, which are resistant to HTLV-1 infection, we investigated a potential protective role of miR-28-3p against de novo HTLV-1 infection. To this end, we developed a new sensitive and quantitative assay on the basis of the detection of products of reverse transcription. We demonstrate that miR-28-3p does not prevent virus receptor interaction or virus entry but, instead, induces a post-entry block at the reverse transcription level. In addition, we found that HTLV-1, subtype 1A isolates corresponding to the Japanese strain ATK-1 present a natural, single-nucleotide polymorphism within the miR-28-3p target site. As a result of this polymorphism, the ATK-1 virus sequence was not inhibited by miR-28. Interestingly, genetic studies on the transmission of the virus has shown that the ATK-1 strain, which carries a Thr-to-Cys transition mutation, is transmitted efficiently between spouses, suggesting that miR-28 may play an important role in HTLV-1 transmission.

  16. Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

    PubMed Central

    Halvas, Elias K.; Svarovskaia, Evguenia S.; Pathak, Vinay K.

    2000-01-01

    Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription. PMID:11044079

  17. Endogenous CD317/Tetherin limits replication of HIV-1 and murine leukemia virus in rodent cells and is resistant to antagonists from primate viruses.

    PubMed

    Goffinet, Christine; Schmidt, Sarah; Kern, Christian; Oberbremer, Lena; Keppler, Oliver T

    2010-11-01

    Human CD317 (BST-2/tetherin) is an intrinsic immunity factor that blocks the release of retroviruses, filoviruses, herpesviruses, and arenaviruses. It is unclear whether CD317 expressed endogenously in rodent cells has the capacity to interfere with the replication of the retroviral rodent pathogen murine leukemia virus (MLV) or, in the context of small-animal model development, contributes to the well-established late-phase restriction of human immunodeficiency virus type 1 (HIV-1). Here, we show that small interfering RNA (siRNA)-mediated knockdown of CD317 relieved a virion release restriction and markedly enhanced the egress of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) in rat cells, including primary macrophages. Moreover, rodent CD317 potently inhibited MLV release, and siRNA-mediated depletion of CD317 in a mouse T-cell line resulted in the accelerated spread of MLV. Several virus-encoded antagonists have recently been reported to overcome the restriction imposed by human or monkey CD317, including HIV-1 Vpu, envelope glycoproteins of HIV-2 and Ebola virus, Kaposi's sarcoma-associated herpesvirus K5, and SIV Nef. In contrast, both rat and mouse CD317 showed a high degree of resistance to these viral antagonists. These data suggest that CD317 is a broadly acting and conserved mediator of innate control of retroviral infection and pathogenesis that restricts the release of retroviruses and lentiviruses in rodents. The high degree of resistance of the rodent CD317 restriction factors to antagonists from primate viruses has implications for HIV-1 small-animal model development and may guide the design of novel antiviral interventions.

  18. Human T-cell leukemia virus type 2 antisense viral protein 2 is dispensable for in vitro immortalization but functions to repress early virus replication in vivo.

    PubMed

    Yin, Han; Kannian, Priya; Dissinger, Nathan; Haines, Robyn; Niewiesk, Stefan; Green, Patrick L

    2012-08-01

    Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are closely related but pathogenically distinct human retroviruses. The antisense strand of the HTLV-1 genome encodes HTLV-1 basic leucine zipper (b-ZIP) protein (HBZ), a protein that inhibits Tax-mediated viral transcription, enhances T-cell proliferation, and promotes viral persistence. Recently, an HTLV-2 antisense viral protein (APH-2) was identified. Despite its lack of a typical b-ZIP domain, APH-2, like HBZ, interacts with cyclic AMP response element binding protein (CREB) and downregulates Tax-mediated viral transcription. Here, we provide evidence that the APH-2 C-terminal LXXLL motif is important for CREB binding and Tax repression. In order to investigate the functional role of APH-2 in the HTLV-2-mediated immortalization of primary T lymphocytes in vitro and in HTLV-2 infection in vivo, we generated APH-2 mutant viruses. In cell cultures, the immortalization capacities of APH-2 mutant viruses were indistinguishable from that of wild-type HTLV-2 (wtHTLV-2), indicating that, like HBZ, APH-2 is dispensable for viral infection and cellular transformation. In vivo, rabbits inoculated with either wtHTLV-2 or APH-2 mutant viruses established a persistent infection. However, the APH-2 knockout virus displayed an increased replication rate, as measured by an increased viral antibody response and a higher proviral load. In contrast to HTLV-1 HBZ, we show that APH-2 is dispensable for the establishment of an efficient infection and persistence in a rabbit animal model. Therefore, antisense proteins of HTLV-1 and HTLV-2 have evolved different functions in vivo, and further comparative studies will provide fundamental insights into the distinct pathobiologies of these two viruses.

  19. DNA Virus Replication Compartments

    PubMed Central

    Schmid, Melanie; Speiseder, Thomas; Dobner, Thomas

    2014-01-01

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

  20. Repression of human T-cell leukemia virus type 1 and type 2 replication by a viral mRNA-encoded posttranscriptional regulator.

    PubMed

    Younis, Ihab; Khair, Lyne; Dundr, Miroslav; Lairmore, Michael D; Franchini, Genoveffa; Green, Patrick L

    2004-10-01

    Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are complex retroviruses that persist in the host, eventually causing leukemia and neurological disease in a small percentage of infected individuals. In addition to structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory (open reading frame I and II) proteins. The viral Tax and Rex proteins positively regulate virus production. Tax activates viral and cellular transcription to promote T-cell growth and, ultimately, malignant transformation. Rex acts posttranscriptionally to facilitate cytoplasmic expression of viral mRNAs that encode the structural and enzymatic gene products, thus positively controlling virion expression. Here, we report that both HTLV-1 and HTLV-2 have evolved accessory genes to encode proteins that act as negative regulators of both Tax and Rex. HTLV-1 p30(II) and the related HTLV-2 p28(II) inhibit virion production by binding to and retaining tax/rex mRNA in the nucleus. Reduction of viral replication in a cell carrying the provirus may allow escape from immune recognition in an infected individual. These data are consistent with the critical role of these proteins in viral persistence and pathogenesis in animal models of HTLV-1 and HTLV-2 infection.

  1. Human T-cell leukemia virus type 2 Rex carboxy terminus is an inhibitory/stability domain that regulates Rex functional activity and viral replication.

    PubMed

    Xie, Li; Kesic, Matthew; Yamamoto, Brenda; Li, Min; Younis, Ihab; Lairmore, Michael D; Green, Patrick L

    2009-05-01

    Human T-cell leukemia virus (HTLV) regulatory protein, Rex, functions to increase the expression of the viral structural and enzymatic gene products. The phosphorylation of two serine residues (S151 and S153) at the C terminus is important for the function of HTLV-2 Rex (Rex-2). The Rex-2 phosphomimetic double mutant (S151D, S153D) is locked in a functionally active conformation. Since rex and tax genes overlap, Rex S151D and S153D mutants were found to alter the Tax oncoprotein coding sequence and transactivation activities. Therefore, additional Rex-2 mutants including P152D, A157D, S151Term, and S158Term were generated and characterized ("Term" indicates termination codon). All Rex-2 mutants and wild-type (wt) Rex-2 localized predominantly to the nucleus/nucleolus, but in contrast to the detection of phosphorylated and unphosphorylated forms of wt Rex-2 (p26 and p24), mutant proteins were detected as a single phosphoprotein species. We found that Rex P152D, A157D, and S158Term mutants are more functionally active than wt Rex-2 and that the Rex-2 C terminus and its specific phosphorylation state are required for stability and optimal expression. In the context of the provirus, the more active Rex mutants (A157D or S158Term) promoted increased viral protein production, increased viral infectious spread, and enhanced HTLV-2-mediated cellular proliferation. Moreover, these Rex mutant viruses replicated and persisted in inoculated rabbits despite higher antiviral antibody responses. Thus, we identified in Rex-2 a novel C-terminal inhibitory domain that regulates functional activity and is positively regulated through phosphorylation. The ability of this domain to modulate viral replication likely plays a key role in the infectious spread of the virus and in virus-induced cellular proliferation.

  2. Human T-cell leukemia virus type 1 expressing nonoverlapping tax and rex genes replicates and immortalizes primary human T lymphocytes but fails to replicate and persist in vivo.

    PubMed

    Younis, Ihab; Yamamoto, Brenda; Phipps, Andrew; Green, Patrick L

    2005-12-01

    Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus associated primarily with adult T-cell leukemia and neurological disease. HTLV-1 encodes the positive trans-regulatory proteins Tax and Rex, both of which are essential for viral replication. Tax activates transcription initiation from the viral long terminal repeat and modulates the transcription or activity of a number of cellular genes. Rex regulates gene expression posttranscriptionally by facilitating the cytoplasmic expression of incompletely spliced viral mRNAs. Tax and Rex mutants have been identified that have defective activities or impaired biochemical properties associated with their function. To ultimately determine the contribution of specific protein activities on viral replication and cellular transformation of primary T cells, mutants need to be characterized in the context of an infectious molecular clone. Since the tax and rex genes are in partially overlapping reading frames, mutation in one gene frequently disrupts the other, confounding interpretation of mutational analyses in the context of the virus. Here we generated and characterized a unique proviral clone (H1IT) in which the tax and rex genes were separated by expressing Tax from an internal ribosome entry site. We showed that H1IT expresses both functional Tax and Rex. In short- and long-term coculture assays, H1IT was competent to infect and immortalize primary human T cells similar to wild-type HTLV-1. In contrast, H1IT failed to efficiently replicate and persist in inoculated rabbits, thus emphasizing the importance of temporal and quantitative regulation of specific mRNA for viral survival in vivo.

  3. Gibbon ape leukemia virus poorly replicates in primary human T lymphocytes: implications for safety testing of primary human T lymphocytes transduced with GALV-pseudotyped vectors.

    PubMed

    Lamers, Cor H J; Willemsen, Ralph A; van Elzakker, Pascal M M L; Gratama, Jan Willem; Debets, Reno

    2009-04-01

    The Food and Drug Administration/Center for Biologics Evaluation and Research has defined that for retroviral gene therapy, the vector-producing cell, the vector preparation, and the ex vivo gene-transduced cells have to be tested for absence of replication-competent retrovirus (RCR) if the transduced cells are cultured for >4 days. We assessed the sensitivity of the "extended PG4(S+L-) assay" to detect gibbon ape leukemia virus (GALV) RCR, and applied this assay to measure GALV RCR spread in retrovirally transduced T cells. To this end, T cells were expanded for 12 days after transduction with a GALV-envelope pseudotyped retroviral vector expressing single chain variable fragment (anticarbonic anhydrase IX) in presence or absence of GALV RCR. Results showed that: (1) the "extended PG4(S+L-) assay" detects 1 focus-forming unit (ffu) GALV RCR and thus is applicable and sufficiently sensitive to screen human T-cell cultures for absence of infectious GALV RCR; (2) although GALV RCR infect human T cells, it very poorly replicate in T cells; (3) GALV RCR, when present at low levels immediately upon gene transduction (ie, 100 ffu/20x10 T cells in 100 mL), did not spread during a 12-day T-cell culture at clinical scale. Our observation that GALV RCR poorly spreads in primary human T-cell cultures questions the relevance of testing T-cell transductants for RCR on top of testing the vector-producing cells and the clinical vector batch for RCR and warrants evaluation of the current policy for safety testing of ex vivo retrovirally transduced T lymphocytes for GALV RCR.

  4. Plant virus replication and movement.

    PubMed

    Heinlein, Manfred

    2015-05-01

    Replication and intercellular spread of viruses depend on host mechanisms supporting the formation, transport and turnover of functional complexes between viral genomes, virus-encoded products and cellular factors. To enhance these processes, viruses assemble and replicate in membrane-associated complexes that may develop into "virus factories" or "viroplasms" in which viral components and host factors required for replication are concentrated. Many plant viruses replicate in association with the cortical ER-actin network that is continuous between cells through plasmodesmata. The replication complexes can be highly organized and supported by network interactions between the viral genome and the virus-encoded proteins. Intracellular PD targeting of replication complexes links the process of movement to replication and provides specificity for transport of the viral genome by the virus-encoded movement proteins. The formation and trafficking of replication complexes and also the development and anchorage of replication factories involves important roles of the cortical cytoskeleton and associated motor proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. A deletion in the proximal untranslated pX region of human T-cell leukemia virus type II decreases viral replication but not infectivity in vivo.

    PubMed

    Cockerell, G L; Rovnak, J; Green, P L; Chen, I S

    1996-02-01

    The function of untranslated (UT) nucleotide sequences in the proximal portion of the pX region of the human T-cell leukemia virus (HTLV) family of retroviruses remains enigmatic. Previous studies have shown that these sequences are not necessary for the expression of viral proteins or for the induction, transmission, or maintenance of the transformed cell type in vitro. To determine the effect of the UT region in vivo, separate groups of rabbits were inoculated with lethally irradiated, stable clones of the human B-lymphoblastoid cell line, 729, transfected with either a full-length wild-type HTLV-II clone (pH6neo) or a mutant clone containing a 324-bp deletion in the proximal UT portion of pX (pH6neo delta UT[6661-6984]), or nontransfected 729 cells. All rabbits inoculated with either wild-type or pX-deleted HTLV-II developed a similar profile and titer of serum antibodies against HTLV-II antigens, as determined by Western immunoblots, by 4 weeks postinoculation (PI). Antibody titers, as determined by enzyme immunoassay, were similar between the two groups of rabbits and increased over the 18-week period of study. All rabbits were killed at 18 weeks PI, and spleen, peripheral blood lymphocytes (PBMC), bone marrow, and mesenteric lymph node were assayed for HTLV-II tax/rex sequences by quantitative polymerase chain reaction. Virus was detected in all tissues tested from all rabbits inoculated with 729pH6neo cells containing wild-type HTLV-II, which contained between 1.4 and 0.3 mean copies of provirus per cell. In contrast, the distribution and number of provirus copies were more limited in rabbits inoculated with 729pH6neo delta UT(6661-6984) cells containing UT-deleted HTLV-II; in most tissues, there was a fivefold to sevenfold reduction in mean provirus copies per cell as compared with rabbits inoculated with wild-type HTLV-II. All rabbits inoculated with control 729 cells remained negative for HTLV-II infection, as determined by the same techniques. It was

  6. Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation.

    PubMed

    Green, P L; Ross, T M; Chen, I S; Pettiford, S

    1995-01-01

    Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.

  7. Development of leukemia in mice transgenic for the tax gene of human T-cell leukemia virus type I.

    PubMed Central

    Grossman, W J; Kimata, J T; Wong, F H; Zutter, M; Ley, T J; Ratner, L

    1995-01-01

    The human T-cell leukemia virus type I Tax protein trans-activates several cellular genes implicated in T-cell replication and activation. To investigate its leukemogenic potential, Tax was targeted to the mature T-lymphocyte compartment in transgenic mice by using the human granzyme B promoter. These mice developed large granular lymphocytic leukemia, demonstrating that expression of Tax in the lymphocyte compartment is sufficient for the development of leukemia. Furthermore, these observations suggest that human T-cell leukemia virus infection may be involved in the development of large granular lymphocytic leukemia. Images Fig. 1 Fig. 2 Fig. 4 PMID:7862633

  8. HSP90 Protects the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Oncoprotein from Proteasomal Degradation To Support NF-κB Activation and HTLV-1 Replication

    PubMed Central

    Gao, Linlin

    2013-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients. PMID:24109220

  9. Pathogenicity of molecularly cloned bovine leukemia virus.

    PubMed Central

    Rovnak, J; Boyd, A L; Casey, J W; Gonda, M A; Jensen, W A; Cockerell, G L

    1993-01-01

    To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis. Images PMID:8230433

  10. LEUKEMIA-ASSOCIATED TRANSPLANTATION ANTIGENS RELATED TO MURINE LEUKEMIA VIRUS

    PubMed Central

    Sato, H.; Boyse, E. A.; Aoki, T.; Iritani, C.; Old, L. J.

    1973-01-01

    Two BALB radiation leukemias are strongly rejected by hybrids of BALB with certain other mouse strains, although BALB mice themselves exhibit no detectable resistance whatever. Hybrids immunized with progressively increased inocula are resistant to 200 x 106 or more leukemia cells; their serum is cytotoxic for the leukemia cells in vitro and protects BALB mice against challenge with these BALB leukemias. The antigenic system thus identified has been named X.1. In (BALB x B6) hybrids the major determinant of resistance was shown to be a B6 gene in the K region of H-2. This is likely to be the Rgv-1 (Resistance to gross virus) locus of Lilly, which may thus be identified in this case as an Ir (Immune response) allele conferring ability to respond to X.1 antigen on MuLV and leukemia cells, and so responsible for production of X.1 antibody and the rejection of X.1+ leukemia cells by hybrid mice. Immunoelectron microscopy with X.1 antiserum (from immunized hybrids) shows labeling both on the cell surface and on virions produced by the leukemia cells. It is not known whether X.1 comprises only one or more than one antigen. Three radiation-induced BALB leukemias, one A strain radiation-induced leukemia, and 15/15 AKR primary spontaneous leukemias were typed X.1+ by the cytotoxicity test. Several other leukemias, including one induced by passage A Gross virus and one long-transplanted AKR ascites leukemia carried in (B6 x AKR)F1 hybrids, were X.1-. Normal mice of strains with a high incidence of leukemia and one other strain (129) express X.1 antigen, but evidently in amounts too small for certain detection in vitro; by the method of absorption in vivo, however, these strains could be typed X.1+ and other strains X.1-. We ascribe the X.1 antigen system tentatively to a sub-type of MuLV that is not passage A Gross virus and is probably not the dominant sub-type in strains with a high incidence of leukemia. After repeated passage in hybrids, one of the BALB leukemias became

  11. Replication-selective viruses for cancer therapy.

    PubMed

    Biederer, Carola; Ries, Stefan; Brandts, Christian H; McCormick, Frank

    2002-03-01

    Advances in our understanding of the molecular basis of cancer and the availability of technology to genetically engineer viruses have led to the development of replication-competent viruses to treat cancer. In theory, replication-selective viruses offer several appealing properties as biological agents for cancer therapy: they kill tumor cells selectively, and their replication leads to amplification of their oncolytic potential. Most preclinical experiments in tissue culture and in animal models support this notion. Clinical data on the first generation of replication-selective viruses are now rapidly accruing. The therapeutic index, and ultimately the clinical outcome, will depend on a complex balance between host and viral factors. This review discusses strategies to kill cancer cells based on our understanding of their molecular defects and the progress being made using replication-competent viruses for tumor therapy. We focus our discussion on a replication-selective adenovirus called ONYX-015 that has recently demonstrated encouraging results in clinical trials

  12. Leukemia Viruses Associated with Mouse Myeloma Cells*

    PubMed Central

    Watson, J.; Ralph, P.; Sarkar, S.; Cohn, Melvin

    1970-01-01

    Myeloma cells derived from BALB/c and C3H mice show evidence of infection by a murine leumemia virus. The immunoglobulin-producing myelomas secrete an RNA-containing virus with a density of 1.20 to 1.22 gm/cm3. RNA with a sedimentation coefficient of 74 S in 0.1 M sodium sodium chloride has been isolated from secreted virus particles and has a base composition similar to that found for other murine leukemia virus RNA. An intracellular virus particle has been partially purified and has a density of 1.29 to 1.32 gm/cm3. Both extracellular and intracellular virus particles contain the leukemia virus group-specific antigen. Images PMID:4317914

  13. [Oncogenes and the origin of leukemia. Acute avian leukemia viruses].

    PubMed

    Graf, T

    1988-03-01

    Oncogenes have been intimately associated with the genesis of human neoplasms. A particularly useful system to study the mechanism of tumorigenesis is a small group of avian retroviruses that carry two oncogenes. These viruses causes acute leukemias and can transform hematopoietic cells in vitro. The mechanisms by which viral oncogenes affect the growth control and differentiation of their target cells is now understood in fair detail for two of these virus strains. In the avian erythroblastosis virus AEV, the v-erbB oncogene deregulates the growth control of erythroid precursors, while verbA blocks their terminal differentiation into erythrocytes. Based on the findings that v-erbB oncogene corresponds to a mutated growth factor receptor gene and that v-erbA corresponds to a mutated hormone receptor gene, models have been developed that explain the function of these two oncogenes on a molecular basis. The myelomonocytic leukemia virus MH2 acts by a completely different mechanism. In this case, the v-myc oncogene stimulates the proliferation of macrophage-like cells, while the v-mil gene stimulates them to produce their own growth factor, thus leading to autocrine growth. It will be interesting to determine whether the type of mechanisms of oncogene cooperativity elucidated for acute leukemia viruses are also operative during leukemogenesis in humans.

  14. Genetic mapping of a mouse chromosomal locus required for mink cell focus-forming virus replication.

    PubMed Central

    Kozak, C A

    1983-01-01

    Mouse-hamster somatic cell hybrids were used to show that the recombinant mink cell focus-forming murine leukemia viruses and their ecotropic virus progenitors require different mouse chromosomes for replication. Mouse chromosome 1 was shown to carry the genetic information necessary for the replication of six different mink cell focus-forming isolates, and this gene, designated Rmc-1, was tentatively positioned at the distal end of the chromosome. PMID:6310150

  15. Uterine adenocarcinoma with feline leukemia virus infection.

    PubMed

    Cho, Sung-Jin; Lee, Hyun-A; Hong, Sunhwa; Kim, Okjin

    2011-12-01

    Feline endometrial adenocarcinomas are uncommon malignant neoplasms that have been poorly characterized to date. In this study, we describe a uterine adenocarcinoma in a Persian cat with feline leukemia virus infection. At the time of presentation, the cat, a female Persian chinchilla, was 2 years old. The cat underwent surgical ovariohystectomy. A cross-section of the uterine wall revealed a thickened uterine horn. The cat tested positive for feline leukemia virus as detected by polymerase chain reaction. Histopathological examination revealed uterine adenocarcinoma that had metastasized to the omentum, resulting in thickening and the formation of inflammatory lesions. Based on the histopathological findings, this case was diagnosed as a uterine adenocarcinoma with abdominal metastasis. To the best of our knowledge, this is the first report of a uterine adenocarcinoma with feline leukemia virus infection.

  16. Methadone enhances human influenza A virus replication.

    PubMed

    Chen, Yun-Hsiang; Wu, Kuang-Lun; Tsai, Ming-Ta; Chien, Wei-Hsien; Chen, Mao-Liang; Wang, Yun

    2017-01-01

    Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs. © 2015 Society for the Study of Addiction.

  17. Replication-Competent Controlled Herpes Simplex Virus.

    PubMed

    Bloom, David C; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria; Voellmy, Richard

    2015-10-01

    We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of

  18. Replication-Competent Controlled Herpes Simplex Virus

    PubMed Central

    Bloom, David C.; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria

    2015-01-01

    ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate

  19. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus

    PubMed Central

    Aida, Yoko; Murakami, Hironobu; Takahashi, Masahiko; Takeshima, Shin-Nosuke

    2013-01-01

    Bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) make up a unique retrovirus family. Both viruses induce chronic lymphoproliferative diseases with BLV affecting the B-cell lineage and HTLV-1 affecting the T-cell lineage. The pathologies of BLV- and HTLV-induced infections are notably similar, with an absence of chronic viraemia and a long latency period. These viruses encode at least two regulatory proteins, namely, Tax and Rex, in the pX region located between the env gene and the 3′ long terminal repeat. The Tax protein is a key contributor to the oncogenic potential of the virus, and is also the key protein involved in viral replication. However, BLV infection is not sufficient for leukemogenesis, and additional events such as gene mutations must take place. In this review, we first summarize the similarities between the two viruses in terms of genomic organization, virology, and pathology. We then describe the current knowledge of the BLV model, which may also be relevant for the understanding of leukemogenesis caused by HTLV-1. In addition, we address our improved understanding of Tax functions through the newly identified BLV Tax mutants, which have a substitution between amino acids 240 and 265. PMID:24265629

  20. The molecular biology of Bluetongue virus replication.

    PubMed

    Patel, Avnish; Roy, Polly

    2014-03-01

    The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention.

  1. Association between endogenous feline leukemia virus loads and exogenous feline leukemia virus infection in domestic cats.

    PubMed

    Tandon, Ravi; Cattori, Valentino; Pepin, Andrea C; Riond, Barbara; Meli, Marina L; McDonald, Mike; Doherr, Marcus G; Lutz, Hans; Hofmann-Lehmann, Regina

    2008-07-01

    Recently, we demonstrated that endogenous feline leukemia virus (enFeLV) loads may vary among cats of different populations and that FeLV-infected cats have higher enFeLV loads than uninfected cats. Thus, we hypothesized that enFeLV might influence the pathogenesis and outcome of FeLV infection. No significant difference in the infection outcome (regressive versus progressive infection) was observed between groups of cats with high or low enFeLV loads following FeLV-A challenge. However, cats with high enFeLV loads showed higher viral replication (plasma viral RNA and p27 antigen levels) than cats with low enFeLV loads in the early phase of the infection. The enFeLV transcription level varied at different time points, but no clear-cut pattern was observed. In conclusion, our results demonstrated an association between enFeLV loads and FeLV replication but not outcome of infection. enFeLV should be considered as an important confounder in experimental FeLV infection or vaccination studies.

  2. Multiscale modeling of virus replication and spread.

    PubMed

    Kumberger, Peter; Frey, Felix; Schwarz, Ulrich S; Graw, Frederik

    2016-07-01

    Replication and spread of human viruses is based on the simultaneous exploitation of many different host functions, bridging multiple scales in space and time. Mathematical modeling is essential to obtain a systems-level understanding of how human viruses manage to proceed through their life cycles. Here, we review corresponding advances for viral systems of large medical relevance, such as human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV). We will outline how the combination of mathematical models and experimental data has advanced our quantitative knowledge about various processes of these pathogens, and how novel quantitative approaches promise to fill remaining gaps. © 2016 Federation of European Biochemical Societies.

  3. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

    PubMed

    Guo, Longjun; Yu, Haidong; Gu, Weihong; Luo, Xiaolei; Li, Ren; Zhang, Jian; Xu, Yunfei; Yang, Lijun; Shen, Nan; Feng, Li; Wang, Yue

    2016-03-31

    Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

  4. Replication of biotinylated human immunodeficiency viruses.

    PubMed

    Belshan, Michael; Matthews, John M; Madson, Christian J

    2011-01-01

    Previous work demonstrated recently the adaptation of the Escherichia coli biotin ligase BirA - biotin acceptor sequence (BAS) labeling system to produce human immunodeficiency virus type 1 viruses with biotinylated integrase (NLXIN(B)) and matrix (NLXMA(B)) proteins (Belshan et al., 2009). This report describes the construction of an HIV permissive cell line stably expressing BirA (SupT1.BirA). Consistent with the results in the previous report, NLXMA(B) replicated similar to wild-type levels and expressed biotinylated Gag and MA proteins in the SupT1.BirA cells, whereas the replication of NLXIN(B) was reduced severely. Three additional HIV type 2 (HIV-2) viruses were constructed with the BAS inserted into the vpx and vpr accessory genes. Two BAS insertions were made into the C-terminal half of the Vpx, including one internal insertion, and one at the N-terminus of Vpr. All three viruses were replication competent in the SupT1.BirA cells and their target proteins biotinylated efficiently and incorporated into virions. These results demonstrate the potential utility of the biotinylation system to label and capture HIV protein complexes in the context of replicating virus.

  5. Methamphetamine Reduces Human Influenza A Virus Replication

    PubMed Central

    Chen, Yun-Hsiang; Wu, Kuang-Lun; Chen, Chia-Hsiang

    2012-01-01

    Methamphetamine (meth) is a highly addictive psychostimulant that is among the most widely abused illicit drugs, with an estimated over 35 million users in the world. Several lines of evidence suggest that chronic meth abuse is a major factor for increased risk of infections with human immunodeficiency virus and possibly other pathogens, due to its immunosuppressive property. Influenza A virus infections frequently cause epidemics and pandemics of respiratory diseases among human populations. However, little is known about whether meth has the ability to enhance influenza A virus replication, thus increasing severity of influenza illness in meth abusers. Herein, we investigated the effects of meth on influenza A virus replication in human lung epithelial A549 cells. The cells were exposed to meth and infected with human influenza A/WSN/33 (H1N1) virus. The viral progenies were titrated by plaque assays, and the expression of viral proteins and cellular proteins involved in interferon responses was examined by Western blotting and immunofluorescence staining. We report the first evidence that meth significantly reduces, rather than increases, virus propagation and the susceptibility to influenza infection in the human lung epithelial cell line, consistent with a decrease in viral protein synthesis. These effects were apparently not caused by meth’s effects on enhancing virus-induced interferon responses in the host cells, reducing viral biological activities, or reducing cell viability. Our results suggest that meth might not be a great risk factor for influenza A virus infection among meth abusers. Although the underlying mechanism responsible for the action of meth on attenuating virus replication requires further investigation, these findings prompt the study to examine whether other structurally similar compounds could be used as anti-influenza agents. PMID:23139774

  6. ANTIGENS OF LEUKEMIAS INDUCED BY NATURALLY OCCURRING MURINE LEUKEMIA VIRUS: THEIR RELATION TO THE ANTIGENS OF GROSS VIRUS AND OTHER MURINE LEUKEMIA VIRUSES

    PubMed Central

    Geering, Gayla; Old, Lloyd J.; Boyse, Edward A.

    1966-01-01

    Leukemias can be induced in W/Fu inbred rats by neonatal inoculation of normal thymus cells of C58 mice. These leukemias are not transplantable to C58 mice or to adult W/Fu rats, but they can be kept in passage in W/Fu rats aged 0 to 7 days. Adult W/Fu rats inoculated repeatedly with these isogenic leukemias produce cytotoxic and precipitating antibodies. These antisera are of particular value in the analysis of the antigens of leukemia cells and of leukemia viruses because their mode of preparation precludes the formation of antibody against any normal constituents of the cell. Analysis based on the cytotoxic test indicates the presence of 2 distinct cell surface antigens in leukemias induced by Passage A Gross virus or occurring spontaneously in mice of high-incidence strains. All leukemias and other tissues known to contain G (Gross) leukemia antigen have both determinants, but certain leukemias of low-incidence strains have only 1 of them and so were previously classified G-. Immunoprecipitation with these antisera reveals the presence of a cellular antigen common to G+ cells and absent from G- cells; the same antigen can be demonstrated in ether-treated Gross virus, but not in intact virus. This antigen is present also in ether-treated preparations of the Friend, Moloney, and Rauscher leukemia viruses, but not in Bittner (mammary tumor) virus. Thus it may be regarded as a group-specific antigen of murine leukemia viruses, in contrast to the type-specific cellular antigens demonstrable by the cytotoxic test. Four additional antigens associated with leukemias induced by wild-type Gross virus have been demonstrated by immunoprecipitation, but their relation to viral and cellular antigens has not been determined. PMID:4288480

  7. [Helper viruses of adeno-associated virus type 4 replication].

    PubMed

    Dreĭzin, R S; Zhuravel', T F; Shalunova, N V; Klenova, A V; Zolotarskaia, E E

    1979-01-01

    In replication of adeno-associated virus type 4 (AAV-4) the helper function may be performed by a non-defective virus from the same group of parvoviruses (Kilham virus). The synthesis of AAV-4 antigen was observed in a pig embryo kidney cell line, SPEV, chronically infected with Kilham virus, strain RV-13, 45--52 passages. A one-day-old SPEV-Kilham culture was infected with AAV-4. The AAV-4 antigen was detected by immunofluorescence at 6, 8, 12, 18 hours, 2, 3, 4, and 5 days after inoculation. During the first 2--4 days after inoculation the AAV-4 antigen was found in the nucleus and perinuclear zone, later in the cytoplasm. A "new" helper virus for AAV-4 replication has been found: simiancytomegalovirus in human embryo fibroblast cell culture permissive for the helper virus. In the systems where AAV-4 replicates, its antigen can be detected in the nucleus and perinuclear zone by IF. AAV-4 did not replicate in a system insensitive to the helper virus or under non-permissive conditions: at the time, the AAV-4 antigen localized only in the cell cytoplasm was detected.

  8. Calsyntenin-1 mediates hepatitis C virus replication.

    PubMed

    Awan, Zunaira; Tay, Enoch S E; Eyre, Nicholas S; Wu, Lindsay E; Beard, Michael R; Boo, Irene; Drummer, Heidi E; George, Jacob; Douglas, Mark W

    2016-08-01

    The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

  9. Ultrastructural Characterization of Zika Virus Replication Factories.

    PubMed

    Cortese, Mirko; Goellner, Sarah; Acosta, Eliana Gisela; Neufeldt, Christopher John; Oleksiuk, Olga; Lampe, Marko; Haselmann, Uta; Funaya, Charlotta; Schieber, Nicole; Ronchi, Paolo; Schorb, Martin; Pruunsild, Priit; Schwab, Yannick; Chatel-Chaix, Laurent; Ruggieri, Alessia; Bartenschlager, Ralf

    2017-02-28

    A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.

  10. Junín Virus Pathogenesis and Virus Replication

    PubMed Central

    Grant, Ashley; Seregin, Alexey; Huang, Cheng; Kolokoltsova, Olga; Brasier, Allan; Peters, Clarence; Paessler, Slobodan

    2012-01-01

    Junín virus, the etiological agent of Argentine hemorrhagic fever, causes significant morbidity and mortality. The virus is spread through the aerosolization of host rodent excreta and endemic to the humid pampas of Argentina. Recently, significant progress has been achieved with the development of new technologies (e.g. reverse genetics) that have expanded knowledge about the pathogenesis and viral replication of Junín virus. We will review the pathogenesis of Junín virus in various animal models and the role of innate and adaptive immunity during infection. We will highlight current research regarding the role of molecular biology of Junín virus in elucidating virus attenuation. We will also summarize current knowledge on Junín virus pathogenesis focusing on the recent development of vaccines and potential therapeutics. PMID:23202466

  11. Replication strategy of human hepatitis B virus

    SciTech Connect

    Will, H.; Reiser, W.; Weimer, T.; Pfaff, E.; Buescher, M.; Sprengel, R.; Cattaneo, R.; Schaller, H.

    1987-03-01

    To study the replication strategy of the human hepatitis B virus, the 5' end of the RNA pregenome and the initiation sites of DNA plus and minus strands have been mapped. The RNA pregenome was found to be terminally redundant by 120 nucleotides; it is initiated within the pre-C region and may also function as mRNA for synthesis of the major core protein and the hepatitis B virus reverse transcriptase. The hepatitis B virus DNA minus strand is initiated within the direct repeat sequence DR1, it contains a terminal redundancy of up to eight nucleotides, and its synthesis does not require any template switch. The DNA plus strand is primed by a short oligoribonucleotide probably derived from the 5' end of the RNA pregenome, and its synthesis is initiated close to the direct repeat sequence DR2. For its elongation to pass the discontinuity in the DNA minus strand an intramolecular template switch occurs using the terminal redundancy of this template. Thus, the route of reverse transcription and DNA replication of hepatitis B viruses is fundamentally different from that of retroviruses.

  12. Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection

    PubMed Central

    Valle-Tenney, Roger; Opazo, Tatiana; Cancino, Jorge; Goff, Stephen P.

    2016-01-01

    ABSTRACT During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm—a crowded environment where diffusion is slow—is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for

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

    NASA Astrophysics Data System (ADS)

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

    1993-12-01

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

  14. Replication and transmission of influenza viruses in Japanese quail

    PubMed Central

    Makarova, Natalia V.; Ozaki, Hiroishi; Kida, Hiroshi; Webster, Robert G.; Perez, Daniel R.

    2015-01-01

    Quail have emerged as a potential intermediate host in the spread of avian influenza A viruses in poultry in Hong Kong. To better understand this possible role, we tested the replication and transmission in quail of influenza A viruses of all 15 HA subtypes. Quail supported the replication of at least 14 subtypes. Influenza A viruses replicated predominantly in the respiratory tract. Transmission experiments suggested that perpetuation of avian influenza viruses in quail requires adaptation. Swine influenza viruses were isolated from the respiratory tract of quail at low levels. There was no evidence of human influenza A or B virus replication. Interestingly, a human–avian recombinant containing the surface glycoprotein genes of a quail virus and the internal genes of a human virus replicated and transmitted readily in quail; therefore, quail could function as amplifiers of influenza virus reassortants that have the potential to infect humans and/or other mammalian species. PMID:12788625

  15. Apparent posttranscriptional block to anaerobic induction of endogenous leukemia virus.

    PubMed Central

    Whitaker-Dowling, P A; Marotti, K R; Anderson, G R

    1979-01-01

    Uninfected Fischer rat cells were induced by anaerobic stress to transcribe high levels of endogenous type C leukemia virus RNA. Complete 35S virus RNA with attached polyadenylic acid sequences was found associated with polysomes, indicating functional mRNA. Since no mature virus was released under these conditions, the presence of a posttranscriptional block to complete virus synthesis is strongly indicated. PMID:232174

  16. Autophagic machinery activated by dengue virus enhances virus replication

    SciTech Connect

    Lee, Y.-R.; Lei, H.-Y.; Liu, M.-T.; Wang, J.-R.; Chen, S.-H.; Jiang-Shieh, Y.-F.; Lin, Y.-S.; Yeh, T.-M.; Liu, C.-C.; Liu, H.-S.

    2008-05-10

    Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.

  17. In Vivo Analysis of Human T-Cell Leukemia Virus Type 1 Reverse Transcription Accuracy

    PubMed Central

    Mansky, Louis M.

    2000-01-01

    Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZα peptide gene region (the mutational target). The vector was replicated by trans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 × 10−6 mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription. PMID:11000222

  18. Rubella Virus Replication and Links to Teratogenicity

    PubMed Central

    Lee, Jia-Yee; Bowden, D. Scott

    2000-01-01

    Rubella virus (RV) is the causative agent of the disease known more popularly as German measles. Rubella is predominantly a childhood disease and is endemic throughout the world. Natural infections of rubella occur only in humans and are generally mild. Complications of rubella infection, most commonly polyarthralgia in adult women, do exist; occasionally more serious sequelae occur. However, the primary public health concern of RV infection is its teratogenicity. RV infection of women during the first trimester of pregnancy can induce a spectrum of congenital defects in the newborn, known as congenital rubella syndrome (CRS). The development of vaccines and implementation of vaccination strategies have substantially reduced the incidence of disease and in turn of CRS in developed countries. The pathway whereby RV infection leads to teratogenesis has not been elucidated, but the cytopathology in infected fetal tissues suggests necrosis and/or apoptosis as well as inhibition of cell division of critical precursor cells involved in organogenesis. In cell culture, a number of unusual features of RV replication have been observed, including mitochondrial abnormalities, and disruption of the cytoskeleton; these manifestations are most probably linked and play some role in RV teratogenesis. Further understanding of the mechanism of RV teratogenesis will be brought about by the investigation of RV replication and virus-host interactions. PMID:11023958

  19. Are endogenous feline leukemia viruses really endogenous?

    PubMed

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

    2011-10-15

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

  20. AKT capture by feline leukemia virus.

    PubMed

    Kawamura, Maki; Umehara, Daigo; Odahara, Yuka; Miyake, Ariko; Ngo, Minh Ha; Ohsato, Yoshiharu; Hisasue, Masaharu; Nakaya, Masa-Aki; Watanabe, Shinya; Nishigaki, Kazuo

    2017-04-01

    Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

  1. Replication-defective viruses as vaccines and vaccine vectors.

    PubMed

    Dudek, Tim; Knipe, David M

    2006-01-05

    The classical viral vaccine approaches using inactivated virus or live-attenuated virus have not been successful for some viruses, such as human immunodeficiency virus or herpes simplex virus. Therefore, new types of vaccines are needed to combat these infections. Replication-defective mutant viruses are defective for one or more functions that are essential for viral genome replication or synthesis and assembly of viral particles. These viruses are propagated in complementing cell lines expressing the missing gene product; however, in normal cells, they express viral gene products but do not replicate to form progeny virions. As vaccines, these mutant viruses have advantages of both classical types of viral vaccines in being as safe as inactivated virus but expressing viral antigens inside infected cells so that MHC class I and class II presentation can occur efficiently. Replication-defective viruses have served both as vaccines for the virus itself and as a vector for the expression of heterologous antigens. The potential advantages and disadvantages of these vaccines are discussed as well as contrasting them with single-cycle mutant virus vaccines and replicon/amplicon versions of vaccines. Replication-defective viruses have also served as important probes of the host immune response in helping to define the importance of the first round of infected cells in the host immune response, the mechanisms of activation of innate immune response, and the role of the complement pathway in humoral immune responses to viruses.

  2. Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture.

    PubMed

    Black, P L; Downs, M B; Lewis, M G; Ussery, M A; Dreyer, G B; Petteway, S R; Lambert, D M

    1993-01-01

    Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polyproteins in cultures of human immunodeficiency virus type 1 (HIV-1)-infected T lymphocytes and, as a result, inhibit the infectivity of HIV-1 for such cultures. The ability of HIV-1 protease inhibitors to suppress replication of the C-type retrovirus Rauscher murine leukemia virus (R-MuLV) and the HIV-related lentivirus simian immunodeficiency virus (SIV) was examined in plaque reduction assays and syncytium reduction assays, respectively. Three of seven compounds examined blocked production of infectious R-MuLV, with 50% inhibitory concentrations of < or = 1 microM. Little or no cellular cytotoxicity was detectable at concentrations up to 100 microM. The same compounds which inhibited the infectivity of HIV-1 also produced activity against SIV and R-MuLV. Electron microscopic examination revealed the presence of many virions with atypical morphologies in cultures treated with the active compounds. Morphometric analysis demonstrated that the active compounds reduced the number of membrane-associated virus particles. These results demonstrate that synthetic peptide analog inhibitors of retroviral proteases significantly inhibit proteolytic processing of the gag polyproteins of R-MuLV and SIV and inhibit the replication of these retroviruses. These results are similar to those for inhibition of HIV-1 infectivity by these compounds, and thus, R-MuLV and SIV might be suitable models for the in vivo evaluation of the antiretroviral activities of these protease inhibitors.

  3. Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture.

    PubMed Central

    Black, P L; Downs, M B; Lewis, M G; Ussery, M A; Dreyer, G B; Petteway, S R; Lambert, D M

    1993-01-01

    Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polyproteins in cultures of human immunodeficiency virus type 1 (HIV-1)-infected T lymphocytes and, as a result, inhibit the infectivity of HIV-1 for such cultures. The ability of HIV-1 protease inhibitors to suppress replication of the C-type retrovirus Rauscher murine leukemia virus (R-MuLV) and the HIV-related lentivirus simian immunodeficiency virus (SIV) was examined in plaque reduction assays and syncytium reduction assays, respectively. Three of seven compounds examined blocked production of infectious R-MuLV, with 50% inhibitory concentrations of < or = 1 microM. Little or no cellular cytotoxicity was detectable at concentrations up to 100 microM. The same compounds which inhibited the infectivity of HIV-1 also produced activity against SIV and R-MuLV. Electron microscopic examination revealed the presence of many virions with atypical morphologies in cultures treated with the active compounds. Morphometric analysis demonstrated that the active compounds reduced the number of membrane-associated virus particles. These results demonstrate that synthetic peptide analog inhibitors of retroviral proteases significantly inhibit proteolytic processing of the gag polyproteins of R-MuLV and SIV and inhibit the replication of these retroviruses. These results are similar to those for inhibition of HIV-1 infectivity by these compounds, and thus, R-MuLV and SIV might be suitable models for the in vivo evaluation of the antiretroviral activities of these protease inhibitors. Images PMID:8381640

  4. ESCRT Requirements for Murine Leukemia Virus Release.

    PubMed

    Bartusch, Christina; Prange, Reinhild

    2016-04-18

    The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements.

  5. ESCRT Requirements for Murine Leukemia Virus Release

    PubMed Central

    Bartusch, Christina; Prange, Reinhild

    2016-01-01

    The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements. PMID:27096867

  6. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates.

    PubMed Central

    Fenyö, E M; Morfeldt-Månson, L; Chiodi, F; Lind, B; von Gegerfelt, A; Albert, J; Olausson, E; Asjö, B

    1988-01-01

    According to their capacity to replicate in vitro, human immunodeficiency virus (HIV) isolates can be divided into two major groups, rapid/high and slow/low. Rapid/high viruses can easily be transmitted to a variety of cell lines of T-lymphoid (CEM, H9, and Jurkat) and monocytoid (U937) origin. In contrast, slow/low viruses replicate transiently, if at all, in these cell lines. Except for a few isolates, the great majority of slow/low viruses replicate in peripheral blood mononuclear cells and Jurkat-tatIII cells constitutively expressing the tatIII gene of HIV-1. The viruses able to replicate efficiently cause syncytium formation and are regularly isolated from immunodeficient patients. Poorly replicating HIV isolates, often obtained from individuals with no or mild disease, show syncytium formation and single-cell killing simultaneously or, with some isolates, cell killing only. Images PMID:2459416

  7. Vaccination against δ-retroviruses: the bovine leukemia virus paradigm.

    PubMed

    Gutiérrez, Gerónimo; Rodríguez, Sabrina M; de Brogniez, Alix; Gillet, Nicolas; Golime, Ramarao; Burny, Arsène; Jaworski, Juan-Pablo; Alvarez, Irene; Vagnoni, Lucas; Trono, Karina; Willems, Luc

    2014-06-20

    Bovine leukemia virus (BLV) and human T-lymphotropic virus type 1 (HTLV-1) are closely related d-retroviruses that induce hematological diseases. HTLV-1 infects about 15 million people worldwide, mainly in subtropical areas. HTLV-1 induces a wide spectrum of diseases (e.g., HTLV-associated myelopathy/tropical spastic paraparesis) and leukemia/lymphoma (adult T-cell leukemia). Bovine leukemia virus is a major pathogen of cattle, causing important economic losses due to a reduction in production, export limitations and lymphoma-associated death. In the absence of satisfactory treatment for these diseases and besides the prevention of transmission, the best option to reduce the prevalence of d-retroviruses is vaccination. Here, we provide an overview of the different vaccination strategies in the BLV model and outline key parameters required for vaccine efficacy.

  8. Discovery of drugs that possess activity against feline leukemia virus.

    PubMed

    Greggs, Willie M; Clouser, Christine L; Patterson, Steven E; Mansky, Louis M

    2012-04-01

    Feline leukemia virus (FeLV) is a gammaretrovirus that is a significant cause of neoplastic-related disorders affecting cats worldwide. Treatment options for FeLV are limited, associated with serious side effects, and can be cost-prohibitive. The development of drugs used to treat a related retrovirus, human immunodeficiency virus type 1 (HIV-1), has been rapid, leading to the approval of five drug classes. Although structural differences affect the susceptibility of gammaretroviruses to anti-HIV drugs, the similarities in mechanism of replication suggest that some anti-HIV-1 drugs may also inhibit FeLV. This study demonstrates the anti-FeLV activity of four drugs approved by the US FDA (Food and Drug Administration) at non-toxic concentrations. Of these, tenofovir and raltegravir are anti-HIV-1 drugs, while decitabine and gemcitabine are approved to treat myelodysplastic syndromes and pancreatic cancer, respectively, but also have anti-HIV-1 activity in cell culture. Our results indicate that these drugs may be useful for FeLV treatment and should be investigated for mechanism of action and suitability for veterinary use.

  9. Amphotropic murine leukemia viruses induce spongiform encephalomyelopathy

    PubMed Central

    Münk, Carsten; Löhler, Jürgen; Prassolov, Vladimir; Just, Ursula; Stockschläder, Marcus; Stocking, Carol

    1997-01-01

    Recombinants of amphotropic murine leukemia virus (A-MuLV) have found widespread use in retroviral vector systems due to their ability to efficiently and stably infect cells of several different species, including human. Previous work has shown that replication-competent recombinants containing the amphotropic env gene, encoding the major SU envelope glycoprotein that determines host tropism, induce lymphomas in vivo. We show here that these viruses also induce a spongiform encephalomyelopathy in mice inoculated perinatally. This fatal central nervous system disease is characterized by noninflammatory spongiform lesions of nerve and glial cells and their processes, and is associated with moderate astro- and microgliosis. The first clinical symptoms are ataxia, tremor, and spasticity, progressing to complete tetraparesis and incontinence, and finally death of the animal. Sequences within the amphotropic env gene are necessary for disease induction. Coinfection of A-MuLV recombinants with nonneuropathogenic ecotropic or polytropic MuLV drastically increases the incidence, degree, and distribution of the neurodegenerative disorder. The consequence of these results in view of the use of A-MuLV recombinants in the clinic is discussed. PMID:9159161

  10. A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing

    PubMed Central

    Déjardin, Jérôme; Bompard-Maréchal, Guillaume; Audit, Muriel; Hope, Thomas J.; Sitbon, Marc; Mougel, Marylène

    2000-01-01

    Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD′) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD′ was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD′ without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells. PMID:10729146

  11. Animals Models of Human T Cell Leukemia Virus Type I Leukemogenesis

    PubMed Central

    Niewiesk, Stefan

    2016-01-01

    Infection with human T cell leukemia virus type I (HTLV-I) causes adult T cell leukemia (ATL) in a minority of infected individuals after long periods of viral persistence. The various stages of HTLV-I infection and leukemia development are studied by using several different animal models: (1) the rabbit (and mouse) model of persistent HTLV-I infection, (2) transgenic mice to model tumorigenesis by HTLV-I specific protein expression, (3) ATL cell transfers into immune-deficient mice, and (4) infection of humanized mice with HTLV-I. After infection, virus replicates without clinical disease in rabbits and to a lesser extent in mice. Transgenic expression of both the transactivator protein (Tax) and the HTLV-I bZIP factor (HBZ) protein have provided insight into factors important in leukemia/lymphoma development. To investigate factors relating to tumor spread and tissue invasion, a number of immune-deficient mice based on the severe combined immunodeficiency (SCID) or non-obese diabetic/SCID background have been used. Inoculation of adult T cell leukemia cell (lines) leads to lymphoma with osteolytic bone lesions and to a lesser degree to leukemia development. These mice have been used extensively for the testing of anticancer drugs and virotherapy. A recent development is the use of so-called humanized mice, which, upon transfer of CD34+ human umbilical cord stem cells, generate human lymphocytes. Infection with HTLV-I leads to leukemia/lymphoma development, thus providing an opportunity to investigate disease development with the aid of molecularly cloned viruses. However, further improvements of this mouse model, particularly in respect to the development of adaptive immune responses, are necessary. PMID:27034390

  12. Animals Models of Human T Cell Leukemia Virus Type I Leukemogenesis.

    PubMed

    Niewiesk, Stefan

    2016-01-01

    Infection with human T cell leukemia virus type I (HTLV-I) causes adult T cell leukemia (ATL) in a minority of infected individuals after long periods of viral persistence. The various stages of HTLV-I infection and leukemia development are studied by using several different animal models: (1) the rabbit (and mouse) model of persistent HTLV-I infection, (2) transgenic mice to model tumorigenesis by HTLV-I specific protein expression, (3) ATL cell transfers into immune-deficient mice, and (4) infection of humanized mice with HTLV-I. After infection, virus replicates without clinical disease in rabbits and to a lesser extent in mice. Transgenic expression of both the transactivator protein (Tax) and the HTLV-I bZIP factor (HBZ) protein have provided insight into factors important in leukemia/lymphoma development. To investigate factors relating to tumor spread and tissue invasion, a number of immune-deficient mice based on the severe combined immunodeficiency (SCID) or non-obese diabetic/SCID background have been used. Inoculation of adult T cell leukemia cell (lines) leads to lymphoma with osteolytic bone lesions and to a lesser degree to leukemia development. These mice have been used extensively for the testing of anticancer drugs and virotherapy. A recent development is the use of so-called humanized mice, which, upon transfer of CD34(+)human umbilical cord stem cells, generate human lymphocytes. Infection with HTLV-I leads to leukemia/lymphoma development, thus providing an opportunity to investigate disease development with the aid of molecularly cloned viruses. However, further improvements of this mouse model, particularly in respect to the development of adaptive immune responses, are necessary.

  13. Feline leukemia virus immunity induced by whole inactivated virus vaccination.

    PubMed

    Torres, Andrea N; O'Halloran, Kevin P; Larson, Laurie J; Schultz, Ronald D; Hoover, Edward A

    2010-03-15

    A fraction of cats exposed to feline leukemia virus (FeLV) effectively contain virus and resist persistent antigenemia/viremia. Using real-time PCR (qPCR) to quantitate circulating viral DNA levels, previously we detected persistent FeLV DNA in blood cells of non-antigenemic cats considered to have resisted FeLV challenge. In addition, previously we used RNA qPCR to quantitate circulating viral RNA levels and determined that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia. A single comparison of all USDA-licensed commercially available FeLV vaccines using these modern sensitive methods has not been reported. To determine whether FeLV vaccination would prevent nucleic acid persistence, we assayed circulating viral DNA, RNA, antigen, infectious virus, and virus neutralizing (VN) antibody in vaccinated and unvaccinated cats challenged with infectious FeLV. We identified challenged vaccinates with undetectable antigenemia and viremia concomitant with persistent FeLV DNA and/or RNA. Moreover, these studies demonstrated that two whole inactivated virus (WIV) adjuvanted FeLV vaccines (Fort Dodge Animal Health's Fel-O-Vax Lv-K) and Schering-Plough Animal Health's FEVAXYN FeLV) provided effective protection against FeLV challenge. In nearly every recipient of these vaccines, neither viral DNA, RNA, antigen, nor infectious virus could be detected in blood after FeLV challenge. Interestingly, this effective viral containment occurred despite a weak to undetectable VN antibody response. The above findings reinforce the precept of FeLV infection as a unique model of effective retroviral immunity elicited by WIV vaccination, and as such holds valuable insights into retroviral immunoprevention and therapy.

  14. (+)RNA viruses rewire cellular pathways to build replication organelles.

    PubMed

    Belov, George A; van Kuppeveld, Frank J M

    2012-12-01

    Positive-strand RNA [(+)RNA] viruses show a significant degree of conservation of their mechanisms of replication. The universal requirement of (+)RNA viruses for cellular membranes for genome replication, and the formation of membranous replication organelles with similar architecture, suggest that they target essential control mechanisms of membrane metabolism conserved among eukaryotes. Recently, significant progress has been made in understanding the role of key host factors and pathways that are hijacked for the development of replication organelles. In addition, electron tomography studies have shed new light on their ultrastructure. Collectively, these studies reveal an unexpected complexity of the spatial organization of the replication membranes and suggest that (+)RNA viruses actively change cellular membrane composition to build their replication organelles. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Insertional Polymorphisms of Endogenous Feline Leukemia Viruses

    PubMed Central

    Roca, Alfred L.; Nash, William G.; Menninger, Joan C.; Murphy, William J.; O'Brien, Stephen J.

    2005-01-01

    The number, chromosomal distribution, and insertional polymorphisms of endogenous feline leukemia viruses (enFeLVs) were determined in four domestic cats (Burmese, Egyptian Mau, Persian, and nonbreed) using fluorescent in situ hybridization and radiation hybrid mapping. Twenty-nine distinct enFeLV loci were detected across 12 of the 18 autosomes. Each cat carried enFeLV at only 9 to 16 of the loci, and many loci were heterozygous for presence of the provirus. Thus, an average of 19 autosomal copies of enFeLV were present per cat diploid genome. Only five of the autosomal enFeLV sites were present in all four cats, and at only one autosomal locus, B4q15, was enFeLV present in both homologues of all four cats. A single enFeLV occurred in the X chromosome of the Burmese cat, while three to five enFeLV proviruses occurred in each Y chromosome. The X chromosome and nine autosomal enFeLV loci were telomeric, suggesting that ectopic recombination between nonhomologous subtelomeres may contribute to enFeLV distribution. Since endogenous FeLVs may affect the infectiousness or pathogenicity of exogenous FeLVs, genomic variation in enFeLVs represents a candidate for genetic influences on FeLV leukemogenesis in cats. PMID:15767400

  16. Apparent feline leukemia virus-induced chronic lymphocytic leukemia and response to treatment.

    PubMed

    Kyle, Kristy N; Wright, Zachary

    2010-04-01

    Chylothorax secondary to chronic lymphocytic leukemia (CLL) was diagnosed in a feline leukemia virus (FeLV)-positive 8-year-old castrated male domestic shorthair feline. The leukemia resolved following therapy with chlorambucil, prednisone, cyclophosphamide, doxorubicin, and lomustine. To our knowledge, this is the first reported case of CLL in an FeLV-positive cat. Although a causative relationship cannot be proven, patients diagnosed with either disease may benefit from diagnostics to rule out the presence of the other concurrent condition. Copyright 2009 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

  17. The Virus-Host Interplay: Biogenesis of +RNA Replication Complexes

    PubMed Central

    Reid, Colleen R.; Airo, Adriana M.; Hobman, Tom C.

    2015-01-01

    Positive-strand RNA (+RNA) viruses are an important group of human and animal pathogens that have significant global health and economic impacts. Notable members include West Nile virus, Dengue virus, Chikungunya, Severe acute respiratory syndrome (SARS) Coronavirus and enteroviruses of the Picornaviridae family.Unfortunately, prophylactic and therapeutic treatments against these pathogens are limited. +RNA viruses have limited coding capacity and thus rely extensively on host factors for successful infection and propagation. A common feature among these viruses is their ability to dramatically modify cellular membranes to serve as platforms for genome replication and assembly of new virions. These viral replication complexes (VRCs) serve two main functions: To increase replication efficiency by concentrating critical factors and to protect the viral genome from host anti-viral systems. This review summarizes current knowledge of critical host factors recruited to or demonstrated to be involved in the biogenesis and stabilization of +RNA virus VRCs. PMID:26287230

  18. Moloney murine leukemia virus activates NF-kappa B.

    PubMed Central

    Pak, J; Faller, D V

    1996-01-01

    Nonacutely transforming retroviruses, such as Moloney murine leukemia virus (M-MuLV), differ from transforming viruses in their mechanisms of tumor induction. While the transforming viruses cause tumors by transduction of oncogenes, the leukemia retroviruses, lacking oncogenes, employ other mechanisms, including promoter insertion and enhancer activation. Although these two mechanisms occur in many tumors induced by leukemia viruses, a substantial proportion of such tumors do not show site-specific proviral insertions. Thus, other, unidentified virus-driven mechanisms may participate in tumorigenesis. In these studies, we show that infection of cells by M-MuLV activates expression of Rel family transcription factors. In murine cells chronically infected with M-MuLV, gel shift analyses with kappaB DNA-binding motifs from the murine immunoglobulin kappa light chain enhancer demonstrated induction of at least two distinct kappaB enhancer-binding complexes. Supershifting and immunoblotting analyses defined p50, p52, RelB, and c-Rel subunits as constituents of these virus-induced protein complexes. Transient transfections performed with kappaB-dependent reporter plasmids showed transcriptional activation in M-MuLV-infected cells relative to uninfected cells. Induction of Rel/NF-kappaB transcription factor activity by M-MuLV infection may prove relevant to the mechanism of M-MuLV-induced leukemia. PMID:8648762

  19. The effect of lithium chloride on the replication of herpes simplex virus.

    PubMed

    Skinner, G R; Hartley, C; Buchan, A; Harper, L; Gallimore, P

    1980-01-01

    Lithium chloride inhibited the replication of type 1 and type 2 Herpes simplex virus at concentrations which permitted host cell replication. Virus polypeptide and antigen synthesis were unaffected while viral DNA synthesis was inhibited. The replication of two other DNA viruses, pseudorabies and vaccinia virus, was inhibited but there was no inhibition of two RNA viruses, namely, EMC and influenze virus.

  20. Replicative intermediates of maize streak virus found during leaf development.

    PubMed

    Erdmann, Julia B; Shepherd, Dionne N; Martin, Darren P; Varsani, Arvind; Rybicki, Edward P; Jeske, Holger

    2010-04-01

    Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombination-dependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR.

  1. Proteome analysis of sheep B lymphocytes in the course of bovine leukemia virus-induced leukemia.

    PubMed

    Reichert, Michal

    2017-07-01

    Presented are the results of a study of the expression pattern of different proteins in the course of bovine leukemia virus-induced leukemia in experimental sheep and I discuss how the obtained data may be useful in gaining a better understanding of the pathogenesis of the disease, diagnosis, and for the selection of possible therapeutic targets. In cattle, the disease is characterized by life-long persistent lymphocytosis leading to leukemia/lymphoma in about 5% of infected animals. In sheep, as opposed to cattle, the course of the disease is always fatal and clinical symptoms usually occur within a three-year period after infection. For this reason, sheep are an excellent experimental model of retrovirus-induced leukemia. This model can be useful for human pathology, as bovine leukemia virus is closely related to human T-lymphotropic virus type 1. The data presented here provide novel insights into the molecular mechanisms of the bovine leukemia virus-induced tumorigenic process and indicate the potential marker proteins both for monitoring progression of the disease and as possible targets of pharmacological intervention. A study of the proteome of B lymphocytes from four leukemic sheep revealed 11 proteins with altered expression. Among them, cytoskeleton and intermediate filament proteins were the most abundant, although proteins belonging to the other functional groups, i.e. enzymes, regulatory proteins, and transcription factors, were also present. It was found that trypsin inhibitor, platelet factor 4, thrombospondin 1, vasodilator-stimulated phosphoprotein, fibrinogen alpha chain, zyxin, filamin-A, and vitamin D-binding protein were downregulated, whereas cleavage and polyadenylation specificity factor subunit 5, non-POU domain-containing octamer-binding protein and small glutamine-rich tetratricopeptide repeat-containing protein alpha were upregulated. Discussed are the possible mechanisms of their altered expression and its significance in the bovine

  2. Glucocorticosteroids enhance replication of respiratory viruses: effect of adjuvant interferon

    PubMed Central

    Thomas, Belinda J.; Porritt, Rebecca A.; Hertzog, Paul J.; Bardin, Philip G.; Tate, Michelle D.

    2014-01-01

    Glucocorticosteroids (GCS) are used on a daily basis to reduce airway inflammation in asthma and chronic obstructive pulmonary disease (COPD). This treatment is usually escalated during acute disease exacerbations, events often associated with virus infections. We examined the impact of GCS on anti-viral defences and virus replication and assessed supplementary interferon (IFN) treatment. Here, we report that treatment of primary human airway cells in vitro with GCS prior to rhinovirus (RV) or influenza A virus (IAV) infection significantly reduces the expression of innate anti-viral genes and increases viral replication. Mice given intranasal treatment with GCS prior to IAV infection developed more severe disease associated with amplified virus replication and elevated inflammation in the airways. Adjuvant IFN treatment markedly reduced GCS-amplified infections in human airway cells and in mouse lung. This study demonstrates that GCS cause an extrinsic compromise in anti-viral defences, enhancing respiratory virus infections and provides a rationale for adjuvant IFN treatment. PMID:25417801

  3. Identification of a New Ribonucleoside Inhibitor of Ebola Virus Replication

    PubMed Central

    Reynard, Olivier; Nguyen, Xuan-Nhi; Alazard-Dany, Nathalie; Barateau, Véronique; Cimarelli, Andrea; Volchkov, Viktor E.

    2015-01-01

    The current outbreak of Ebola virus (EBOV) in West Africa has claimed the lives of more than 15,000 people and highlights an urgent need for therapeutics capable of preventing virus replication. In this study we screened known nucleoside analogues for their ability to interfere with EBOV replication. Among them, the cytidine analogue β-d-N4-hydroxycytidine (NHC) demonstrated potent inhibitory activities against EBOV replication and spread at non-cytotoxic concentrations. Thus, NHC constitutes an interesting candidate for the development of a suitable drug treatment against EBOV. PMID:26633464

  4. Eradication of bovine leukemia virus infection in commercial dairy herds using the agar gel immunodiffusion test.

    PubMed Central

    Shettigara, P T; Samagh, B S; Lobinowich, E M

    1986-01-01

    Demands for bovine leukemia virus test negative breeding cattle and for semen from bovine leukemia virus test negative bulls by several countries have encouraged the eradication of bovine leukemia virus infection from selected herds in Canada. This project was undertaken to evaluate the suitability of the agar gel immunodiffusion test, standardized to detect anti-bovine leukemia virus glycoprotein antibodies, for eradication of bovine leukemia virus from commercial dairy herds. Of nine participating herds, the prevalence rate of bovine leukemia virus infection was low (less than 10%) in three, medium (11-30%) in four and high (greater than 30%) in two. The herds were tested by the agar gel immunodiffusion test, reactors were removed and the herds were then retested at regular intervals. The results indicate that it is possible to eliminate bovine leukemia virus infection from the herds after two to three cycles of agar gel immunodiffusion tests and prompt removal of the reactors. PMID:3019498

  5. Ultrastructural study of Mayaro virus replication in BHK-21 cells.

    PubMed

    Mezencio, J M; de Souza, W; Fonseca, M E; Rebello, M A

    1990-01-01

    The replication of Mayaro virus in BHK-21 cells was studied by electron microscopy. The infected cells show an intense vacuolization and proliferation of membranous structures. At 5 h post-infection, precursor virus particles were seen in the cytoplasm of infected cells. Later, mature virus particles were found outside the cells and budding from the plasma membrane. Enveloped virus particles were also observed inside the vesicles and budding across their membrane. The release of virus particles into the extracellular space by exocytosis was also observed. In a later stage of the infection, inclusion bodies were sometimes present in the cytoplasm of infected cells. We conclude that in BHK-21 cells, budding from the plasma membrane is the main process of Mayaro virus maturation, and in this kind of cell replication differs significantly from that observed in Aedes albopictus cells.

  6. Herpes simplex virus induces the replication of foreign DNA

    SciTech Connect

    Danovich, R.M.; Frenkel, N.

    1988-08-01

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

  7. Replication-Competent Influenza A Viruses Expressing Reporter Genes

    PubMed Central

    Breen, Michael; Nogales, Aitor; Baker, Steven F.; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. PMID:27347991

  8. Replication-Competent Influenza A Viruses Expressing Reporter Genes.

    PubMed

    Breen, Michael; Nogales, Aitor; Baker, Steven F; Martínez-Sobrido, Luis

    2016-06-23

    Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo.

  9. Interferon action on Mayaro virus replication.

    PubMed

    Rebello, M C; Fonseca, M E; Marinho, J O; Rebello, M A

    1993-08-01

    Treatment of TC7 cells with interferon (IFN) drastically reduced the yield of infectious Mayaro virus under experimental conditions that virus attachment and penetration into the cells were not affected. In IFN-treated cells, synthesis of Mayaro virus proteins was inhibited and cellular protein synthesis was restored. This phenomenon is dependent on IFN concentration and multiplicity of infection. Electron microscopy of these cells revealed normal and anomalous viral particles inside cytoplasmic vacuoles. This suggests that IFN also interferes with Mayaro virus morphogenesis and inhibits the release of virions from cells.

  10. T-cell lymphoma induction by radiation leukemia virus in athymic nude mice

    PubMed Central

    1978-01-01

    We report the development of extrathymic lymphoblastic lymphomas in RadLV-inoculated congenitally athymic nude mice. Thus, a leukemogenic virus which appears to require the presence of a thymus for its replication in normothymic mice can infect and transform target cells in the absence of this organ in the athymic host. The cells of one of these lymphomas have been established in vitro as a permanent cell line, BALB/Nu1. This cell line as well as a lymphoma induced in NIH/Swiss nude mice exhibit several T-cell markers, including terminal deoxynucleotidyl transferase activity, Thy-1.2, and Ly-2.2, but not Ly- 1.2 nor TL. Ig determinants were not detected. The characteristics of the tumor cells support the view that cells with T-cell markers may normally exist in nude mice and undergo neoplastic transformation and clonal expansion after infection with a leukemogenic virus. The alternative possibility that virus-induced differentiation of prothymocytes may lead to the expression of Thy-1.2 and Ly-2.2 antigens is also considered. BALB/Nu1 cells release large numbers of type C viral particles. The virus, designated radiation leukemia virus (RadLV)/Nu1, has RTase activity and the protein profile characteristic of murine leukemia virus (MuLV). In radioimmunoassays, it cross-reacts completely with RadLV/VL3, a virus obtained from RadLV-induced C57BL/Ka thymic lymphoma cells in culture, and slightly with a xenotropic virus (BALB:virus-2) and with AKR MuLV. On inoculation into C57BL/Ka mice it has thymotropic and leukemogenic activity. In vitro it is B-tropic, poorly fibrotropic, and has limited xenotropic activity. Thus, RadLV/Nu1 appears to be biologically and serologically similar or identical to its parent virus, RadLV. PMID:214507

  11. Vaccinia Virus Requires Glutamine but Not Glucose for Efficient Replication

    PubMed Central

    Fontaine, Krystal A.; Camarda, Roman

    2014-01-01

    ABSTRACT Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Vaccinia virus (VACV) is a member of the Poxviridae family, and its use as a vaccine enabled the eradication of variola virus, the etiologic agent of smallpox. A global metabolic screen of VACV-infected primary human foreskin fibroblasts suggested that glutamine metabolism is altered during infection. Glutamine and glucose represent the two main carbon sources for mammalian cells. Depriving VACV-infected cells of exogenous glutamine led to a substantial decrease in infectious virus production, whereas starving infected cells of exogenous glucose had no significant impact on replication. Viral yield in glutamine-deprived cells or in cells treated with an inhibitor of glutaminolysis, the pathway of glutamine catabolism, could be rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Thus, VACV infection induces a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle. VACV protein synthesis, but not viral transcription, was decreased in glutamine-deprived cells, which corresponded with a dramatic reduction in all VACV morphogenetic intermediates. This study reveals the unique carbon utilization program implemented during poxvirus infection and provides a potential metabolic pathway to target viral replication. IMPORTANCE Viruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for critical processes including genome replication, viral protein synthesis, and membrane production. This study investigates how vaccinia virus (VACV) infection alters global cellular metabolism, providing the first metabolomic analysis for a member of the poxvirus family. Unlike most viruses examined to date, VACV does not activate glycolysis, and exogenous glucose is not required for maximal virus production. Instead, VACV

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

    PubMed Central

    Pfüller, R; Hammerschmidt, W

    1996-01-01

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

  13. Inhibitors of the Interferon Response Enhance Virus Replication In Vitro

    PubMed Central

    Stewart, Claire E.; Randall, Richard E.; Adamson, Catherine S.

    2014-01-01

    Virus replication efficiency is influenced by two conflicting factors, kinetics of the cellular interferon (IFN) response and induction of an antiviral state versus speed of virus replication and virus-induced inhibition of the IFN response. Disablement of a virus's capacity to circumvent the IFN response enables both basic research and various practical applications. However, such IFN-sensitive viruses can be difficult to grow to high-titer in cells that produce and respond to IFN. The current default option for growing IFN-sensitive viruses is restricted to a limited selection of cell-lines (e.g. Vero cells) that have lost their ability to produce IFN. This study demonstrates that supplementing tissue-culture medium with an IFN inhibitor provides a simple, effective and flexible approach to increase the growth of IFN-sensitive viruses in a cell-line of choice. We report that IFN inhibitors targeting components of the IFN response (TBK1, IKK2, JAK1) significantly increased virus replication. More specifically, the JAK1/2 inhibitor Ruxolitinib enhances the growth of viruses that are sensitive to IFN due to (i) loss of function of the viral IFN antagonist (due to mutation or species-specific constraints) or (ii) mutations/host cell constraints that slow virus spread such that it can be controlled by the IFN response. This was demonstrated for a variety of viruses, including, viruses with disabled IFN antagonists that represent live-attenuated vaccine candidates (Respiratory Syncytial Virus (RSV), Influenza Virus), traditionally attenuated vaccine strains (Measles, Mumps) and a slow-growing wild-type virus (RSV). In conclusion, supplementing tissue culture-medium with an IFN inhibitor to increase the growth of IFN-sensitive viruses in a cell-line of choice represents an approach, which is broadly applicable to research investigating the importance of the IFN response in controlling virus infections and has utility in a number of practical applications including

  14. MicroRNA Regulation of RNA Virus Replication and Pathogenesis.

    PubMed

    Trobaugh, Derek W; Klimstra, William B

    2017-01-01

    microRNAs (miRNAs) are non-coding RNAs that regulate many processes within a cell by manipulating protein levels through direct binding to mRNA and influencing translation efficiency, or mRNA abundance. Recent evidence demonstrates that miRNAs can also affect RNA virus replication and pathogenesis through direct binding to the RNA virus genome or through virus-mediated changes in the host transcriptome. Here, we review the current knowledge on the interaction between RNA viruses and cellular miRNAs. We also discuss how cell and tissue-specific expression of miRNAs can directly affect viral pathogenesis. Understanding the role of cellular miRNAs during viral infection may lead to the identification of novel mechanisms to block RNA virus replication or cell-specific regulation of viral vector targeting. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Molecular biology and replication of hepatitis E virus

    PubMed Central

    Cao, Dianjun; Meng, Xiang-Jin

    2012-01-01

    Hepatitis E virus (HEV), a single-stranded, positive-sense RNA virus, is responsible for acute hepatitis E epidemics in many developing countries, and the virus is also endemic in some industrialized countries. Hepatitis E is a recognized zoonotic disease, and several animal species, including pigs, are potential reservoirs for HEV. The genome of HEV contains three open reading frames (ORFs). ORF1 encodes the nonstructural proteins, ORF2 encodes the capsid protein, and ORF3 encodes a small multifunctional protein. The ORF2 and ORF3 proteins are translated from a single, bicistronic mRNA. The coding sequences for these two ORFs overlap each other, but neither overlaps with ORF1. Whereas the mechanisms underlying HEV replication are poorly understood, the construction of infectious viral clones, the identification of cell lines that support HEV replication, and the development of small animal models have allowed for more detailed study of the virus. As result of these advances, recently, our understanding of viral entry, genomic replication and viral egress has improved. Furthermore, the determination of the T=1 and T=3 structure of HEV virus-like particles has furthered our understanding of the replication of HEV. This article reviews the latest developments in the molecular biology of HEV with an emphasis on the genomic organization, the expression and function of genes, and the structure and replication of HEV. PMID:26038426

  16. Saikosaponin A inhibits influenza A virus replication and lung immunopathology

    PubMed Central

    Zhao, Yaqin; Ling, Fangfang; Xiao, Kun; Li, Qian; Li, Bin; Lu, Chunni; Qi, Wenbao; Zeng, Zhenling; Liao, Ming; Liu, Yahong; Chen, Weisan

    2015-01-01

    Fatal influenza outcomes result from a combination of rapid virus replication and collateral lung tissue damage caused by exaggerated pro-inflammatory host immune cell responses. There are few therapeutic agents that target both biological processes for the attenuation of influenza-induced lung pathology. We show that Saikosaponin A, a bioactive triterpene saponin with previouslyestablished anti-inflammatory effects, demonstrates both in vitro and in vivo anti-viral activity against influenza A virus infections. Saikosaponin A attenuated the replication of three different influenza A virus strains, including a highly pathogenic H5N1 strain, in human alveolar epithelial A549 cells. This anti-viral activity occurred through both downregulation of NF-κB signaling and caspase 3-dependent virus ribonucleoprotein nuclear export as demonstrated by NF-κB subunit p65 and influenza virus nucleoprotein nuclear translocation studies in influenza virus infected A549 cells. Critically, Saikosaponin A also attenuated viral replication, aberrant pro-inflammatory cytokine production and lung histopathology in the widely established H1N1 PR8 model of influenza A virus lethality in C57BL/6 mice. Flow cytometry studies of mouse bronchoalveolar lavage cells revealed that SSa exerted immunomodulatory effects through a selective attenuation of lung neutrophil and monocyte recruitment during the early peak of the innate immune response to PR8 infection. Altogether, our results indicate that Saikosaponin A possesses novel therapeutic potential for the treatment of pathological influenza virus infections. PMID:26637810

  17. Bovine leukemia virus integration site selection in cattle that develop leukemia.

    PubMed

    Murakami, Hironobu; Yamada, Takahito; Suzuki, Miho; Nakahara, Yusuke; Suzuki, Kazuhiko; Sentsui, Hiroshi

    2011-03-01

    It is essential for efficient replication of retroviruses that the viral genome is integrated into the host genome after reverse transcription. Some retroviruses are preferentially integrated into certain genomic regions that may differ depending on the disease. In this study, we analyzed the integration site of bovine leukemia virus (BLV) in leukemic cells and 55 integration sites were determined. Although the integration sites were not located in a particular chromosome, the BLV provirus was integrated into transcription units at a frequency of 43.6% (24/55) and the transcriptional direction of the provirus was in accordance with that of the integrated host genes in 62.5% (15/24). The integration sites were located in introns of the host gene, excluding only one site, which was located in downstream from a stop codon. BLV provirus was never found in a protein coding sequence (CDS) in this study. Moreover, the BLV provirus did not favor integration near transcription start sites and CpG islands, or repetitive sequences such as transposons. Therefore, the possibility that the integration of the BLV provirus disrupts the host gene is very low. Although a hot spot was not found in the BLV provirus integration sites, the provirus favored the integration into regions disadvantageous for viral gene expression since no integration site was preferentially located into/near CDS, transcription start site or CpG island. It is suggested that the integration site of the BLV provirus in leukemic cells is related to the suppression of viral gene expression. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Vaccinia virus as a subhelper for AAV replication and packaging.

    PubMed

    Moore, Andrea R; Dong, Biao; Chen, Lingxia; Xiao, Weidong

    2015-01-01

    Adeno-associated virus (AAV) has been widely used as a gene therapy vector to treat a variety of disorders. While these vectors are increasingly popular and successful in the clinic, there is still much to learn about the viruses. Understanding the biology of these viruses is essential in engineering better vectors and generating vectors more efficiently for large-scale use. AAV requires a helper for production and replication making this aspect of the viral life cycle crucial. Vaccinia virus (VV) has been widely cited as a helper virus for AAV. However, to date, there are no detailed analyses of its helper function. Here, the helper role of VV was studied in detail. In contrast to common belief, we demonstrated that VV was not a sufficient helper virus for AAV replication. Vaccinia failed to produce rAAV and activate AAV promoters. While this virus could not support rAAV production, Vaccinia could initiate AAV replication and packaging when AAV promoter activation is not necessary. This activity is due to the ability of Vaccinia-driven Rep78 to transcribe in the cytoplasm and subsequently translate in the nucleus and undergo typical functions in the AAV life cycle. As such, VV is subhelper for AAV compared to complete helper functions of adenovirus.

  19. NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

    SciTech Connect

    Furukawa, Ayako; Okamura, Hideyasu; Morishita, Ryo; Matsunaga, Satoko; Kobayashi, Naohiro; Ikegami, Takahisa; Kodaki, Tsutomu; Takaori-Kondo, Akifumi; Ryo, Akihide; Nagata, Takashi; Katahira, Masato

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Protease (PR) of XMR virus (XMRV) was successfully synthesized with cell-free system. Black-Right-Pointing-Pointer Interface of XMRV PR with an inhibitor, amprenavir (APV), was identified with NMR. Black-Right-Pointing-Pointer Structural heterogeneity is induced for two PR protomers in the APV:PR = 1:2 complex. Black-Right-Pointing-Pointer Structural heterogeneity is transmitted even to distant regions from the interface. Black-Right-Pointing-Pointer Long-range transmission of structural change may be utilized for drug discovery. -- Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially {sup 15}N-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [{sup 1}H, {sup 15}N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) . A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR = 1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has

  20. Inhibitors of nucleotidyltransferase superfamily enzymes suppress herpes simplex virus replication.

    PubMed

    Tavis, John E; Wang, Hong; Tollefson, Ann E; Ying, Baoling; Korom, Maria; Cheng, Xiaohong; Cao, Feng; Davis, Katie L; Wold, William S M; Morrison, Lynda A

    2014-12-01

    Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 μM, with suppression at 50 μM reaching ∼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 μM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family.

  1. Involvement of FKBP6 in hepatitis C virus replication

    PubMed Central

    Kasai, Hirotake; Kawakami, Kunihiro; Yokoe, Hiromasa; Yoshimura, Kentaro; Matsuda, Masanori; Yasumoto, Jun; Maekawa, Shinya; Yamashita, Atsuya; Tanaka, Tomohisa; Ikeda, Masanori; Kato, Nobuyuki; Okamoto, Toru; Matsuura, Yoshiharu; Sakamoto, Naoya; Enomoto, Nobuyuki; Takeda, Sen; Fujii, Hideki; Tsubuki, Masayoshi; Kusunoki, Masami; Moriishi, Kohji

    2015-01-01

    The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val121 of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N′, N′-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A. PMID:26567527

  2. Gene profiling of Graffi murine leukemia virus-induced lymphoid leukemias: identification of leukemia markers and Fmn2 as a potential oncogene.

    PubMed

    Charfi, Cyndia; Voisin, Véronique; Levros, Louis-Charles; Edouard, Elsy; Rassart, Eric

    2011-02-10

    The Graffi murine leukemia virus induces a large spectrum of leukemias in mice and thus provides a good model to compare the transcriptome of all types of leukemias. We analyzed the gene expression profiles of both T and B leukemias induced by the virus with DNA microarrays. Given that we considered that a 4-fold change in expression level was significant, 388 probe sets were associated to B, to T, or common to both leukemias. Several of them were not yet associated with lymphoid leukemia. We confirmed specific deregulation of Fmn2, Arntl2, Bfsp2, Gfra2, Gpm6a, and Gpm6b in B leukemia, of Nln, Fbln1, and Bmp7 in T leukemias, and of Etv5 in both leukemias. More importantly, we show that the mouse Fmn2 induced an anchorage-independent growth, a drastic modification in cell shape with a concomitant disruption of the actin cytoskeleton. Interestingly, we found that human FMN2 is overexpressed in approximately 95% of pre-B acute lymphoblastic leukemia with the highest expression levels in patients with a TEL/AML1 rearrangement. These results, surely related to the role of FMN2 in meiotic spindle maintenance, suggest its important role in leukemogenesis. Finally, we propose a new panel of genes potentially involved in T and/or B leukemias.

  3. No involvement of bovine leukemia virus in childhood acute lymphoblastic leukemia and non-Hodgkin's lymphoma

    SciTech Connect

    Bender, A.P.; Robison, L.L.; Kashmiri, S.V.; McClain, K.L.; Woods, W.G.; Smithson, W.A.; Heyn, R.; Finlay, J.; Schuman, L.M.; Renier, C.

    1988-05-15

    Bovine leukemia virus (BLV) is the causative agent of enzootic bovine lymphosarcoma. Much speculation continues to be directed at the role of BLV in human leukemia. To test this hypothesis rigorously, a case-control study of childhood acute lymphoblastic leukemia and non-Hodgkin's lymphoma was conducted between December 1983 and February 1986. Cases (less than or equal to 16 years at diagnosis) derived from patients diagnosed at the primary institutions and affiliated hospitals were matched (age, sex, and race) with regional population controls. DNA samples from bone marrow or peripheral blood from 157 cases (131 acute lymphoblastic leukemia, 26 non-Hodgkin's lymphoma) and peripheral blood from 136 controls were analyzed by Southern blot technique, under highly stringent conditions, using cloned BLV DNA as a probe. None of the 157 case or 136 control DNA samples hybridized with the probe. The high statistical power and specificity of this study provide the best evidence to date that genomic integration of BLV is not a factor in childhood acute lymphoblastic leukemia/non-Hodgkin's lymphoma.

  4. Novel Polyanions Inhibiting Replication of Influenza Viruses

    PubMed Central

    Ciejka, Justyna; Milewska, Aleksandra; Wytrwal, Magdalena; Wojarski, Jacek; Golda, Anna; Ochman, Marek; Nowakowska, Maria

    2016-01-01

    Novel sulfonated derivatives of poly(allylamine hydrochloride) (NSPAHs) and N-sulfonated chitosan (NSCH) have been synthesized, and their activity against influenza A and B viruses has been studied and compared with that of a series of carrageenans, marine polysaccharides of well-documented anti-influenza activity. NSPAHs were found to be nontoxic and very soluble in water, in contrast to gel-forming and thus generally poorly soluble carrageenans. In vitro and ex vivo studies using susceptible cells (Madin-Darby canine kidney epithelial cells and fully differentiated human airway epithelial cultures) demonstrated the antiviral effectiveness of NSPAHs. The activity of NSPAHs was proportional to the molecular mass of the chain and the degree of substitution of amino groups with sulfonate groups. Mechanistic studies showed that the NSPAHs and carrageenans inhibit influenza A and B virus assembly in the cell. PMID:26729490

  5. Bovine Leukemia Virus DNA in Human Breast Tissue

    PubMed Central

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

    2014-01-01

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

  6. Bovine leukemia virus DNA in human breast tissue.

    PubMed

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

    2014-05-01

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

  7. Inhibition of activity of the protease from bovine leukemia virus.

    PubMed

    Ménard, A; Leonard, R; Llido, S; Geoffre, S; Picard, P; Berteau, F; Precigoux, G; Hospital, M; Guillemain, B

    1994-06-13

    In view of the close similarity between bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I) we investigated the possibility of developing specific inhibitors of the proteases of these retroviruses using the purified enzyme from BLV. We tested the ability of this protease to specifically cleave various short oligopeptide substrates containing cleavage sites of BLV and HTLV-I proteases, as well as a recombinant BLV Gag precursor. The best substrate, a synthetic decapeptide bearing the natural cleavage site between the matrix and the capsid proteins of BLV Gag precursor polyprotein, was used to develop an inhibition assay. We determined the relative inhibitory effect of synthetic Gag precursor-like peptides in which the cleavable site was replaced by a non-hydrolyzable moiety. The encouraging inhibitory effect of these compounds indicates that potent non-peptidic inhibitors for retroviral proteases are not unattainable.

  8. Lytic Replication of Epstein-Barr Virus During Space Flight

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Pierson, D. L.; Barrett, A. D. T.

    1999-01-01

    Reactivation of latent Epstein-Barr virus (EBV) may be an important threat to crew health during extended space missions. Cellular immunity, which is decreased during and after space flight, is responsible for controlling EBV replication in vivo. In this study, we investigated the effects of short-term space flight on latent EBV reactivation.

  9. VIRUS: a massively replicated IFU spectrograph for HET

    NASA Astrophysics Data System (ADS)

    Hill, Gary J.; MacQueen, Phillip J.; Tejada, Carlos; Cobos, Francisco J.; Palunas, Povilas; Gebhardt, Karl; Drory, Niv

    2004-09-01

    We investigate the role of industrial replication in the construction of the next generation of spectrographs for large telescopes. In this paradigm, a simple base spectrograph unit is replicated to provide multiplex advantage, while the engineering costs are amortized over many copies. We argue that this is a cost-effective approach when compared to traditional spectrograph design, where each instrument is essentially a one-off prototype with heavy expenditure on engineering effort. As an example of massive replication, we present the design of, and the science drivers for, the Visible IFU Replicable Ultra-cheap Spectrograph (VIRUS). This instrument is made up of 132 individually small and simple spectrographs, each fed by a fiber integral field unit. The total VIRUS-132 instrument covers ~29 sq. arcminutes per observation, providing integral field spectroscopy from 340 to 570 nm, simultaneously, of 32,604 spatial elements, each 1 sq. arcsecond on the sky. VIRUS-132 will be mounted on the 9.2 m Hobby-Eberly Telescope and fed by a new wide-field corrector with a science field in excess of 16.5 arcminutes diameter. VIRUS represents a new approach to spectrograph design, offering the science multiplex advantage of huge sky coverage for an integral field spectrograph, coupled with the engineering multiplex advantage of >102 spectrographs making up a whole.

  10. Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides.

    PubMed Central

    Goodchild, J; Agrawal, S; Civeira, M P; Sarin, P S; Sun, D; Zamecnik, P C

    1988-01-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs. PMID:3041414

  11. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    NASA Astrophysics Data System (ADS)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

    1988-08-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  12. Leukemia

    MedlinePlus

    ... a combination of genetic and environmental factors. How leukemia forms In general, leukemia is thought to occur ... causing the signs and symptoms of leukemia. How leukemia is classified Doctors classify leukemia based on its ...

  13. Leukemia

    MedlinePlus

    ... version of this page please turn Javascript on. Leukemia What Is Leukemia? Leukemia is a cancer of the blood cells. ... diagnosed with leukemia are over 50 years old. Leukemia Starts in Bone Marrow Click for more information ...

  14. Replication-competent fluorescent-expressing influenza B virus

    PubMed Central

    Nogales, Aitor; Rodríguez-Sánchez, Irene; Monte, Kristen; Lenschow, Deborah J.; Perez, Daniel R.; Martínez-Sobrido, Luis

    2016-01-01

    Influenza B viruses (IBVs) cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated morbidity and mortality. Studying influenza viruses requires the use of secondary methodologies to identify virus-infected cells. To this end, replication-competent influenza A viruses (IAVs) expressing easily traceable fluorescent proteins have been recently developed. In contrast, similar approaches for IBV are mostly lacking. In this report, we describe the generation and characterization of replication-competent influenza B/Brisbane/60/2008 viruses expressing fluorescent mCherry or GFP fused to the C-terminal of the viral non-structural 1 (NS1) protein. Fluorescent-expressing IBVs display similar growth kinetics and plaque phenotype to wild-type IBV, while fluorescent protein expression allows for the easy identification of virus-infected cells. Without the need of secondary approaches to monitor viral infection, fluorescent-expressing IBVs represent an ideal approach to study the biology of IBV and an excellent platform for the rapid identification and characterization of antiviral therapeutics or neutralizing antibodies using high-throughput screening approaches. Lastly, fluorescent-expressing IBVs can be combined with the recently described reporter-expressing IAVs for the identification of novel therapeutics to combat these two important human respiratory pathogens. PMID:26590325

  15. Delayed-onset enzootic bovine leukosis possibly caused by superinfection with bovine leukemia virus mutated in the pol gene.

    PubMed

    Watanabe, Tadaaki; Inoue, Emi; Mori, Hiroshi; Osawa, Yoshiaki; Okazaki, Katsunori

    2015-08-01

    Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), to which animals are most susceptible at 4-8 years of age. In this study, we examined tumor cells associated with EBL in an 18-year-old cow to reveal that the cells carried at least two different copies of the virus, one of which was predicted to encode a reverse transcriptase (RT) lacking ribonuclease H activity and no integrase. Such a deficient enzyme may exhibit a dominant negative effect on the wild-type RT and cause insufficient viral replication, resulting in delayed tumor development in this cow.

  16. Dengue virus replicates and accumulates in Aedes aegypti salivary glands.

    PubMed

    Raquin, Vincent; Lambrechts, Louis

    2017-07-01

    Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Replication of Oral BK Virus in Human Salivary Gland Cells

    PubMed Central

    Burger-Calderon, Raquel; Madden, Victoria; Hallett, Ryan A.; Gingerich, Aaron D.; Nickeleit, Volker

    2014-01-01

    BK polyomavirus (BKPyV) is the most common viral pathogen among allograft patients. Increasing evidence links BKPyV to the human oral compartment and to HIV-associated salivary gland disease (HIVSGD). To date, few studies have analyzed orally derived BKPyV. This study aimed to characterize BKPyV isolated from throat wash (TW) samples from HIVSGD patients. The replication potential of HIVSGD-derived clinical isolates HIVSGD-1 and HIVSGD-2, both containing the noncoding control region (NCCR) architecture OPQPQQS, were assessed and compared to urine-derived virus. The BKPyV isolates displayed significant variation in replication potential. Whole-genome alignment of the two isolates revealed three nucleotide differences that were analyzed for a potential effect on the viral life cycle. Analysis revealed a negligible difference in NCCR promoter activity despite sequence variation and emphasized the importance of functional T antigen (Tag) for efficient replication. HIVSGD-1 encoded full-length Tag, underwent productive infection in both human salivary gland cells and kidney cells, and expressed viral DNA and Tag protein. Additionally, HIVSGD-1 generated DNase-resistant particles and by far surpassed the replication potential of the kidney-derived isolate in HSG cells. HIVSGD-2 encoded a truncated form of Tag and replicated much less efficiently. Quantitation of infectious virus, via the fluorescent forming unit assay, suggested that HIVSGD BKPyV had preferential tropism for salivary gland cells over kidney cells. Similarly, the results suggested that kidney-derived virus had preferential tropism for kidney cells over salivary gland cells. Evidence of HIVSGD-derived BKPyV oral tropism and adept viral replication in human salivary gland cells corroborated the potential link between HIVSGD pathogenesis and BKPyV. PMID:24173219

  18. Activities of proteasome and m-calpain are essential for Chikungunya virus replication.

    PubMed

    Karpe, Yogesh A; Pingale, Kunal D; Kanade, Gayatri D

    2016-10-01

    Replication of many viruses is dependent on the ubiquitin proteasome system. The present study demonstrates that Chikungunya virus replication increases proteasome activity and induces unfolded protein response (UPR) in cultured cells. Further, it was seen that the virus replication was dependent on the activities of proteasomes and m-calpain. Proteasome inhibition induced accumulation of polyubiquitinated proteins and earlier visualization of UPR.

  19. Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses.

    PubMed

    Gushchin, Vladimir A; Solovyev, Andrey G; Erokhina, Tatyana N; Morozov, Sergey Y; Agranovsky, Alexey A

    2013-01-01

    In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of "virus factories" in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).

  20. Comparative analysis of radiation- and virus-induced leukemias in BALB/c mice

    SciTech Connect

    Newcomb, E.W.; Binari, R.; Fleissner, E.

    1985-01-15

    Endogenous murine leukemia virus (MuLV) proviral copies were analyzed in thymomas induced in normal BALB/c (Fv-1b) and in Fv-1n congenic mice by X-irradiation. Both strains of mice developed leukemia with similar kinetics, indicating that N-tropism of endogenous MuLV was not a rate-limiting factor in development of disease. Southern blot analysis, using a probe specific for ecotropic virus and for ecotropic-specific sequences retained in pathogenic, env-recombinant viruses, showed that the majority of radiation leukemias lacked newly acquired, clonally integrated, proviruses. This was in contrast to virus-induced leukemias, which routinely exhibited several new proviral integration sites. When an internal proviral DNA restriction fragment was monitored, some radiation leukemias showed evidence of nonclonal infection, accounting for more frequent isolation of infectious virus from such leukemias. Differences in expression of T-cell surface antigens were found in X-ray-induced and virus-induced leukemias. All radiation leukemias were TL positive, whereas virus-induced leukemias were primarily negative for TL. Some differences were also found in Lyt-1 and Lyt-2 expression. The data as a whole suggest that, in the majority of cases, radiation leukemogenesis is not initiated by a viral route--that is, the sort of viral mechanism for which exogenous infection by known pathogenic MuLV is the paradigm.

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

    PubMed Central

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

    2013-01-01

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

  2. DNA intercalator stimulates influenza transcription and virus replication.

    PubMed

    Li, Olive T W; Poon, Leo L M

    2011-03-15

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

  3. Extract of Scutellaria baicalensis inhibits dengue virus replication

    PubMed Central

    2013-01-01

    Background Scutellaria baicalensis (S. baicalensis) is one of the traditional Chinese medicinal herbs that have been shown to possess many health benefits. In the present study, we evaluated the in vitro antiviral activity of aqueous extract of the roots of S. baicalensis against all the four dengue virus (DENV) serotypes. Methods Aqueous extract of S. baicalensis was prepared by microwave energy steam evaporation method (MEGHE™), and the anti-dengue virus replication activity was evaluated using the foci forming unit reduction assay (FFURA) in Vero cells. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to determine the actual dengue virus RNA copy number. The presence of baicalein, a flavonoid known to inhibit dengue virus replication was determined by mass spectrometry. Results The IC50 values for the S. baicalensis extract on Vero cells following DENV adsorption ranged from 86.59 to 95.19 μg/mL for the different DENV serotypes. The IC50 values decreased to 56.02 to 77.41 μg/mL when cells were treated with the extract at the time of virus adsorption for the different DENV serotypes. The extract showed potent direct virucidal activity against extracellular infectious virus particles with IC50 that ranged from 74.33 to 95.83 μg/mL for all DENV serotypes. Weak prophylactic effects with IC50 values that ranged from 269.9 to 369.8 μg/mL were noticed when the cells were pre-treated 2 hours prior to virus inoculation. The concentration of baicalein in the S. baicalensis extract was ~1% (1.03 μg/gm dried extract). Conclusions Our study demonstrates the in vitro anti-dengue virus replication property of S. baicalensis against all the four DENV serotypes investigated. The extract reduced DENV infectivity and replication in Vero cells. The extract was rich in baicalein, and could be considered for potential development of anti-DENV therapeutics. PMID:23627436

  4. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication

    PubMed Central

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  5. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

    PubMed Central

    Levy, D N; Refaeli, Y; Weiner, D B

    1995-01-01

    The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics. PMID:7815499

  6. Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts.

    PubMed

    Sakuma, Toshie; Davila, Jaime I; Malcolm, Jessica A; Kocher, Jean-Pierre A; Tonne, Jason M; Ikeda, Yasuhiro

    2014-04-01

    Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is involved in the

  7. Porcine Epidemic Diarrhea Virus Induces Autophagy to Benefit Its Replication

    PubMed Central

    Guo, Xiaozhen; Zhang, Mengjia; Zhang, Xiaoqian; Tan, Xin; Guo, Hengke; Zeng, Wei; Yan, Guokai; Memon, Atta Muhammad; Li, Zhonghua; Zhu, Yinxing; Zhang, Bingzhou; Ku, Xugang; Wu, Meizhou; Fan, Shengxian; He, Qigai

    2017-01-01

    The new porcine epidemic diarrhea (PED) has caused devastating economic losses to the swine industry worldwide. Despite extensive research on the relationship between autophagy and virus infection, the concrete role of autophagy in porcine epidemic diarrhea virus (PEDV) infection has not been reported. In this study, autophagy was demonstrated to be triggered by the effective replication of PEDV through transmission electron microscopy, confocal microscopy, and Western blot analysis. Moreover, autophagy was confirmed to benefit PEDV replication by using autophagy regulators and RNA interference. Furthermore, autophagy might be associated with the expression of inflammatory cytokines and have a positive feedback loop with the NF-κB signaling pathway during PEDV infection. This work is the first attempt to explore the complex interplay between autophagy and PEDV infection. Our findings might accelerate our understanding of the pathogenesis of PEDV infection and provide new insights into the development of effective therapeutic strategies. PMID:28335505

  8. Porcine Epidemic Diarrhea Virus Induces Autophagy to Benefit Its Replication.

    PubMed

    Guo, Xiaozhen; Zhang, Mengjia; Zhang, Xiaoqian; Tan, Xin; Guo, Hengke; Zeng, Wei; Yan, Guokai; Memon, Atta Muhammad; Li, Zhonghua; Zhu, Yinxing; Zhang, Bingzhou; Ku, Xugang; Wu, Meizhou; Fan, Shengxian; He, Qigai

    2017-03-19

    The new porcine epidemic diarrhea (PED) has caused devastating economic losses to the swine industry worldwide. Despite extensive research on the relationship between autophagy and virus infection, the concrete role of autophagy in porcine epidemic diarrhea virus (PEDV) infection has not been reported. In this study, autophagy was demonstrated to be triggered by the effective replication of PEDV through transmission electron microscopy, confocal microscopy, and Western blot analysis. Moreover, autophagy was confirmed to benefit PEDV replication by using autophagy regulators and RNA interference. Furthermore, autophagy might be associated with the expression of inflammatory cytokines and have a positive feedback loop with the NF-κB signaling pathway during PEDV infection. This work is the first attempt to explore the complex interplay between autophagy and PEDV infection. Our findings might accelerate our understanding of the pathogenesis of PEDV infection and provide new insights into the development of effective therapeutic strategies.

  9. Membranous Replication Factories Induced by Plus-Strand RNA Viruses

    PubMed Central

    Romero-Brey, Inés; Bartenschlager, Ralf

    2014-01-01

    In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. PMID:25054883

  10. Low Oxygen Tension Enhances Hepatitis C Virus Replication

    PubMed Central

    Kalliampakou, K. I.; Kotta-Loizou, I.; Befani, C.; Liakos, P.; Simos, G.; Mentis, A. F.; Kalliaropoulos, A.; Doumba, P. P.; Smirlis, D.; Foka, P.; Bauhofer, O.; Poenisch, M.; Windisch, M. P.; Lee, M. E.; Koskinas, J.; Bartenschlager, R.

    2013-01-01

    Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction. PMID:23269812

  11. Xenotropic murine leukemia virus-related virus establishes an efficient spreading infection and exhibits enhanced transcriptional activity in prostate carcinoma cells.

    PubMed

    Rodriguez, Jason J; Goff, Stephen P

    2010-03-01

    Xenotropic murine leukemia virus-related virus (XMRV) is a novel human gammaretrovirus discovered in association with human prostate tumors. XMRV was first identified in prostate stromal cells surrounding the tumors of patients carrying a mutation in the HPC1 gene locus. To determine the tropism of XMRV in cell culture, we tested the ability of XMRV to spread and replicate in various prostate and nonprostate cell lines. We found that although the expression of XMRV viral proteins and the spread of infectious virus were minimal in a variety of cell lines, XMRV displayed robust expression and infection in LNCaP prostate tumor cells. The transcriptional activity of the XMRV long terminal repeat (LTR) was found to be higher than the Moloney murine leukemia virus LTRs in both LNCaP and WPMY-1 (simian virus 40-transformed prostate stromal cells). The U3 promoter of XMRV and a glucocorticoid response element (GRE) within the U3 were required for the transcriptional activity in LNCaP cells. Coexpression of the androgen receptor and stimulation with dihydrotestosterone stimulated XMRV-LTR-dependent transcription in 293T cells, and the GRE was required for this activity. These data suggest that XMRV may replicate more efficiently in LNCaP cells in part due to the transcriptional environment in LNCaP cells.

  12. Dengue virus induces and requires glycolysis for optimal replication.

    PubMed

    Fontaine, Krystal A; Sanchez, Erica L; Camarda, Roman; Lagunoff, Michael

    2015-02-01

    Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future. Approximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of

  13. Dengue Virus Induces and Requires Glycolysis for Optimal Replication

    PubMed Central

    Fontaine, Krystal A.; Sanchez, Erica L.; Camarda, Roman

    2014-01-01

    ABSTRACT Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future. IMPORTANCE Approximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block

  14. Acute Myeloid Leukemia Targeting by Myxoma Virus In Vivo Depends on Cell Binding But Not Permissiveness to Infection In Vitro

    PubMed Central

    Madlambayan, Gerard J.; Bartee, Eric; Kim, Manbok; Rahman, Masmudur M.; Meacham, Amy; Scott, Edward W.; McFadden, Grant; Cogle, Christopher R.

    2012-01-01

    Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo. PMID:22341701

  15. Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro.

    PubMed

    Madlambayan, Gerard J; Bartee, Eric; Kim, Manbok; Rahman, Masmudur M; Meacham, Amy; Scott, Edward W; McFadden, Grant; Cogle, Christopher R

    2012-05-01

    Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.

  16. Protein Phosphatase-1 Regulates Rift Valley Fever Virus Replication

    PubMed Central

    Baer, Alan; Shafagati, Nazly; Benedict, Ashwini; Ammosova, Tatiana; Ivanov, Andrey; Hakami, Ramin M.; Terasaki, Kaori; Makino, Shinji; Nekhai, Sergei; Kehn-Hall, Kylene

    2016-01-01

    Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target. PMID:26801627

  17. Protein Phosphatase-1 regulates Rift Valley fever virus replication.

    PubMed

    Baer, Alan; Shafagati, Nazly; Benedict, Ashwini; Ammosova, Tatiana; Ivanov, Andrey; Hakami, Ramin M; Terasaki, Kaori; Makino, Shinji; Nekhai, Sergei; Kehn-Hall, Kylene

    2016-03-01

    Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target.

  18. Silver nanoparticles impair Peste des petits ruminants virus replication.

    PubMed

    Khandelwal, Nitin; Kaur, Gurpreet; Chaubey, Kundan Kumar; Singh, Pushpendra; Sharma, Shalini; Tiwari, Archana; Singh, Shoor Vir; Kumar, Naveen

    2014-09-22

    In the present study, we evaluated the antiviral efficacy of the silver nanoparticles (SNPs) against Peste des petits ruminants virus (PPRV), a prototype Morbillivirus. The leaf extract of the Argemone maxicana was used as a reducing agent for biological synthesis of the SNPs from silver nitrate. The SNPs were characterized using UV-vis absorption spectroscopy, X-ray diffraction (XRD) and transmission electron microscopy (TEM). The TEM analysis revealed particle size of 5-30 nm and the XRD analysis revealed their characteristic silver structure. The treatment of Vero cells with the SNPs at a noncytotoxic concentration significantly inhibited PPRV replication in vitro. The time-course and virus step-specific assays showed that the SNPs impair PPRV replication at the level of virus entry. The TEM analysis showed that the SNPs interact with the virion surface as well with the virion core. However, this interaction has no direct virucidal effect, instead exerts a blocking effect on viral entry into the target cells. This is the first documented evidence indicating that the SNPs are capable of inhibiting a Morbillivirus replication in vitro. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Monkey Viperin Restricts Porcine Reproductive and Respiratory Syndrome Virus Replication.

    PubMed

    Fang, Jianyu; Wang, Haiyan; Bai, Juan; Zhang, Qiaoya; Li, Yufeng; Liu, Fei; Jiang, Ping

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen which causes huge economic damage globally in the swine industry. Current vaccination strategies provide only limited protection against PRRSV infection. Viperin is an interferon (IFN) stimulated protein that inhibits some virus infections via IFN-dependent or IFN-independent pathways. However, the role of viperin in PRRSV infection is not well understood. In this study, we cloned the full-length monkey viperin (mViperin) complementary DNA (cDNA) from IFN-α-treated African green monkey Marc-145 cells. It was found that the mViperin is up-regulated following PRRSV infection in Marc-145 cells along with elevated IRF-1 gene levels. IFN-α induced mViperin expression in a dose- and time-dependent manner and strongly inhibits PRRSV replication in Marc-145 cells. Overexpression of mViperin suppresses PRRSV replication by blocking the early steps of PRRSV entry and genome replication and translation but not inhibiting assembly and release. And mViperin co-localized with PRRSV GP5 and N protein, but only interacted with N protein in distinct cytoplasmic loci. Furthermore, it was found that the 13-16 amino acids of mViperin were essential for inhibiting PRRSV replication, by disrupting the distribution of mViperin protein from the granular distribution to a homogeneous distribution in the cytoplasm. These results could be helpful in the future development of novel antiviral therapies against PRRSV infection.

  20. Human cytomegalovirus function inhibits replication of herpes simplex virus

    SciTech Connect

    Cockley, K.D.; Shiraki, K.; Rapp, F.

    1988-01-01

    Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 ..mu..g/ml) for 3 or 24 h was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5/sup 0/C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with (/sup 3/H)thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection.

  1. Investigation of the HIV-1 matrix interactome during virus replication.

    PubMed

    Li, Yan; Frederick, Kristin M; Haverland, Nicole A; Ciborowski, Pawel; Belshan, Michael

    2016-02-01

    Like all viruses, human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. A Strep-tag was inserted into the C-terminus of the matrix (MA) region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. MA complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS, data were log2 -transformed, and Student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. X-ray repair cross-complementing protein 6 (Ku70), X-ray repair cross-complementing protein 5 (Ku80), and Y-box binding protein 1 (YB-1) were confirmed to interact with MA by immunoblot. Knockdown of two candidates, EZRIN and Y-box binding protein 1, enhanced HIV infection in vitro. The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV host -cell interactions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Seroprevalence of Toxoplasma gondii and concurrent Bartonella spp., feline immunodeficiency virus, feline leukemia virus, and Dirofilaria immitis infections in Egyptian cats

    USDA-ARS?s Scientific Manuscript database

    Toxoplasma gondii and Bartonella spp. are zoonotic pathogens of cats. Feline Immunodeficiency Virus (FIV), and Feline Leukemia Virus (FeLv) are related to Human Immunodeficiency Virus, and Human Leukemia Virus, respectively, and these viruses are immunosuppressive. In the present study, the prevalen...

  3. Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia.

    PubMed

    Betancourt, Dillon; Ramos, Juan Carlos; Barber, Glen N

    2015-12-01

    Adult T cell leukemia/lymphoma (ATL) is an aggressive cancer of CD4/CD25(+) T lymphocytes, the etiological agent of which is human T-cell lymphotropic virus type 1 (HTLV-1). ATL is highly refractory to current therapies, making the development of new treatments a high priority. Oncolytic viruses such as vesicular stomatitis virus (VSV) are being considered as anticancer agents since they readily infect transformed cells compared to normal cells, the former appearing to exhibit defective innate immune responses. Here, we have evaluated the efficacy and safety of a recombinant VSV that has been retargeted to specifically infect and replicate in transformed CD4(+) cells. This was achieved by replacing the single VSV glycoprotein (G) with human immunodeficiency virus type 1 (HIV-1) gp160 to create a hybrid fusion protein, gp160G. The resultant virus, VSV-gp160G, was found to only target cells expressing CD4 and retained robust oncolytic activity against HTLV-1 actuated ATL cells. VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4(+) T cells. Accordingly, VSV-gp160G did not elicit any evidence of neurotoxicity even in severely immunocompromised animals such as NOD/Shi-scid, IL-2Rγ-c-null (NSG) mice. Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit. Our data indicate that VSV-gp160G exerts potent oncolytic efficacy against CD4(+) malignant cells and either alone or in conjunction with established therapies may provide an effective treatment in patients displaying ATL. Adult T cell leukemia (ATL) is a serious form of cancer with a high mortality rate. HTLV-1 infection is the etiological agent of ATL and, unfortunately, most patients succumb to the disease within a few years. Current treatment options have failed to significantly improve survival rate. In this study, we

  4. Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia

    PubMed Central

    Betancourt, Dillon; Ramos, Juan Carlos

    2015-01-01

    ABSTRACT Adult T cell leukemia/lymphoma (ATL) is an aggressive cancer of CD4/CD25+ T lymphocytes, the etiological agent of which is human T-cell lymphotropic virus type 1 (HTLV-1). ATL is highly refractory to current therapies, making the development of new treatments a high priority. Oncolytic viruses such as vesicular stomatitis virus (VSV) are being considered as anticancer agents since they readily infect transformed cells compared to normal cells, the former appearing to exhibit defective innate immune responses. Here, we have evaluated the efficacy and safety of a recombinant VSV that has been retargeted to specifically infect and replicate in transformed CD4+ cells. This was achieved by replacing the single VSV glycoprotein (G) with human immunodeficiency virus type 1 (HIV-1) gp160 to create a hybrid fusion protein, gp160G. The resultant virus, VSV-gp160G, was found to only target cells expressing CD4 and retained robust oncolytic activity against HTLV-1 actuated ATL cells. VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4+ T cells. Accordingly, VSV-gp160G did not elicit any evidence of neurotoxicity even in severely immunocompromised animals such as NOD/Shi-scid, IL-2Rγ-c-null (NSG) mice. Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit. Our data indicate that VSV-gp160G exerts potent oncolytic efficacy against CD4+ malignant cells and either alone or in conjunction with established therapies may provide an effective treatment in patients displaying ATL. IMPORTANCE Adult T cell leukemia (ATL) is a serious form of cancer with a high mortality rate. HTLV-1 infection is the etiological agent of ATL and, unfortunately, most patients succumb to the disease within a few years. Current treatment options have failed to significantly improve survival rate. In

  5. Generation of influenza A viruses as live but replication-incompetent virus vaccines.

    PubMed

    Si, Longlong; Xu, Huan; Zhou, Xueying; Zhang, Ziwei; Tian, Zhenyu; Wang, Yan; Wu, Yiming; Zhang, Bo; Niu, Zhenlan; Zhang, Chuanling; Fu, Ge; Xiao, Sulong; Xia, Qing; Zhang, Lihe; Zhou, Demin

    2016-12-02

    The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)-harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell-mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus.

  6. Tumor viruses and replicative immortality--avoiding the telomere hurdle.

    PubMed

    Chen, Xinsong; Kamranvar, Siamak Akbari; Masucci, Maria G

    2014-06-01

    Tumor viruses promote cell proliferation in order to gain access to an environment suitable for persistence and replication. The expression of viral products that promote growth transformation is often accompanied by the induction of multiple signs of telomere dysfunction, including telomere shortening, damage of telomeric DNA and chromosome instability. Long-term survival and progression to full malignancy require the bypassing of senescence programs that are triggered by the damaged telomeres. Here we review different strategies by which tumor viruses interfere with telomere homeostasis during cell transformation. This frequently involves the activation of telomerase, which assures both the integrity and functionality of telomeres. In addition, recent evidence suggests that oncogenic viruses may activate a recombination-based mechanism for telomere elongation known as Alternative Lengthening of Telomeres (ALT). This error-prone strategy promotes genomic instability and could play an important role in viral oncogenesis.

  7. Rectal transmission of bovine leukemia virus in cattle and sheep.

    PubMed

    Henry, E T; Levine, J F; Coggins, L

    1987-04-01

    Bovine leukemia virus (BLV) was transmitted by rectal inoculation of BLV-infective whole blood into cattle and sheep. Two cows and 2 sheep each were given 500 ml and 50 ml of blood, respectively, by rectal infusion. Two sheep which served as positive controls each were given 1 ml of the same blood, IV. All animals became seropositive to BLV by postinoculation week 5. Although relatively large volumes of blood were used for rectal inoculation, a base line for infectivity was established for the rectal route.

  8. Receptor tyrosine kinase signaling regulates replication of the peste des petits ruminants virus.

    PubMed

    Chaudhary, K; Chaubey, K K; Singh, S V; Kumar, N

    2015-03-01

    In this study, we found out that blocking the receptor tyrosine kinase (RTK) signaling in Vero cells by tryphostin AG879 impairs the in vitro replication of the peste des petits ruminants virus (PPRV). A reduced virus replication in Trk1-knockdown (siRNA) Vero cells confirmed the essential role of RTK in the virus replication, in particular a specific regulation of viral RNA synthesis. These data represent the first evidence that the RTK signaling regulates replication of a morbillivirus.

  9. Promotion of Hendra Virus Replication by MicroRNA 146a

    PubMed Central

    Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.; Foord, Adam J.; Bean, Andrew G.; Wang, Lin-Fa

    2013-01-01

    Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease. PMID:23345523

  10. Ferret airway epithelial cell cultures support efficient replication of influenza B virus but not mumps virus.

    PubMed

    Elderfield, Ruth A; Parker, Lauren; Stilwell, Peter; Roberts, Kim L; Schepelmann, Silke; Barclay, Wendy S

    2015-08-01

    Ferrets have become the model animal of choice for influenza pathology and transmission experiments as they are permissive and susceptible to human influenza A viruses. However, inoculation of ferrets with mumps virus (MuV) did not lead to successful infections. We evaluated the use of highly differentiated ferret tracheal epithelium cell cultures, FTE, for predicting the potential of ferrets to support respiratory viral infections. FTE cultures supported productive replication of human influenza A and B viruses but not of MuV, whereas analogous cells generated from human airways supported replication of all three viruses. We propose that in vitro strategies using these cultures might serve as a method of triaging viruses and potentially reducing the use of ferrets in viral studies.

  11. The tax gene of human T-cell leukemia virus type 2 is essential for transformation of human T lymphocytes.

    PubMed

    Ross, T M; Pettiford, S M; Green, P L

    1996-08-01

    The mechanism of human T-cell leukemia virus (HTLV)-mediated transformation and induction of malignancy is unknown; however, several studies have implicated the viral gene product, Tax. Conclusive evidence for the role of Tax in the HTLV malignant process has been impeded by the inability to mutate tax in the context of an infectious virus and dissociate viral replication from cellular transformation. To circumvent this problem we constructed a mutant of HTLV type 2 (HTLV-2) that replicates by a Tax-independent mechanism. For these studies, the Tax response element in the viral long terminal repeat was replaced with the cytomegalovirus immediate-early promoter enhancer (C-enh). Transcription of the chimeric HTLV-2 (HTLVC-enh) was efficiently directed by this heterologous promoter. Also, the chimeric virus transformed primary human T lymphocytes with an efficiency similar to that of wild-type HTLV-2. A tax-knockout virus, termed HTLVC-enhDeltaTax, was constructed to directly assess the importance of Tax in cellular transformation. Transfection and infection studies indicated that HTLVC-enhDeltaTax was replication competent; however, HTLVC-enhDeltaTax failed to transform primary human T lymphocytes. We conclude that Tax is essential for HTLV-mediated transformation of human T lymphocytes. Furthermore, this chimeric HTLV, that replicates in the absence of Tax, should facilitate studies to determine the precise mechanism of T-lymphocyte transformation by HTLV.

  12. Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

    PubMed Central

    Bawa, Bhupinder; Wang, Wei; Shabman, Reed S.; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B.; Richt, Juergen A.; Wentworth, David E.; Ma, Wenjun

    2014-01-01

    Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses. PMID:25275541

  13. Characterization of uncultivable bat influenza virus using a replicative synthetic virus.

    PubMed

    Zhou, Bin; Ma, Jingjiao; Liu, Qinfang; Bawa, Bhupinder; Wang, Wei; Shabman, Reed S; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B; Richt, Juergen A; Wentworth, David E; Ma, Wenjun

    2014-10-01

    Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

  14. Genotyping of feline leukemia virus in Mexican housecats.

    PubMed

    Ramírez, Hugo; Autran, Marcela; García, M Martha; Carmona, M Ángel; Rodríguez, Cecilia; Martínez, H Alejandro

    2016-04-01

    Feline leukemia virus (FeLV) is a retrovirus with variable rates of infection globally. DNA was obtained from cats' peripheral blood mononuclear cells, and proviral DNA of pol and env genes was detected using PCR. Seventy-six percent of cats scored positive for FeLV using env-PCR; and 54 %, by pol-PCR. Phylogenetic analysis of both regions identified sequences that correspond to a group that includes endogenous retroviruses. They form an independent branch and, therefore, a new group of endogenous viruses. Cat gender, age, outdoor access, and cohabitation with other cats were found to be significant risk factors associated with the disease. This strongly suggests that these FeLV genotypes are widely distributed in the studied feline population in Mexico.

  15. Measles virus induces persistent infection by autoregulation of viral replication

    PubMed Central

    Doi, Tomomitsu; Kwon, Hyun-Jeong; Honda, Tomoyuki; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

    2016-01-01

    Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity. PMID:27883010

  16. Correlation between Virus Replication and Antibody Responses in Macaques following Infection with Pandemic Influenza A Virus

    PubMed Central

    Koopman, Gerrit; Dekking, Liesbeth; Mortier, Daniëlla; Nieuwenhuis, Ivonne G.; van Heteren, Melanie; Kuipers, Harmjan; Remarque, Edmond J.; Radošević, Katarina; Bogers, Willy M. J. M.

    2015-01-01

    ABSTRACT Influenza virus infection of nonhuman primates is a well-established animal model for studying pathogenesis and for evaluating prophylactic and therapeutic intervention strategies. However, usually a standard dose is used for the infection, and there is no information on the relation between challenge dose and virus replication or the induction of immune responses. Such information is also very scarce for humans and largely confined to evaluation of attenuated virus strains. Here, we have compared the effect of a commonly used dose (4 × 106 50% tissue culture infective doses) versus a 100-fold-higher dose, administered by intrabronchial installation, to two groups of 6 cynomolgus macaques. Animals infected with the high virus dose showed more fever and had higher peak levels of gamma interferon in the blood. However, virus replication in the trachea was not significantly different between the groups, although in 2 out of 6 animals from the high-dose group it was present at higher levels and for a longer duration. The virus-specific antibody response was not significantly different between the groups. However, antibody enzyme-linked immunosorbent assay, virus neutralization, and hemagglutination inhibition antibody titers correlated with cumulative virus production in the trachea. In conclusion, using influenza virus infection in cynomolgus macaques as a model, we demonstrated a relationship between the level of virus production upon infection and induction of functional antibody responses against the virus. IMPORTANCE There is only very limited information on the effect of virus inoculation dose on the level of virus production and the induction of adaptive immune responses in humans or nonhuman primates. We found only a marginal and variable effect of virus dose on virus production in the trachea but a significant effect on body temperature. The induction of functional antibody responses, including virus neutralization titer, hemagglutination inhibition

  17. A Replication-incompetent Rift Valley Fever Vaccine: Chimeric Virus-like Particles Protect Mice and Rats Against Lethal Challenge

    PubMed Central

    Mandell, Robert B.; Koukuntla, Ramesh; Mogler, Laura J. K.; Carzoli, Andrea K.; Freiberg, Alexander N.; Holbrook, Michael R.; Martin, Brian K.; Staplin, William R.; Vahanian, Nicholas N.; Link, Charles J.; Flick, Ramon

    2009-01-01

    Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins GN and GC, nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate. PMID:19932911

  18. A Multicenter Blinded Analysis Indicates No Association between Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and either Xenotropic Murine Leukemia Virus-Related Virus or Polytropic Murine Leukemia Virus

    PubMed Central

    Alter, Harvey J.; Mikovits, Judy A.; Switzer, William M.; Ruscetti, Francis W.; Lo, Shyh-Ching; Klimas, Nancy; Komaroff, Anthony L.; Montoya, Jose G.; Bateman, Lucinda; Levine, Susan; Peterson, Daniel; Levin, Bruce; Hanson, Maureen R.; Genfi, Afia; Bhat, Meera; Zheng, HaoQiang; Wang, Richard; Li, Bingjie; Hung, Guo-Chiuan; Lee, Li Ling; Sameroff, Stephen; Heneine, Walid; Coffin, John; Hornig, Mady; Lipkin, W. Ian

    2012-01-01

    ABSTRACT The disabling disorder known as chronic fatigue syndrome or myalgic encephalomyelitis (CFS/ME) has been linked in two independent studies to infection with xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (pMLV). Although the associations were not confirmed in subsequent studies by other investigators, patients continue to question the consensus of the scientific community in rejecting the validity of the association. Here we report blinded analysis of peripheral blood from a rigorously characterized, geographically diverse population of 147 patients with CFS/ME and 146 healthy subjects by the investigators describing the original association. This analysis reveals no evidence of either XMRV or pMLV infection. PMID:22991430

  19. Detection and molecular characterization of bovine leukemia viruses from Jordan.

    PubMed

    Ababneh, Mustafa M; Al-Rukibat, Raida K; Hananeh, Wael M; Nasar, Abdelrahman T; Al-Zghoul, Mohammad B

    2012-12-01

    Bovine leukemia virus (BLV) is distributed worldwide. BLV has many effects on the health status and productivity of infected animals and is a potential risk for humans. In this study, we aimed to investigate the presence of and genotype bovine leukemia viruses on Jordanian dairy farms. Nested PCR coupled with RFLP and direct sequencing of a partial fragment of the env gene were carried out. Two BLV genotypes were found, genotypes 1 and 6. These genotypes were identified by nested PCR-RFLP of 444 bp of the env gene by restriction digestion with HaeIII, Bcl I and Pvu II. However, BLV-Jordan-10 seems to represent an entirely new genotype in our phylogenetic analysis. The nucleotide sequence identity between these two Jordanian BLV genotypes (1 and 6) was 96.2 %. The nucleotide sequence identity between Jordanian BLV genotype 1 and other reference BLV genotype 1 strains ranged from 99 % to 99.5 %. The nucleotide sequence similarity of the Jordanian BLV genotype 6 to other BLV genotypes ranged from 90 % to 96.7 %. A neutralizing motif and CD8(+) T-cell epitope were found in the env protein of both Jordanian isolates. In this study, we documented the presence of two BLV genotypes (1 and 6) on Jordanian dairy farms.

  20. Bovine leukemia virus: purification and characterization of the aspartic protease.

    PubMed

    Menard, A; Mamoun, R Z; Geoffre, S; Castroviejo, M; Raymond, S; Precigoux, G; Hospital, M; Guillemain, B

    1993-04-01

    To develop efficient bovine leukemia virus (BLV) protease (PR) inhibitors, pure enzyme is required. For this, we have developed a two-step chromatographic nondenaturing purification protocol of PR from virions. As expected, the purified protein presents a molecular weight (14 kDa) and a NH2 terminal end fitting with previously reported data. The enzymatic activity of BLV PR was characterized using a synthetic peptide containing a potential cleavage site of the BLV gag-pro polypeptide precursor as substrate. The protease was most active at pH 6, 40 degrees, and high salt concentration (1-2 M NaCl or ammonium sulfate). In contrast, using a natural substrate such as a human T-cell leukemia virus recombinant gag precursor, BLV PR activity was higher at a low salt concentration (0.5 M NaCl). Besides, the use of different potentially cleavable molecules revealed that PR activity may be influenced by the substrate conformational structure around the cleavage site. Replacement of the two amino acids of a synthetic substrate cleavable site by a statin residue completely inhibited the enzymatic activity of the BLV PR.

  1. Effect of temperature on replication of epizootic hemorrhagic disease viruses in Culicoides sonorensis (Diptera: Ceratopogonidae)

    USDA-ARS?s Scientific Manuscript database

    Replication of many arboviruses, including some orbiviruses, within the vector has been shown to be temperature-dependent. In general, cooler ambient temperatures slow virus replication in arthropod vectors, whereas viruses replicate faster and to higher titers at warmer ambient temperatures. Prev...

  2. Friend leukemia virus integration-1 (FLI-1) expression in gastrointestinal stromal tumors.

    PubMed

    Kaygusuz, Gülşah; Kuzu, Işinsu

    2009-06-01

    Friend leukemia virus integration-1 expression has been shown in a variety of tumors, including vascular tumors, desmoplastic small round cell tumor, Merkel cell carcinoma, and lymphoblastic lymphoma, in addition to Ewing's sarcoma/primitive neuroectodermal tumor. The aim of the current study was to examine Friend leukemia virus integration-1 protein expression in a series of gastrointestinal stromal tumors and also to assess if Friend leukemia virus integration-1 has any role in the disease process. It is the first study analyzing Friend leukemia virus integration-1 expression in gastrointestinal stromal tumors in the English literature. A tissue microarray block containing 52 cases of gastrointestinal stromal tumors was done. Immunohistochemical analysis was performed for Friend leukemia virus integration-1 polyclonal antibody. Immunohistochemically, Friend leukemia virus integration-1 was negative in all cases. Friend leukemia virus integration-1 can be expressed in a variety of tumors, and is helpful in making the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor. We think that this protein is not expressed in gastrointestinal stromal tumors and it is not a part of the pathogenesis of this disease.

  3. Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication

    PubMed Central

    Kawamura-Nagaya, Kazue; Ishibashi, Kazuhiro; Huang, Ying-Ping; Miyashita, Shuhei; Ishikawa, Masayuki

    2014-01-01

    Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided. PMID:24711385

  4. Mutation of a Single Envelope N-Linked Glycosylation Site Enhances the Pathogenicity of Bovine Leukemia Virus

    PubMed Central

    Bouzar, Amel Baya; Jacques, Jean-Rock; Cosse, Jean-Philippe; Gillet, Nicolas; Callebaut, Isabelle; Reichert, Michal

    2015-01-01

    ABSTRACT Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing. PMID:26085161

  5. Curcumin inhibits Rift Valley fever virus replication in human cells.

    PubMed

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-09-28

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.

  6. Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells*

    PubMed Central

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-01-01

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets. PMID:22847000

  7. Effect of monensin on Mayaro virus replication in monkey kidney and Aedes albopictus cells.

    PubMed

    De Campos, R M; Ferreira, D F; Da Veiga, V F; Rebello, M A; Rebello, M C S

    2003-01-01

    The effect of a cationic ionophore, monensin, on the replication of Mayaro virus in monkey kidney TC7 and Aedes albopictus cells has been studied. Treatment of these cells with 1 micromol/l monensin during infection did not affect the virus protein synthesis but inhibited severely the virus replication. Electron microscopy of the cells infected with Mayaro virus and treated with monensin revealed that the morphogenesis of Mayaro virus was impaired in TC7 but not in A. albopictus cells.

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

    PubMed

    Chetverin, A B

    2011-01-01

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

  9. Trigocherrierin A, a potent inhibitor of chikungunya virus replication.

    PubMed

    Bourjot, Mélanie; Leyssen, Pieter; Neyts, Johan; Dumontet, Vincent; Litaudon, Marc

    2014-03-24

    Trigocherrierin A (1) and trigocherriolide E (2), two new daphnane diterpenoid orthoesters (DDOs), and six chlorinated analogues, trigocherrins A, B, F and trigocherriolides A-C, were isolated from the leaves of Trigonostemon cherrieri. Their structures were identified by mass spectrometry, extensive one- and two-dimensional NMR spectroscopy and through comparison with data reported in the literature. These compounds are potent and selective inhibitors of chikungunya virus (CHIKV) replication. Among the DDOs isolated, compound 1 exhibited the strongest anti-CHIKV activity (EC₅₀ = 0.6 ± 0.1 µM, SI = 71.7).

  10. Clinical aspects of feline immunodeficiency and feline leukemia virus infection.

    PubMed

    Hartmann, Katrin

    2011-10-15

    Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are retroviruses with a global impact on the health of domestic cats. The two viruses differ in their potential to cause disease. FIV can cause an acquired immunodeficiency syndrome that increases the risk of developing opportunistic infections, neurological diseases, and tumors. In most naturally infected cats, however, FIV itself does not cause severe clinical signs, and FIV-infected cats may live many years without any health problems. FeLV is more pathogenic, and was long considered to be responsible for more clinical syndromes than any other agent in cats. FeLV can cause tumors (mainly lymphoma), bone marrow suppression syndromes (mainly anemia) and lead to secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. Today, FeLV is less important as a deadly infectious agent as in the last 20 years prevalence has been decreasing in most countries. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

    SciTech Connect

    Eyre, Nicholas S.; Hampton-Smith, Rachel J.; Aloia, Amanda L.; Eddes, James S.; Simpson, Kaylene J.; Hoffmann, Peter; Beard, Michael R.

    2016-04-15

    Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

  12. Cytoskeletal Dynamics: Concepts in Measles Virus Replication and Immunomodulation

    PubMed Central

    Avota, Elita; Gassert, Evelyn; Schneider-Schaulies, Sibylle

    2011-01-01

    In common with most viruses, measles virus (MV) relies on the integrity of the cytoskeleton of its host cells both with regard to efficient replication in these cells, but also retention of their motility which favors viral dissemination. It is, however, the surface interaction of the viral glycoprotein (gp) complex with receptors present on lymphocytes and dendritic cells (DCs), that signals effective initiation of host cell cytoskeletal dynamics. For DCs, these may act to regulate processes as diverse as viral uptake and sorting, but also the ability of these cells to successfully establish and maintain functional immune synapses (IS) with T cells. In T cells, MV signaling causes actin cytoskeletal paralysis associated with a loss of polarization, adhesion and motility, which has been linked to activation of sphingomyelinases and subsequent accumulation of membrane ceramides. MV modulation of both DC and T cell cytoskeletal dynamics may be important for the understanding of MV immunosuppression at the cellular level. PMID:22049305

  13. Cyclophilin function in Cancer; lessons from virus replication.

    PubMed

    Lavin, Paul T M; Mc Gee, Margaret M

    2015-01-01

    Cyclophilins belong to a group of proteins that possess peptidyl prolyl isomerase activity and catalyse the cis-trans conversion of proline peptide bonds. Cyclophilin members play important roles in protein folding and as molecular chaperones, in addition to a well-established role as host factors required for completion of the virus life cycle. Members of the cyclophilin family are overexpressed in a range of human malignancies including hepatocellular cancer, pancreatic cancer, nonsmall cell lung cancer, gastric cancer, colorectal cancer and glioblastoma multiforme, however, their precise role in tumourigenesis remains unclear. In recent years, mounting evidence supports a role for prolyl isomerisation during mammalian cell division; a process with striking similarity to plasma membrane remodelling during virus replication. Here, we summarise our current understanding of the role of cyclophilins in cancer. We review the function of cyclophilins during mammalian cell division and during HIV-1 infection, and highlight common processes involving members of the ESCRT and Rab GTPase families.

  14. The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication

    PubMed Central

    Sacramento, Carolina Q.; de Melo, Gabrielle R.; de Freitas, Caroline S.; Rocha, Natasha; Hoelz, Lucas Villas Bôas; Miranda, Milene; Fintelman-Rodrigues, Natalia; Marttorelli, Andressa; Ferreira, André C.; Barbosa-Lima, Giselle; Abrantes, Juliana L.; Vieira, Yasmine Rangel; Bastos, Mônica M.; de Mello Volotão, Eduardo; Nunes, Estevão Portela; Tschoeke, Diogo A.; Leomil, Luciana; Loiola, Erick Correia; Trindade, Pablo; Rehen, Stevens K.; Bozza, Fernando A.; Bozza, Patrícia T.; Boechat, Nubia; Thompson, Fabiano L.; de Filippis, Ana M. B.; Brüning, Karin; Souza, Thiago Moreno L.

    2017-01-01

    Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV. PMID:28098253

  15. The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication.

    PubMed

    Sacramento, Carolina Q; de Melo, Gabrielle R; de Freitas, Caroline S; Rocha, Natasha; Hoelz, Lucas Villas Bôas; Miranda, Milene; Fintelman-Rodrigues, Natalia; Marttorelli, Andressa; Ferreira, André C; Barbosa-Lima, Giselle; Abrantes, Juliana L; Vieira, Yasmine Rangel; Bastos, Mônica M; de Mello Volotão, Eduardo; Nunes, Estevão Portela; Tschoeke, Diogo A; Leomil, Luciana; Loiola, Erick Correia; Trindade, Pablo; Rehen, Stevens K; Bozza, Fernando A; Bozza, Patrícia T; Boechat, Nubia; Thompson, Fabiano L; de Filippis, Ana M B; Brüning, Karin; Souza, Thiago Moreno L

    2017-01-18

    Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

  16. Differential diagnosis of feline leukemia virus subgroups using pseudotype viruses expressing green fluorescent protein.

    PubMed

    Nakamura, Megumi; Sato, Eiji; Miura, Tomoyuki; Baba, Kenji; Shimoda, Tetsuya; Miyazawa, Takayuki

    2010-06-01

    Feline leukemia virus (FeLV) is classified into three receptor interference subgroups, A, B and C. In this study, to differentiate FeLV subgroups, we developed a simple assay system using pseudotype viruses expressing green fluorescent protein (GFP). We prepared gfp pseudotype viruses, named gfp(FeLV-A), gfp(FeLV-B) and gfp(FeLV-C) harboring envelopes of FeLV-A, B and C, respectively. The gfp pseudotype viruses completely interfered with the same subgroups of FeLV reference strains on FEA cells (a feline embryonic fibroblast cell line). We also confirmed that the pseudotype viruses could differentiate FeLV subgroups in field isolates. The assay will be useful for differential diagnosis of FeLV subgroups in veterinary diagnostic laboratories in the future.

  17. Viral determinants that control the neuropathogenicity of PVC-211 murine leukemia virus in vivo determine brain capillary endothelial cell tropism of the virus in vitro.

    PubMed Central

    Masuda, M; Hoffman, P M; Ruscetti, S K

    1993-01-01

    PVC-211 murine leukemia virus (MuLV) is a neuropathogenic, weakly leukemogenic variant of the nonneuropathogenic, highly leukemogenic Friend MuLV (F-MuLV). Chimeric viruses constructed from PVC-211 MuLV clone 3d and F-MuLV clone 57 indicate that the env gene of PVC-211 MuLV contains the determinant(s) responsible for pathological changes in the central nervous system. However, sequences within the 5' one-third (AatII-EcoRI region) of the PVC-211 MuLV genome, which include the 5' leader sequence, the gag gene, and the 5' quarter of the pol gene, are also needed in conjunction with the env gene determinant(s) to cause clinically evident neurological disease in the majority of virus-infected animals after a short latency. In the presence of the AatII-EcoRI region of the PVC-211 MuLV genome, the PVC-211 MuLV env gene sequences encoding the amino-terminal half of the SU protein, which contains the receptor-binding region of the protein, were sufficient to cause rapidly progressive neurological disease. When PVC-211 MuLV, F-MuLV, and various chimeric viruses were tested for their ability to replicate in cultured brain capillary endothelial cells (BCEC), the primary site of PVC-211 MuLV replication within the central nervous system, there was a direct correlation between the replication efficiency of a virus in BCEC in vitro and its ability to cause neurological disease in vivo. This observation indicates that the sequences in PVC-211 MuLV that render it neuropathogenic affect its replication in BCEC and suggests that rapid and efficient replication of the virus in BCEC is crucial for the pathological changes in the central nervous system that result in development of neurological disease. Images PMID:8392599

  18. Expression of Bovine Leukemia Virus Genome is Blocked by a Nonimmunoglobulin Protein in Plasma from Infected Cattle

    NASA Astrophysics Data System (ADS)

    Gupta, P.; Ferrer, J. F.

    1982-01-01

    Plasma of cattle infected with bovine leukemia virus contains a soluble factor that blocks the expression of the viral genome in cultured lymphocytes. The blocking factor is not present in plasma of bovine leukemia virus-free cattle or of cattle infected with common bovine viruses. Blocking of bovine leukemia virus expression by the plasma factor is reversible, and seems to be mediated by a nonimmunoglobulin protein molecule.

  19. HTLV-1 (Human T-Cell Leukemia Virus 1) Seroconversion Study

    DTIC Science & Technology

    1989-01-17

    block number) FIELD GROUP SUB_-GROUP RA 1: IITLV- I .croccnversion;--tpidemiology; Leukemia ; 06 03 Okinawa; texually Transmitted Disease". 06 13 19... Leukemia Virus 1 (HTLV-I) transmission is occurring in active duty forces stationed on Okinawa, Japan. HTLV-I, the first human retrovirus isolated, has been...identified as the etiologic agent for Adult T-Cell Leukemia /Lymphoma (ATLL). In addition, HTLV-I has been linked etioloyically to a subset of patients

  20. Biochemical analysis of murine leukemia viruses isolated from radiation-induced leukemias of strain BALB/c

    SciTech Connect

    Ellis, R.W.; Hopkins, N.; Fleissner, E.

    1980-02-01

    Murine leukemia viruses isolated from radiation-induced BALB/c leukemias were characterized with respect to viral proteins and RNA. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral structural proteins revealed that for p12, p15, p30, and gp70, three to four electrophoretic variants of each could be detected. There was no correlation found between any of these mobilities and N- or B-tropism of the viruses. Proteins of all xenotropic viral isolates were identical in their gel electrophoretic profiles. The similar phenotypes of multiple viral clones from individual leukemias and of isolates grown in different cells suggest that the polymorphism of ecotropic viruses was generated in vivo rather than during in vitro virus growth. By two-dimensional fingerprinting of RNase T1-resistant oligonucleotides from 70S viral RNA, the previously reported association of N- and B-tropism with two distinct oligonucleotides was confirmed. The presence of two other oligonucleotides was correlated with positive and negative phenotypes of the virus-coded G/sub IX/ cell surface antigen. The RNAs of two B-tropic isolates with distinctive p15 and p12 phenotypes differed from the RNA of a prototype N-tropic virus by the absence of three oligonucleotides mapping in the 5' portion (gag region) of the prototype RNA. In addition, one small-plaque B-tropic virus displayed extensive changes in the RNA sequences associated with the env region of the prototype.

  1. Methamphetamine enhances Hepatitis C virus replication in human hepatocytes.

    PubMed

    Ye, L; Peng, J S; Wang, X; Wang, Y J; Luo, G X; Ho, W Z

    2008-04-01

    Very little is known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV infection. This study examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Further investigation of mechanism(s) responsible for the methamphetamine action revealed that methamphetamine was able to inhibit the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses. Methamphetamine also down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicate that methamphetamine may have a cofactor role in the immunopathogenesis of HCV disease.

  2. Methamphetamine enhances Hepatitis C virus replication in human hepatocytes

    PubMed Central

    Ye, L.; Peng, J. S.; Wang, X.; Wang, Y. J.; Luo, G. X.; Ho, W. Z.

    2009-01-01

    SUMMARY Very little is known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV infection. This study examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Further investigation of mechanism(s) responsible for the methamphetamine action revealed that methamphetamine was able to inhibit the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses. Methamphetamine also down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicate that methamphetamine may have a cofactor role in the immunopathogenesis of HCV disease. PMID:18307590

  3. Genetic resistance in Japanese wild mice (Mus musculus molossinus) to an NB-tropic Friend murine leukemia virus.

    PubMed

    Odaka, T; Ikeda, H; Moriwaki, K; Matsuzawa, A; Mizuno, M; Kondo, K

    1978-11-01

    Wild mice (Sk, Hz-Vl, Hz-IV Om, Mol.A, Fu, Te, and Sn) trapped in various areas of Japan were crossed with mice of inbred strains (C57BL/6, C57L, BALB/c, and C57BL/10), and their progeny were infected with NB-tropic Friend nurine leukemia virus. Ten days after infection, the spleens were weighed, examined for macroscopic focal lesions, and assayed for infectious virus by the XC test. Genetic analysis indicated that 4 of 8 mice tested had a dominant gene that suppresses the virus replication; the gene resembles the Fv-4' allele. No mice with the Fv-2' allele were found.

  4. TRAF2 Facilitates Vaccinia Virus Replication by Promoting Rapid Virus Entry

    PubMed Central

    Haga, Ismar R.; Pechenick Jowers, Tali; Griffiths, Samantha J.; Haas, Juergen

    2014-01-01

    ABSTRACT Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is a pivotal intracellular mediator of signaling pathways downstream of TNFR1 and -2 with known pro- and antiviral effects. We investigated its role in the replication of the prototype poxvirus vaccinia virus (VACV). Loss of TRAF2 expression, either through small interfering RNA treatment of HeLa cells or through genetic knockout in murine embryonic fibroblasts (MEFs), led to significant reductions in VACV growth following low-multiplicity infection. In single-cycle infections, there was delayed production of both early and late VACV proteins as well as accelerated virus-induced alterations to cell morphology, indicating that TRAF2 influences early stages of virus replication. Consistent with an early role, uncoating assays showed normal virus attachment but delayed virus entry in the absence of TRAF2. Although alterations to c-Jun N-terminal kinase (JNK) signaling were apparent in VACV-infected TRAF2−/− MEFs, treatment of wild-type cells with a JNK inhibitor did not affect virus entry. Instead, treatment with an inhibitor of endosomal acidification greatly reduced virus entry into TRAF2−/− MEFs, suggesting that VACV is reliant on the endosomal route of entry in the absence of TRAF2. Thus, TRAF2 is a proviral factor for VACV that plays a role in promoting efficient viral entry, most likely via the plasma membrane. IMPORTANCE Tumor necrosis factor receptor-associated factors (TRAFs) are key facilitators of intracellular signaling with roles in innate and adaptive immunity and stress responses. We have discovered that TRAF2 is a proviral factor in vaccinia virus replication in both HeLa cells and mouse embryonic fibroblasts and that its influence is exercised through promotion of efficient virus entry. PMID:24429366

  5. Low-pH Stability of Influenza A Virus Sialidase Contributing to Virus Replication and Pandemic.

    PubMed

    Takahashi, Tadanobu; Suzuki, Takashi

    2015-01-01

    The spike glycoprotein neuraminidase (NA) of influenza A virus (IAV) has sialidase activity that cleaves the terminal sialic acids (viral receptors) from oligosaccharide chains of glycoconjugates. A new antigenicity of viral surface glycoproteins for humans has pandemic potential. We found "low-pH stability of sialidase activity" in NA. The low-pH stability can maintain sialidase activity under acidic conditions of pH 4-5. For human IAVs, NAs of all pandemic viruses were low-pH-stable, whereas those of almost all human seasonal viruses were not. The low-pH stability was dependent on amino acid residues near the active site, the calcium ion-binding site, and the subunit interfaces of the NA homotetramer, suggesting effects of the active site and the homotetramer on structural stability. IAVs with the low-pH-stable NA showed much higher virus replication rates than those of IAVs with low-pH-unstable NA, which was correlated with maintenance of sialidase activity under an endocytic pathway of the viral cell entry mechanism, indicating contribution of low-pH stability to high replication rates of pandemic viruses. The low-pH-stable NA of the 1968 H3N2 pandemic virus was derived from the low-pH-stable NA of H2N2 human seasonal virus, one of two types classified by both low-pH stability in N2 NA and a phylogenetic tree of N2 NA genes. The 2009 H1N1 pandemic virus acquired low-pH-stable NA by two amino acid substitutions at the early stage of the 2009 pandemic. It is thought that low-pH stability contributes to infection spread in a pandemic through enhancement of virus replication.

  6. A Dynamic View of Hepatitis C Virus Replication Complexes▿ ‡

    PubMed Central

    Wölk, Benno; Büchele, Benjamin; Moradpour, Darius; Rice, Charles M.

    2008-01-01

    Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle. PMID:18715913

  7. Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus.

    PubMed

    Roush, David J; Myrold, Adam; Burnham, Michael S; And, Joseph V; Hughes, Joseph V

    2015-01-01

    Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10(10) PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4-5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3-101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79-85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic-Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of

  8. Interferon lambda inhibits dengue virus replication in epithelial cells.

    PubMed

    Palma-Ocampo, Helen K; Flores-Alonso, Juan C; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Flores-Mendoza, Lilian; Herrera-Camacho, Irma; Rosas-Murrieta, Nora H; Santos-López, Gerardo

    2015-09-28

    In viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection. Dengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR. We found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG's peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression. Evidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

  9. Bagaza virus inhibits Japanese encephalitis & West Nile virus replication in Culex tritaeniorhynchus & Cx. quinquefasciatus mosquitoes.

    PubMed

    Sudeep, A B; Bondre, V P; George, R; Ghodke, Y S; Aher, R V; Gokhale, M D

    2015-12-01

    Studies have shown that certain flaviviruses influence susceptibility of mosquitoes by inhibiting/enhancing replication of important flaviviruses. Hence, a study was designed to determine whether Bagaza virus (BAGV), a flavivirus isolated from Culex tritaeniorhynchus mosquitoes in India, alters susceptibility of Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes to Japanese encephalitis (JEV) and West Nile viruses (WNV). JEV and WNV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes in the presence of BAGV was carried out by intrathoracic (IT) inoculation and oral feeding methods. Mosquitoes were infected with BAGV and WNV/JEV either simultaneously or in a phased manner, in which mosquitoes were infected with BAGV by IT inoculation followed by super-infection with JEV/WNV after eight days post-infection (PI). JEV and WNV yield on 7 [th] and 14 [th] day PI after super-infection was determined by 50 per cent tissue culture infective dose (TCID 50 ) method. In Cx. tritaeniorhynchus mosquitoes, prior infection with BAGV significantly reduced JEV and WNV replication while in Cx. quinquefasciatus, BAGV influence was only seen with WNV. Reduction in virus titre was observed in IT inoculated and oral fed mosquitoes irrespective of the infection mode. JEV replication was also found reduced in Cx. tritaeniorhynchus mosquitoes persistently infected with BAGV at passage four. BAGV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes altered their susceptibility to JEV and WNV producing low virus yield. However, the role of BAGV in inhibiting JEV/WNV replication in field mosquitoes needs further investigations.

  10. Genetic diversity in the feline leukemia virus gag gene.

    PubMed

    Kawamura, Maki; Watanabe, Shinya; Odahara, Yuka; Nakagawa, So; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

    2015-06-02

    Feline leukemia virus (FeLV) belongs to the Gammaretrovirus genus and is horizontally transmitted among cats. FeLV is known to undergo recombination with endogenous retroviruses already present in the host during FeLV-subgroup A infection. Such recombinant FeLVs, designated FeLV-subgroup B or FeLV-subgroup D, can be generated by transduced endogenous retroviral env sequences encoding the viral envelope. These recombinant viruses have biologically distinct properties and may mediate different disease outcomes. The generation of such recombinant viruses resulted in structural diversity of the FeLV particle and genetic diversity of the virus itself. FeLV env diversity through mutation and recombination has been studied, while gag diversity and its possible effects are less well understood. In this study, we investigated recombination events in the gag genes of FeLVs isolated from naturally infected cats and reference isolates. Recombination and phylogenetic analyses indicated that the gag genes often contain endogenous FeLV sequences and were occasionally replaced by entire endogenous FeLV gag genes. Phylogenetic reconstructions of FeLV gag sequences allowed for classification into three distinct clusters, similar to those previously established for the env gene. Analysis of the recombination junctions in FeLV gag indicated that these variants have similar recombination patterns within the same genotypes, indicating that the recombinant viruses were horizontally transmitted among cats. It remains to be investigated whether the recombinant sequences affect the molecular mechanism of FeLV transmission. These findings extend our understanding of gammaretrovirus evolutionary patterns in the field. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Genetic characterization of feline leukemia virus from Florida panthers.

    PubMed

    Brown, Meredith A; Cunningham, Mark W; Roca, Alfred L; Troyer, Jennifer L; Johnson, Warren E; O'Brien, Stephen J

    2008-02-01

    From 2002 through 2005, an outbreak of feline leukemia virus (FeLV) occurred in Florida panthers (Puma concolor coryi). Clinical signs included lymphadenopathy, anemia, septicemia, and weight loss; 5 panthers died. Not associated with FeLV outcome were the genetic heritage of the panthers (pure Florida vs. Texas/Florida crosses) and co-infection with feline immunodeficiency virus. Genetic analysis of panther FeLV, designated FeLV-Pco, determined that the outbreak likely came from 1 cross-species transmission from a domestic cat. The FeLV-Pco virus was closely related to the domestic cat exogenous FeLV-A subgroup in lacking recombinant segments derived from endogenous FeLV. FeLV-Pco sequences were most similar to the well-characterized FeLV-945 strain, which is highly virulent and strongly pathogenic in domestic cats because of unique long terminal repeat and envelope sequences. These unique features may also account for the severity of the outbreak after cross-species transmission to the panther.

  12. Genetic Characterization of Feline Leukemia Virus from Florida Panthers

    PubMed Central

    Brown, Meredith A.; Cunningham, Mark W.; Roca, Alfred L.; Troyer, Jennifer L.; Johnson, Warren E.

    2008-01-01

    From 2002 through 2005, an outbreak of feline leukemia virus (FeLV) occurred in Florida panthers (Puma concolor coryi). Clinical signs included lymphadenopathy, anemia, septicemia, and weight loss; 5 panthers died. Not associated with FeLV outcome were the genetic heritage of the panthers (pure Florida vs. Texas/Florida crosses) and co-infection with feline immunodeficiency virus. Genetic analysis of panther FeLV, designated FeLV-Pco, determined that the outbreak likely came from 1 cross-species transmission from a domestic cat. The FeLV-Pco virus was closely related to the domestic cat exogenous FeLV-A subgroup in lacking recombinant segments derived from endogenous FeLV. FeLV-Pco sequences were most similar to the well-characterized FeLV-945 strain, which is highly virulent and strongly pathogenic in domestic cats because of unique long terminal repeat and envelope sequences. These unique features may also account for the severity of the outbreak after cross-species transmission to the panther. PMID:18258118

  13. Focus assay on FeLIX-dependent feline leukemia virus.

    PubMed

    Nakaya, Yuki; Shojima, Takayuki; Hoshino, Shigeki; Miyazawa, Takayuki

    2010-01-01

    T-lymphotropic feline leukemia virus (FeLV-T) induces immunodeficiency in cats. FeLV-T is fusion-defective and requires a cofactor, termed FeLIX, for infection. FeLIX is a truncated envelope glycoprotein of an endogenous FeLV and mediates infection by binding a phosphate transporter Pit-1. In this study, we established a feline sarcoma-positive leukemia-negative cell line expressing FeLIX, named QN/FeLIX cells. Upon infection, FeLV-T induced prominent foci with syncytia in QN/FeLIX cells and could be titrated by the focus assay. In addition, we established a FeLIX-expressing feline fibroblast cell line, named AH/FeLIX cells. FeLV-T productively infected AH/FeLIX cells and induced severe CPE with syncytia. QN/FeLIX and AH/FeLIX cells will be useful for the study of FeLIX-dependent mutants in FeLV-infected cats.

  14. Differential Susceptibility of Spleen Focus-Forming Virus and Murine Leukemia Viruses to Ansamycin Antibiotics

    PubMed Central

    Horoszewicz, Julius S.; Leong, Susan S.; Carter, William A.

    1977-01-01

    The streptovaricin complex (SvCx) and rifamycin SV derivatives display potent antiviral activity against the polycythemic strain of Friend leukemia virus (FV-P), as measured by a reduction in the number of spleen foci produced in mice. Such reductions may be explained by inactivation of functions of (i) the spleen focus-forming virus (SFFV), (ii) its “helper” murine leukemia virus (MuLV), or (iii) both viruses normally present in FV-P. We noted that preincubation of FV-P with fractionation products of SvCx, or derivatives of rifamycin SV, at low concentrations (3 to 5 μg/ml) reduces the number of spleen foci 80 to 97%, whereas titers of MuLV (from the same inoculum) remain unaffected (MuLV titers were measured by XC, S+L−, and “helper activity” assays). Our findings indicate a remarkable biological selectivity of ansamycins, as well as nonansamycin components of SvCx, against the transforming and defective spleen focus-forming virus as compared to MuLV. Thus, the drugs might be useful in distinguishing other types of oncornaviruses. PMID:18986

  15. Suppression of infectious murine leukemia virus in wild mice (Mus musculus) by passive immunization.

    PubMed

    Gardner, M B; Klement, V; Estes, J D; Gilden, R V; Toni, R; Huebner, R J

    1977-06-01

    Passive immunization with heterologous antivirus antiserum beginning at birth successfully suppressed infectious murine leukemia virus expression in Lake Casitas wild mice (Musmusculus) at 5-7 weeks of age.

  16. Bovine leukemia virus structural gene vectors are immunogenic and lack pathogenicity in a rabbit model.

    PubMed

    Kucerova, L; Altanerova, V; Altaner, C; Boris-Lawrie, K

    1999-10-01

    Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV gag, pol, and env and related cis-acting sequences but lacks tax, rex, RIII, and GIV and most of the BLV long terminal repeat sequences, including the cis-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV.

  17. Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

    PubMed Central

    Kucerova, Lucia; Altanerova, Veronika; Altaner, Cestmir; Boris-Lawrie, Kathleen

    1999-01-01

    Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV gag, pol, and env and related cis-acting sequences but lacks tax, rex, RIII, and GIV and most of the BLV long terminal repeat sequences, including the cis-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV. PMID:10482566

  18. The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

    NASA Astrophysics Data System (ADS)

    Shimakami, Tetsuro; Honda, Masao; Shirasaki, Takayoshi; Takabatake, Riuta; Liu, Fanwei; Murai, Kazuhisa; Shiomoto, Takayuki; Funaki, Masaya; Yamane, Daisuke; Murakami, Seishi; Lemon, Stanley M.; Kaneko, Shuichi

    2014-04-01

    Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

  19. Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-mink cell focus-forming and Moloney murine leukemia viruses.

    PubMed Central

    Ishimoto, A; Takimoto, M; Adachi, A; Kakuyama, M; Kato, S; Kakimi, K; Fukuoka, K; Ogiu, T; Matsuyama, M

    1987-01-01

    Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced. PMID:3033317

  20. Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro.

    PubMed

    van Gennip, René G P; van de Water, Sandra G P; Potgieter, Christiaan A; van Rijn, Piet A

    2017-02-15

    The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family.

  1. Construction and characterization of the recombinant Moloney murine leukemia viruses bearing the mouse Fv-4 env gene.

    PubMed Central

    Masuda, M; Yoshikura, H

    1990-01-01

    A nucleotide sequence of the mouse Fv-4 env gene was completed. Structural comparison revealed a close relationship of Fv-4 to the ecotropic Cas-Br-E murine leukemia virus isolated from a wild mouse in southern California. Various portions of the env gene of Moloney murine leukemia virus were replaced by the corresponding Fv-4 env sequence to construct recombinant murine leukemia virus clones. Infectivity of these recombinants was checked by the S+L- cell focus induction assay and the XC cell syncytium formation assay. Recombinants bearing the following Fv-4 env sequence retained ecotropic infectivity; the AccI-BamHI and BamHI-BalI regions coding for the N- and C-terminal halves of Fv-4 gp70SU, respectively; and the BalI-NcoI region encoding the cleavage site between gp70SU and p15(E)TM of the Fv-4 env. However, when the Fv-4 sequence was substituted for the p15(E)TM-coding NcoI-EcoRV region or the AccI-EcoRV region covering almost the entire env gene, infectivity was undetectable in our assays. The recombinant clone containing the Fv-4 AccI-EcoRV region, i.e., almost the entire Fv-4 env sequence, was introduced with pSV2neo into NIH 3T3 cells, and a G418r cell line named NIH(Fv4)-2 was isolated. The NIH(Fv4)-2 cell released viral particles that contained reverse transcriptase, Fv-4 env molecules as well as the other viral proteins, and viral genomic RNA. However, proviral DNA synthesis was not detected upon inoculation of this virus in NIH 3T3 cells. The loss of infectivity of the recombinant virus bearing the Fv-4 AccI-EcoRV region appeared to be caused by failure in an early step of replication. Images PMID:2304138

  2. Foot and mouth disease virus non structural protein 2C interacts with Beclin1 modulating virus replication

    USDA-ARS?s Scientific Manuscript database

    Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease (FMD), is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in th...

  3. Fidelity of Target Site Duplication and Sequence Preference during Integration of Xenotropic Murine Leukemia Virus-Related Virus

    PubMed Central

    Kim, Sanggu; Rusmevichientong, Alice; Dong, Beihua; Remenyi, Roland; Silverman, Robert H.; Chow, Samson A.

    2010-01-01

    Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity. PMID:20421928

  4. Noncytopathic Replication of Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus Replicons in Mammalian Cells

    PubMed Central

    Petrakova, Olga; Volkova, Eugenia; Gorchakov, Rodion; Paessler, Slobodan; Kinney, Richard M.; Frolov, Ilya

    2005-01-01

    Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5′ untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins. PMID:15919912

  5. Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells.

    PubMed

    Petrakova, Olga; Volkova, Eugenia; Gorchakov, Rodion; Paessler, Slobodan; Kinney, Richard M; Frolov, Ilya

    2005-06-01

    Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.

  6. Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    PubMed Central

    Terry, Anne; Kilbey, Anna; Naseer, Asif; Levy, Laura S.; Ahmad, Shamim; Watts, Ciorsdaidh; Mackay, Nancy; Cameron, Ewan; Wilson, Sam

    2016-01-01

    ABSTRACT The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV. IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level

  7. Murine Leukemia Virus Uses NXF1 for Nuclear Export of Spliced and Unspliced Viral Transcripts

    PubMed Central

    Sakuma, Toshie; Davila, Jaime I.; Malcolm, Jessica A.; Kocher, Jean-Pierre A.; Tonne, Jason M.

    2014-01-01

    ABSTRACT Intron-containing mRNAs are subject to restricted nuclear export in higher eukaryotes. Retroviral replication requires the nucleocytoplasmic transport of both spliced and unspliced RNA transcripts, and RNA export mechanisms of gammaretroviruses are poorly characterized. Here, we report the involvement of the nuclear export receptor NXF1/TAP in the nuclear export of gammaretroviral RNA transcripts. We identified a conserved cis-acting element in the pol gene of gammaretroviruses, including murine leukemia virus (MLV) and xenotropic murine leukemia virus (XMRV), named the CAE (cytoplasmic accumulation element). The CAE enhanced the cytoplasmic accumulation of viral RNA transcripts and the expression of viral proteins without significantly affecting the stability, splicing, or translation efficiency of the transcripts. Insertion of the CAE sequence also facilitated Rev-independent HIV Gag expression. We found that the CAE sequence interacted with NXF1, whereas disruption of NXF1 ablated CAE function. Thus, the CAE sequence mediates the cytoplasmic accumulation of gammaretroviral transcripts in an NXF1-dependent manner. Disruption of NXF1 expression impaired cytoplasmic accumulations of both spliced and unspliced RNA transcripts of XMRV and MLV, resulting in their nuclear retention or degradation. Thus, our results demonstrate that gammaretroviruses use NXF1 for the cytoplasmic accumulation of both spliced and nonspliced viral RNA transcripts. IMPORTANCE Murine leukemia virus (MLV) has been studied as one of the classic models of retrovirology. Although unspliced host messenger RNAs are rarely exported from the nucleus, MLV actively exports unspliced viral RNAs to the cytoplasm. Despite extensive studies, how MLV achieves this difficult task has remained a mystery. Here, we have studied the RNA export mechanism of MLV and found that (i) the genome contains a sequence which supports the efficient nuclear export of viral RNAs, (ii) the cellular factor NXF1 is

  8. Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis.

    PubMed

    Terry, Anne; Kilbey, Anna; Naseer, Asif; Levy, Laura S; Ahmad, Shamim; Watts, Ciorsdaidh; Mackay, Nancy; Cameron, Ewan; Wilson, Sam; Neil, James C

    2017-03-01

    The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV.IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most

  9. Human immunodeficiency virus infection of monoblastoid cells: cellular differentiation determines the pattern of virus replication.

    PubMed Central

    Pauza, C D; Galindo, J; Richman, D D

    1988-01-01

    Stringent control of human immunodeficiency virus (HIV) replication was observed in the human monoblastoid cell line U937. A low-multiplicity infection of these cells by the LAV1 strain of HIV was productive for 2.5 days; then virus replication became restricted and no further evidence of virion production was observed. The dramatic decrease in HIV production was due in part of reduced accumulation of cytoplasmic viral RNA and occurred in the absence of evident cytopathic effects. In contrast, infected cells induced to differentiate by phorbol ester, vitamin D3, or lymphokine supernatant did not release markers of HIV despite the accumulation of significant levels of cytoplasmic viral RNA. HIV infection altered the pattern of c-myc RNA accumulation in U937 cells. Expression of this gene changes normally in response to the state of cellular differentiation; in infected cells the level of c-myc expression was correlated to the levels of viral RNA accumulation and not to cellular differentiation. These results suggest that restricted replication of HIV in monocytes might be an important mechanism of virus persistence and demonstrate a relationship between HIV replication and monocyte differentiation. Images PMID:2458483

  10. Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies.

    PubMed

    Silva, Laurie A; Dermody, Terence S

    2017-03-01

    Chikungunya virus (CHIKV), a reemerging arbovirus, causes a crippling musculoskeletal inflammatory disease in humans characterized by fever, polyarthralgia, myalgia, rash, and headache. CHIKV is transmitted by Aedes species of mosquitoes and is capable of an epidemic, urban transmission cycle with high rates of infection. Since 2004, CHIKV has spread to new areas, causing disease on a global scale, and the potential for CHIKV epidemics remains high. Although CHIKV has caused millions of cases of disease and significant economic burden in affected areas, no licensed vaccines or antiviral therapies are available. In this Review, we describe CHIKV epidemiology, replication cycle, pathogenesis and host immune responses, and prospects for effective vaccines and highlight important questions for future research.

  11. Modulation of innate immune responses during human T-cell leukemia virus (HTLV-1) pathogenesis.

    PubMed

    Olière, Stéphanie; Douville, Renée; Sze, Alexandre; Belgnaoui, S Mehdi; Hiscott, John

    2011-08-01

    Infection with the Human T-cell Leukemia virus type I (HTLV-1) retrovirus results in a number of diverse pathologies, including the aggressive, fatal T-cell malignancy adult T-cell leukemia (ATL) and the chronic, progressive neurologic disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Worldwide, it is estimated there are 15-20 million HTLV-1-infected individuals; although the majority of HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% of AC develops either ATL or HAM/TSP, but never both. Regardless of asymptomatic status or clinical outcome, HTLV-1 carriers are at high risk of opportunistic infection. The progression to pathological HTLV-1 disease is in part attributed to the failure of the innate and adaptive immune system to control virus spread. The innate immune response against retroviral infection requires recognition of viral pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRR) dependent pathways, leading to the induction of host antiviral and inflammatory responses. Recent studies have begun to characterize the interplay between HTLV-1 infection and the innate immune response and have identified distinct gene expression profiles in patients with ATL or HAM/TSP--upregulation of growth regulatory pathways in ATL and constitutive activation of antiviral and inflammatory pathways in HAM/STP. In this review, we provide an overview of the replicative lifecycle of HTLV-1 and the distinct pathologies associated with HTLV-1 infection. We also explore the innate immune mechanisms that respond to HTLV-1 infection, the strategies used by HTLV-1 to subvert these defenses and their contribution to HTLV-1-associated diseases.

  12. Replication of type 2 herpes simplex virus in human endocervical tissue in organ culture.

    PubMed

    Birch, J; Fink, C G; Skinner, G R; Thomas, G H; Jordan, J A

    1976-08-01

    The replication of type 2 herpes simplex virus in human endocervical tissue in organ culture was investigated. The temporal profile of virus replication was related to the initial virus inoculum; high input inocula induced a rapid increase in virus titre while lower multiplicities induced a more slow-rising increase in virus titre. Our evidence suggested that explants were capable of initiating and supporting virus replication for at least 2 weeks following establishment of the culture. Virus yields were optimal when explants were cultured at 37 degrees and in serum-supplemented medium. Explants also supported the replication of type 1 herpes simplex virus and a "non-human" herpes simplex virus (pseudo-rabies virus). The optimal conditions for replication of type 2 herpes simplex virus in human endocervical explants have been established and will provide a model permitting precise investigation of lytic or other virus-cervical cell interactions and their possible relationship to herpes virus-induced pre-invasive carcinoma of this organ.

  13. Replication of type 2 herpes simplex virus in human endocervical tissue in organ culture.

    PubMed Central

    Birch, J.; Fink, C. G.; Skinner, G. R.; Thomas, G. H.; Jordan, J. A.

    1976-01-01

    The replication of type 2 herpes simplex virus in human endocervical tissue in organ culture was investigated. The temporal profile of virus replication was related to the initial virus inoculum; high input inocula induced a rapid increase in virus titre while lower multiplicities induced a more slow-rising increase in virus titre. Our evidence suggested that explants were capable of initiating and supporting virus replication for at least 2 weeks following establishment of the culture. Virus yields were optimal when explants were cultured at 37 degrees and in serum-supplemented medium. Explants also supported the replication of type 1 herpes simplex virus and a "non-human" herpes simplex virus (pseudo-rabies virus). The optimal conditions for replication of type 2 herpes simplex virus in human endocervical explants have been established and will provide a model permitting precise investigation of lytic or other virus-cervical cell interactions and their possible relationship to herpes virus-induced pre-invasive carcinoma of this organ. Images Fig. 1 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 PMID:183806

  14. Rabies viruses leader RNA interacts with host Hsc70 and inhibits virus replication.

    PubMed

    Zhang, Ran; Liu, Chuangang; Cao, Yunzi; Jamal, Muhammad; Chen, Xi; Zheng, Jinfang; Li, Liang; You, Jing; Zhu, Qi; Liu, Shiyong; Dai, Jinxia; Cui, Min; Fu, Zhen F; Cao, Gang

    2017-03-23

    Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication.

  15. Human APOBEC3G incorporation into murine leukemia virus particles

    SciTech Connect

    Kremer, Melanie; Schnierle, Barbara S. . E-mail: schba@pei.de

    2005-06-20

    The human APOBEC3G protein exhibits broad antiretroviral activity against a variety of retroviruses. It is packaged into viral particles and executes its antiviral function in the target cell. The packaging of APOBEC3G into different viral particles requires a mechanism that confers this promiscuity. Here, APOBEC3G incorporation into murine leukemia virus (MLV) was studied using retroviral vectors. APOBEC3G uptake did not require either its cytidine deaminase activity or the presence of a retroviral vector genome. Results from immunoprecipitation and co-localization studies of APOBEC3G with a MLV Gag-CFP (cyan fluorescent protein) fusion protein imply an interaction between both proteins. RNase A treatment did not inhibit the co-precipitation of Gag-CFP and APOBEC3G, suggesting that the interaction is RNA independent. Like human immunodeficiency virus (HIV) Gag, the MLV Gag precursor protein appears to interact with APOBEC3G, indicating that Gag contains conserved structures which are used to encapsidate APOBEC3G into different retroviral particles.

  16. Novel antiviral activity of bromocriptine against dengue virus replication.

    PubMed

    Kato, Fumihiro; Ishida, Yuki; Oishi, Shinya; Fujii, Nobutaka; Watanabe, Satoru; Vasudevan, Subhash G; Tajima, Shigeru; Takasaki, Tomohiko; Suzuki, Youichi; Ichiyama, Koji; Yamamoto, Naoki; Yoshii, Kentaro; Takashima, Ikuo; Kobayashi, Takeshi; Miura, Tomoyuki; Igarashi, Tatsuhiko; Hishiki, Takayuki

    2016-07-01

    Dengue virus (DENV) infectious disease is a major public health problem worldwide; however, licensed vaccines or specific antiviral drugs against this infection are not available. To identify novel anti-DENV compounds, we screened 1280 pharmacologically active compounds using focus reduction assay. Bromocriptine (BRC) was found to have potent anti-DENV activity and low cytotoxicity (half maximal effective concentration [EC50], 0.8-1.6 μM; and half maximal cytotoxicity concentration [CC50], 53.6 μM). Time-of-drug-addition and time-of-drug-elimination assays suggested that BRC inhibits translation and/or replication steps in the DENV life cycle. A subgenomic replicon system was used to verify that BRC restricts RNA replication step. Furthermore, a single amino acid substitution (N374H) was detected in the NS3 protein that conferred resistance to BRC. In summary, BRC was found to be a novel DENV inhibitor and a potential candidate for the treatment of DENV infectious disease. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  17. Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs.

    PubMed

    Bogerd, Hal P; Skalsky, Rebecca L; Kennedy, Edward M; Furuse, Yuki; Whisnant, Adam W; Flores, Omar; Schultz, Kimberly L W; Putnam, Nicole; Barrows, Nicholas J; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A; Griffin, Diane E; Cullen, Bryan R

    2014-07-01

    The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of

  18. Replication of Many Human Viruses Is Refractory to Inhibition by Endogenous Cellular MicroRNAs

    PubMed Central

    Bogerd, Hal P.; Skalsky, Rebecca L.; Kennedy, Edward M.; Furuse, Yuki; Whisnant, Adam W.; Flores, Omar; Schultz, Kimberly L. W.; Putnam, Nicole; Barrows, Nicholas J.; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A.; Griffin, Diane E.

    2014-01-01

    ABSTRACT The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of

  19. Vaccination against δ−Retroviruses: The Bovine Leukemia Virus Paradigm

    PubMed Central

    Gutiérrez, Gerónimo; Rodríguez, Sabrina M.; de Brogniez, Alix; Gillet, Nicolas; Golime, Ramarao; Burny, Arsène; Jaworski, Juan-Pablo; Alvarez, Irene; Vagnoni, Lucas; Trono, Karina; Willems, Luc

    2014-01-01

    Bovine leukemia virus (BLV) and human T-lymphotropic virus type 1 (HTLV-1) are closely related δ-retroviruses that induce hematological diseases. HTLV-1 infects about 15 million people worldwide, mainly in subtropical areas. HTLV-1 induces a wide spectrum of diseases (e.g., HTLV-associated myelopathy/tropical spastic paraparesis) and leukemia/lymphoma (adult T-cell leukemia). Bovine leukemia virus is a major pathogen of cattle, causing important economic losses due to a reduction in production, export limitations and lymphoma-associated death. In the absence of satisfactory treatment for these diseases and besides the prevention of transmission, the best option to reduce the prevalence of δ-retroviruses is vaccination. Here, we provide an overview of the different vaccination strategies in the BLV model and outline key parameters required for vaccine efficacy. PMID:24956179

  20. CD8+ Lymphocytes Can Control HIV Infection in vitro by Suppressing Virus Replication

    NASA Astrophysics Data System (ADS)

    Walker, Christopher M.; Moody, Dewey J.; Stites, Daniel P.; Levy, Jay A.

    1986-12-01

    Lymphocytes bearing the CD8 marker were shown to suppress replication of human immunodeficiency virus (HIV) in peripheral blood mononuclear cells. The effect was dose-dependent and most apparent with autologous lymphocytes; it did not appear to be mediated by a cytotoxic response. This suppression of HIV replication could be demonstrated by the addition of CD8+ cells at the initiation of virus production as well as after several weeks of virus replication by cultured cells. The observations suggest a potential approach to therapy in which autologous CD8 lymphocytes could be administered to individuals to inhibit HIV replication and perhaps progression of disease.

  1. Feline leukemia virus and feline immunodeficiency virus in Canada: Recommendations for testing and management

    PubMed Central

    Little, Susan; Bienzle, Dorothee; Carioto, Lisa; Chisholm, Hugh; O’Brien, Elizabeth; Scherk, Margie

    2011-01-01

    Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are common and important infectious disease agents of cats in Canada. Seroprevalence data for FeLV and FIV in various populations of Canadian cats are reviewed and recommendations for testing and management of infections by these viruses in cats in Canada are presented. Retrovirus testing in Canada is infrequent in comparison with the United States, and efforts should be focused on reducing physical and other barriers to testing, and on education of veterinarians, veterinary team members, and cat owners regarding the importance of testing. New test methodologies for FeLV and FIV are emerging, and should be independently evaluated in order to provide practitioners with information on test reliability. Finally, more information is needed on FIV subtypes in Canada to improve diagnostics and vaccines, and to provide information on disease outcomes. PMID:22294790

  2. Seroprevalence of feline leukemia virus and feline immunodeficiency virus infection among cats in Canada.

    PubMed

    Little, Susan; Sears, William; Lachtara, Jessica; Bienzle, Dorothee

    2009-06-01

    The purposes of this study were to determine the seroprevalence of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) infection among cats in Canada and to identify risk factors for seropositivity. Signalment, lifestyle factors, and test results for FeLV antigen and FIV antibody were analyzed for 11 144 cats from the 10 Canadian provinces. Seroprevalence for FIV antibody was 4.3% and seroprevalence for FeLV antigen was 3.4%. Fifty-eight cats (0.5%) were seropositive for both viruses. Seroprevalence varied geographically. Factors such as age, gender, health status, and lifestyle were significantly associated with risk of FeLV and FIV seropositivity. The results suggest that cats in Canada are at risk of retrovirus infection and support current recommendations that the retrovirus status of all cats should be known.

  3. Feline leukemia virus and feline immunodeficiency virus in Canada: recommendations for testing and management.

    PubMed

    Little, Susan; Bienzle, Dorothee; Carioto, Lisa; Chisholm, Hugh; O'Brien, Elizabeth; Scherk, Margie

    2011-08-01

    Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are common and important infectious disease agents of cats in Canada. Seroprevalence data for FeLV and FIV in various populations of Canadian cats are reviewed and recommendations for testing and management of infections by these viruses in cats in Canada are presented. Retrovirus testing in Canada is infrequent in comparison with the United States, and efforts should be focused on reducing physical and other barriers to testing, and on education of veterinarians, veterinary team members, and cat owners regarding the importance of testing. New test methodologies for FeLV and FIV are emerging, and should be independently evaluated in order to provide practitioners with information on test reliability. Finally, more information is needed on FIV subtypes in Canada to improve diagnostics and vaccines, and to provide information on disease outcomes.

  4. Hexagonal organization of Moloney murine leukemia virus capsid proteins.

    PubMed

    Mayo, Keith; McDermott, Jason; Barklis, Eric

    2002-06-20

    To help elucidate the mechanisms by which retrovirus structural proteins associate to form virus particles, we have examined membrane-bound assemblies of Moloney murine leukemia virus (M-MuLV) capsid (CA) proteins. Electron microscopy and image reconstruction techniques showed that CA dimers appear to function as organizational subunits of the cage-like, membrane-bound protein arrays. However, new three-dimensional (3D) data also were consistent with hexagonal (p6) assembly models. The p6 3D reconstructions of membrane-bound M-MuLV CA proteins gave unit cells of a = b = 80.3 A, c = 110 A, gamma = 120 degrees, in which six dimer units formed a cage lattice. Neighbor cage hole-to-hole distances were 45 A, while distances between hexagonal cage holes corresponded to unit cell lengths (80.3 A). The hexagonal model predicts two types of cage holes (trimer and hexamer holes), uses symmetric head-to-head dimer building blocks, and permits the introduction of lattice curvature by conversion of hexamer to pentamer units. The M-MuLV CA lattice is similar to those formed in helical tubes by HIV CA in that hexamer units surround cage holes of 25-30 A, but differs in that M-MuLV hexamer units appear to be CA dimers, whereas HIV CA units appear to be monomers. These results suggest that while general assembly principles apply to different retroviruses, clear assembly distinctions exist between these virus types. (c) 2002 Elsevier Science (USA).

  5. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication.

    PubMed

    Portela, Agustín; Digard, Paul

    2002-04-01

    All viruses with negative-sense RNA genomes encode a single-strand RNA-binding nucleoprotein (NP). The primary function of NP is to encapsidate the virus genome for the purposes of RNA transcription, replication and packaging. The purpose of this review is to illustrate using the influenza virus NP as a well-studied example that the molecule is much more than a structural RNA-binding protein, but also functions as a key adapter molecule between virus and host cell processes. It does so through the ability to interact with a wide variety of viral and cellular macromolecules, including RNA, itself, two subunits of the viral RNA-dependent RNA polymerase and the viral matrix protein. NP also interacts with cellular polypeptides, including actin, components of the nuclear import and export apparatus and a nuclear RNA helicase. The evidence for the existence of each of these activities and their possible roles in transcription, replication and intracellular trafficking of the virus genome is considered.

  6. Massive depletion of bovine leukemia virus proviral clones located in genomic transcriptionally active sites during primary infection.

    PubMed

    Gillet, Nicolas A; Gutiérrez, Gerónimo; Rodriguez, Sabrina M; de Brogniez, Alix; Renotte, Nathalie; Alvarez, Irene; Trono, Karina; Willems, Luc

    2013-01-01

    Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed

  7. Massive Depletion of Bovine Leukemia Virus Proviral Clones Located in Genomic Transcriptionally Active Sites during Primary Infection

    PubMed Central

    Gillet, Nicolas A.; Renotte, Nathalie; Alvarez, Irene; Trono, Karina; Willems, Luc

    2013-01-01

    Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed

  8. Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication.

    PubMed Central

    Luciw, P A; Cheng-Mayer, C; Levy, J A

    1987-01-01

    Mutations were made by recombinant DNA techniques in an infectious molecular clone of the human immunodeficiency virus San Francisco isolate 2 (HIVSF2) [formerly the prototype isolate of the acquired immunodeficiency syndrome-associated retrovirus (ARV-2)]. The effect of these changes on the replicative and cytopathologic properties of the virus was studied by transfecting modified virus clones into cultured human cells. Mutations in the gag, pol, env, and tat regions precluded virus replication and cytopathology in lymphoid cells. A mutation in orf-A dramatically reduced but did not abolish virus replication. Mutant viruses with deletions in the orf-B region were highly cytopathic and replicated to approximately 5-fold higher levels than wild-type virus. They also produced approximately 5-fold more viral DNA in infected lymphoid cells than did wild-type virus. Thus, the orf-B region may function to down-regulate virus replication. This mutational analysis of the HIVSF2 genome is a means of assessing genes regulating viral replication and cytopathology. Images PMID:2434956

  9. Effects of dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency virus replication

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; McGrath, M. S.; Hanks, D.; Erickson, S.; Pulliam, L.

    1992-01-01

    We have investigated the direct effect of dimethyl prostaglandin A1 (dmPGA1) on the replication of herpes simplex virus (HSV) and human immunodeficiency virus type 1 (HIV-1). dmPGA1 significantly inhibited viral replication in both HSV and HIV infection systems at concentrations of dmPGA1 that did not adversely alter cellular DNA synthesis. The 50% inhibitory concentration (ID50) for several HSV type 1 (HSV-1) strains ranged from 3.8 to 5.6 micrograms/ml for Vero cells and from 4.6 to 7.3 micrograms/ml for human foreskin fibroblasts. The ID50s for two HSV-2 strains varied from 3.8 to 4.5 micrograms/ml for Vero cells; the ID50 was 5.7 micrograms/ml for human foreskin fibroblasts. We found that closely related prostaglandins did not have the same effect on the replication of HSV; dmPGE2 and dmPGA2 caused up to a 60% increase in HSV replication compared with that in untreated virus-infected cells. HIV-1 replication in acutely infected T cells (VB line) and chronically infected macrophages was assessed by quantitative decreases in p24 concentration. The effective ID50s were 2.5 micrograms/ml for VB cells acutely infected with HIV-1 and 5.2 micrograms/m for chronically infected macrophages. dmPGA1 has an unusual broad-spectrum antiviral activity against both HSV and HIV-1 in vitro and offers a new class of potential therapeutic agents for in vivo use.

  10. Effects of dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency virus replication

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; McGrath, M. S.; Hanks, D.; Erickson, S.; Pulliam, L.

    1992-01-01

    We have investigated the direct effect of dimethyl prostaglandin A1 (dmPGA1) on the replication of herpes simplex virus (HSV) and human immunodeficiency virus type 1 (HIV-1). dmPGA1 significantly inhibited viral replication in both HSV and HIV infection systems at concentrations of dmPGA1 that did not adversely alter cellular DNA synthesis. The 50% inhibitory concentration (ID50) for several HSV type 1 (HSV-1) strains ranged from 3.8 to 5.6 micrograms/ml for Vero cells and from 4.6 to 7.3 micrograms/ml for human foreskin fibroblasts. The ID50s for two HSV-2 strains varied from 3.8 to 4.5 micrograms/ml for Vero cells; the ID50 was 5.7 micrograms/ml for human foreskin fibroblasts. We found that closely related prostaglandins did not have the same effect on the replication of HSV; dmPGE2 and dmPGA2 caused up to a 60% increase in HSV replication compared with that in untreated virus-infected cells. HIV-1 replication in acutely infected T cells (VB line) and chronically infected macrophages was assessed by quantitative decreases in p24 concentration. The effective ID50s were 2.5 micrograms/ml for VB cells acutely infected with HIV-1 and 5.2 micrograms/m for chronically infected macrophages. dmPGA1 has an unusual broad-spectrum antiviral activity against both HSV and HIV-1 in vitro and offers a new class of potential therapeutic agents for in vivo use.

  11. Simian varicella virus open reading frame 63/70 expression is required for efficient virus replication in culture

    PubMed Central

    Brazeau, Elizabeth; Wellish, Mary; Kaufer, Benedict B.; Tischer, B. Karsten; Gray, Wayne; Zhou, Fuchun; Osterrieder, Nikolaus; Hanlon, Teri; Golive, Anjani; Hall, Travis; Nair, Sreekala; Owens, Gregory P.; Mueller, Niklaus H.; Cohrs, Randall J.; Pugazhenthi, Subbiah; Gilden, Don

    2011-01-01

    Simian varicella virus (SVV) open reading frame (ORF) 63, duplicated in the virus genome as ORF 70, is homologous to varicella zoster virus ORF 63/70. Transfection of bacterial artificial chromosome clones containing the wild-type SVV genome and mutants with stop codons in ORF 70, in both ORFs 63 and 70 and the repaired virus DNA sequences into Vero cells produced a cytopathic effect (CPE). The onset of CPE was much slower with the double-mutant transfectants (10 days vs. 3 days) and plaques were smaller. While SVV ORF 63 is not required for replication in culture, its expression leads to robust virus replication. PMID:21479719

  12. Inhibition of respiratory syncytial virus replication and virus-induced p38 kinase activity by berberine.

    PubMed

    Shin, Han-Bo; Choi, Myung-Soo; Yi, Chae-Min; Lee, Jun; Kim, Nam-Jung; Inn, Kyung-Soo

    2015-07-01

    Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection and poses a major public health threat worldwide. No effective vaccines or therapeutics are currently available; berberine, an isoquinoline alkaloid from various medicinal plants, has been shown to exert antiviral and several other biological effects. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) activity is implicated in infection by and replication of viruses such as RSV and the influenza virus. Because berberine has previously been implicated in modulating the activity of p38 MAPK, its effects on RSV infection and RSV-mediated p38 MAPK activation were examined. Replication of RSV in epithelial cells was significantly reduced by treatment with berberine. Berberine treatment caused decrease in viral protein and mRNA syntheses. Similar to previously reported findings, RSV infection caused phosphorylation of p38 MAPK at a very early time point of infection, and phosphorylation was dramatically reduced by berberine treatment. In addition, production of interleukin-6 mRNA upon RSV infection was significantly suppressed by treatment with berberine, suggesting the anti-inflammatory role of berberine during RSV infection. Taken together, we showed that berberine, a natural compound already proven to be safe for human consumption, suppresses the replication of RSV. In addition, the current study suggests that inhibition of RSV-mediated early p38 MAPK activation, which has been implicated as an early step in viral infection, as a potential molecular mechanism. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence.

    PubMed

    McPherson, Robert Lyle; Abraham, Rachy; Sreekumar, Easwaran; Ong, Shao-En; Cheng, Shang-Jung; Baxter, Victoria K; Kistemaker, Hans A V; Filippov, Dmitri V; Griffin, Diane E; Leung, Anthony K L

    2017-02-14

    Chikungunya virus (CHIKV), an Old World alphavirus, is transmitted to humans by infected mosquitoes and causes acute rash and arthritis, occasionally complicated by neurologic disease and chronic arthritis. One determinant of alphavirus virulence is nonstructural protein 3 (nsP3) that contains a highly conserved MacroD-type macrodomain at the N terminus, but the roles of nsP3 and the macrodomain in virulence have not been defined. Macrodomain is a conserved protein fold found in several plus-strand RNA viruses that binds to the small molecule ADP-ribose. Prototype MacroD-type macrodomains also hydrolyze derivative linkages on the distal ribose ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated proteins. Using mass spectrometry, we unambiguously defined its substrate specificity as mono(ADP-ribosyl)ated aspartate and glutamate but not lysine residues. Mutant viruses lacking hydrolase activity were unable to replicate in mammalian BHK-21 cells or mosquito Aedes albopictus cells and rapidly reverted catalytically inactivating mutations. Mutants with reduced enzymatic activity had slower replication in mammalian neuronal cells and reduced virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hydrolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice.

  14. The V protein of canine distemper virus is required for virus replication in human epithelial cells.

    PubMed

    Otsuki, Noriyuki; Nakatsu, Yuichiro; Kubota, Toru; Sekizuka, Tsuyoshi; Seki, Fumio; Sakai, Kouji; Kuroda, Makoto; Yamaguchi, Ryoji; Takeda, Makoto

    2013-01-01

    Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

  15. Leukemia

    MedlinePlus

    Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, the bone marrow produces abnormal white blood cells. ...

  16. Replication of Tomato Yellow Leaf Curl Virus in Its Whitefly Vector, Bemisia tabaci.

    PubMed

    Pakkianathan, Britto Cathrin; Kontsedalov, Svetlana; Lebedev, Galina; Mahadav, Assaf; Zeidan, Muhammad; Czosnek, Henryk; Ghanim, Murad

    2015-10-01

    Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted exclusively by the whitefly Bemisia tabaci in a persistent, circulative manner. Replication of TYLCV in its vector remains controversial, and thus far, the virus has been considered to be nonpropagative. Following 8 h of acquisition on TYLCV-infected tomato plants or purified virions and then transfer to non-TYLCV-host cotton plants, the amounts of virus inside whitefly adults significantly increased (>2-fold) during the first few days and then continuously decreased, as measured by the amounts of genes on both virus DNA strands. Reported alterations in insect immune and defense responses upon virus retention led us to hypothesize a role for the immune response in suppressing virus replication. After virus acquisition, stress conditions were imposed on whiteflies, and the levels of three viral gene sequences were measured over time. When whiteflies were exposed to TYLCV and treatment with two different pesticides, the virus levels continuously increased. Upon exposure to heat stress, the virus levels gradually decreased, without any initial accumulation. Switching of whiteflies between pesticide, heat stress, and control treatments caused fluctuating increases and decreases in virus levels. Fluorescence in situ hybridization analysis confirmed these results and showed virus signals inside midgut epithelial cell nuclei. Combining the pesticide and heat treatments with virus acquisition had significant effects on fecundity. Altogether, our results demonstrate for the first time that a single-stranded DNA plant virus can replicate in its hemipteran vector. Plant viruses in agricultural crops are of great concern worldwide. Many of them are transmitted from infected to healthy plants by insects. Persistently transmitted viruses often have a complex association with their vectors; however, most are believed not to replicate within these vectors. Such replication is important, as it contributes to the

  17. Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro

    PubMed Central

    van de Water, Sandra G. P.; Potgieter, Christiaan A.; van Rijn, Piet A.

    2016-01-01

    ABSTRACT The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro. In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro. Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has

  18. [Submicroscopic features of cells in the microenvironment of hematopoietic development of virus-induced Rauscher leukemia].

    PubMed

    Butenko, Z A; Naumenko, O I

    1993-06-01

    The study was made of submicroscopic changes in the cells of bone marrow and splenic microenvironment in mice developing virus-induced Rauscher leukemia. As shown by electron microscopy, ultrastructural cytochemistry and immunocytochemistry, ultrastructure of the complexes from the stromal and hemopoietic cells underwent noticeable alterations as early as the first days after the virus introduction. This suggests that bone marrow is the primary target of the virus in Rauscher leukemia. Affections of the macrophages, dendrite, interdigital and lymphoid cells of the spleen reflect their participation in the body defenses against the virus. Progressive shift of erythropoiesis from the bone marrow into the spleen is related to morphofunctional changes in the microenvironmental cells. The findings may be useful in consideration of cellular pathogenetic aspects of acute leukemia.

  19. Zika Virus RNA Replication and Persistence in Brain and Placental Tissue.

    PubMed

    Bhatnagar, Julu; Rabeneck, Demi B; Martines, Roosecelis B; Reagan-Steiner, Sarah; Ermias, Yokabed; Estetter, Lindsey B C; Suzuki, Tadaki; Ritter, Jana; Keating, M Kelly; Hale, Gillian; Gary, Joy; Muehlenbachs, Atis; Lambert, Amy; Lanciotti, Robert; Oduyebo, Titilope; Meaney-Delman, Dana; Bolaños, Fernando; Saad, Edgar Alberto Parra; Shieh, Wun-Ju; Zaki, Sherif R

    2017-03-01

    Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections.

  20. Molecular characterization of bovine leukemia virus from Moldovan dairy cattle.

    PubMed

    Pluta, Aneta; Rola-Łuszczak, Marzena; Kubiś, Piotr; Balov, Svetlana; Moskalik, Roman; Choudhury, Bhudipa; Kuźmak, Jacek

    2017-06-01

    Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a disease that has worldwide distribution. Whilst it has been eradicated in most of Western Europe and Scandinavia, it remains a problem in other regions, particularly Eastern Europe and South America. For this study, in 2013, 24 cattle from three farms in three regions of Moldova were screened by ELISA and nested PCR. Of these cattle, 14 which were PCR positive, and these were molecularly characterized based on the nucleotide sequence of the env gene and the deduced amino acid sequence of the encoded gp51 protein. Our results demonstrated a low level of genetic variability (0-2.9%) among BLV field strains from Moldova, in contrast to that observed for other retroviruses, including human immunodeficiency virus (HIV) (20-38%) Mason IL (Trudy vologod moloch Inst 146-164, 1970) and equine infectious anemia virus (EIAV) (~40%) Willems L et al (AIDS Res Hum Retroviruses 16(16):1787-1795, 2000), where the envelope gene exhibits high levels of variation Polat M et al (Retrovirology 13(1):4, 2016). Sequence comparisons and phylogenetic analysis revealed that BLV genotype 7 (G7) is predominant in Moldova and that the BLV population in Moldovan cattle is a mixture of at least three new sub-genotypes: G7D, G7E and G4C. Neutrality tests revealed that negative selection was the major force operating upon the 51-kDa BLV envelope surface glycoprotein subunit gp51, although one positively selected site within conformational epitope G was detected in the N-terminal part of gp51. Furthermore, two functional domains, linear epitope B and the zinc-binding domain, were found to have an elevated ratio of nonsynonymous to synonymous codon differences. Together, these data suggest that the evolutionary constraints on epitopes G and B and the zinc-binding domains of gp51 differ from those on the other domains, with a tendency towards formation of homogenous genetic groups, which is a common concept of

  1. Inhibition of Mayaro virus replication by cerulenin in Aedes albopictus cells.

    PubMed

    Pereira, H S; Rebello, M A

    1998-12-01

    The antibiotic cerulenin, an inhibitor of lipid synthesis, was shown to suppress Mayaro virus replication in Aedes albopictus cells at non-cytotoxic doses. Cerulenin blocked the incorporation of [3H]glycerol into lipids when present at any time post infection (p.i.). Cerulenin added at the beginning of infection inhibited the synthesis of virus proteins. However, when this antibiotic was added at later stages of infection, it had only a mild effect on the virus protein synthesis. The possibility that cerulenin acts by blocking an initial step in the Mayaro virus replication after virus entry and before late viral translation is discussed.

  2. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

    SciTech Connect

    Papaneri, Amy B.; Wirblich, Christoph; Cann, Jennifer A.; Cooper, Kurt; Jahrling, Peter B.; Schnell, Matthias J.; Blaney, Joseph E.

    2012-12-05

    We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RV{Delta}G-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RV{Delta}G-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RV{Delta}G-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RV{Delta}G-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

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

    PubMed Central

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

    1988-01-01

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

  4. CD11c controls herpes simplex virus 1 responses to limit virus replication during primary infection.

    PubMed

    Allen, Sariah J; Mott, Kevin R; Chentoufi, Aziz A; BenMohamed, Lbachir; Wechsler, Steven L; Ballantyne, Christie M; Ghiasi, Homayon

    2011-10-01

    CD11c is expressed on the surface of dendritic cells (DCs) and is one of the main markers for identification of DCs. DCs are the effectors of central innate immune responses, but they also affect acquired immune responses to infection. However, how DCs influence the efficacy of adaptive immunity is poorly understood. Here, we show that CD11c(+) DCs negatively orchestrate both adaptive and innate immunity against herpes simplex virus type 1 (HSV-1) ocular infection. The effectiveness and quantity of virus-specific CD8(+) T cell responses are increased in CD11c-deficient animals. In addition, the levels of CD83, CD11b, alpha interferon (IFN-α), and IFN-β, but not IFN-γ, were significantly increased in CD11c-deficient animals. Higher levels of IFN-α, IFN-β, and CD8(+) T cells in the CD11c-deficient mice may have contributed to lower virus replication in the eye and trigeminal ganglia (TG) during the early period of infection than in wild-type mice. However, the absence of CD11c did not influence survival, severity of eye disease, or latency. Our studies provide for the first time evidence that CD11c expression may abrogate the ability to reduce primary virus replication in the eye and TG via higher activities of type 1 interferon and CD8(+) T cell responses.

  5. Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.

    PubMed

    Farley, Daniel C; Bannister, Richard; Leroux-Carlucci, Marie A; Evans, Nerys E; Miskin, James E; Mitrophanous, Kyriacos A

    2012-10-01

    The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical "equine-tropic" RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems.

  6. Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site.

    PubMed

    Cuesta-Geijo, Miguel Ángel; Barrado-Gil, Lucía; Galindo, Inmaculada; Muñoz-Moreno, Raquel; Alonso, Covadonga

    2017-06-01

    African swine fever virus (ASFV) infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns) synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection.

  7. Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site

    PubMed Central

    Cuesta-Geijo, Miguel Ángel; Barrado-Gil, Lucía; Galindo, Inmaculada; Muñoz-Moreno, Raquel; Alonso, Covadonga

    2017-01-01

    African swine fever virus (ASFV) infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns) synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection. PMID:28587154

  8. Impact of the MRN Complex on Adeno-Associated Virus Integration and Replication during Coinfection with Herpes Simplex Virus 1

    PubMed Central

    Millet, Rachel; Jolinon, Nelly; Nguyen, Xuan-Nhi; Berger, Gregory; Cimarelli, Andrea; Greco, Anna; Bertrand, Pascale; Odenthal, Margarete; Büning, Hildegard

    2015-01-01

    ABSTRACT Adeno-associated virus (AAV) is a helper-dependent parvovirus that requires coinfection with adenovirus (AdV) or herpes simplex virus 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during coinfection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study, we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after coinfection with wild-type (wt) HSV-1 or HSV-1 with the polymerase deleted. This effect was specific to wt AAV, since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1. IMPORTANCE Viral DNA genomes trigger a DNA damage response (DDR), which can be either detrimental or beneficial for virus replication. Adeno-associated virus (AAV) is a defective parvovirus that requires the help of an unrelated virus such as adenovirus (AdV) or herpes simplex virus 1 (HSV-1) for productive replication. Previous studies have demonstrated that the cellular Mre11-Rad50-Nbs1 (MRN) complex, a

  9. Interactions of Host Proteins with the Murine Leukemia Virus Integrase

    PubMed Central

    Studamire, Barbara; Goff, Stephen P.

    2010-01-01

    Retroviral infections cause a variety of cancers in animals and a number of diverse diseases in humans such as leukemia and acquired immune deficiency syndrome. Productive and efficient proviral integration is critical for retroviral function and is the key step in establishing a stable and productive infection, as well as the mechanism by which host genes are activated in leukemogenesis. Host factors are widely anticipated to be involved in all stages of the retroviral life cycle, and the identification of integrase interacting factors has the potential to increase our understanding of mechanisms by which the incoming virus might appropriate cellular proteins to target and capture host DNA sequences. Identification of MoMLV integrase interacting host factors may be key to designing efficient and benign retroviral-based gene therapy vectors; key to understanding the basic mechanism of integration; and key in designing efficient integrase inhibitors. In this review, we discuss current progress in the field of MoMLV integrase interacting proteins and possible roles for these proteins in integration. PMID:21637732

  10. Inhibition by streptovaricins of Rauscher leukemia virus splenomegaly.

    PubMed

    Borden, E C; Carter, W A; Sensenbrenner, L L; Owens, A H; Lichtenstein, J; Gray, G D; Neil, G L; Nichol, F R; Li, L H

    1974-12-15

    Streptovaricins (Sv), ansa macrolide antibiotics, inhibited Rauscher leukemia virus (RLV) splenomegaly by 25-50%. All streptovaricins tested were effective when administered orally either by diet ad lib or by intubation from infection to time of killing. When delivered by intubation, Sv was measurable in plasma for up to 6 h. SvC, at 300 mg/kg/day, reduced mean spleen weight of infected mice from 478 plus or minus 51 (SE) mg to 300 plus or minus 55 (SE) mg. Rifampicin, at 250 mg/kg/day, had no similar activity. Decrease in caloric intake and in body-weight gain also resulted in an inhibition of RLV splenomegaly; although Sv-treated mice gained weight, the increase was usually slightly less than controls. However, mice treated with a Sv diet for a week prior to infection, after an initial period of weight loss, gained at a rate equivalent to control group, and when killed had a marked reduction in splenomegaly. The selectivity of streptovaricins and specificity for viral events was suggested by several observations: (1) Splenomegaly and mortality, induced by L1210 or a non-infective transplantable tumor of RLV origin, was not inhibited. (2) No inhibition of normal hematopoietic spleen colonies was observed. (3) Host immune responses, including cellular and humoral immunity and interferon production and action, were not inhibited. Thus, although the effect of slightly decreased weight and intake could not be unequivocally established, the findings were most compatible with a selective inhibition of RLV splenomegaly by Sv.

  11. Bovine leukemia virus and cow longevity in Michigan dairy herds.

    PubMed

    Bartlett, P C; Norby, B; Byrem, T M; Parmelee, A; Ledergerber, J T; Erskine, R J

    2013-03-01

    To determine the association between infection with bovine leukemia virus (BLV) and cow longevity, a stratified random sample of 3,849 Holsteins in 112 Michigan dairy herds was followed for an average of 597 d following testing for BLV antibodies with an ELISA milk test. The hazard ratio of 1.23 indicates that BLV-positive cows were 23% more likely than their BLV-negative herd mates to die or be culled during the monitoring period. This result is adjusted for lactation number, which is also positively associated with an increased risk of leaving the herd. Because herd was included in models, the effect of BLV ELISA on cow longevity was a within-herd comparison in which BLV-infected cattle were compared with their uninfected herd mates. The analysis of 4 ELISA optical density (OD) groups demonstrated a dose response such that cows with higher OD values had decreased survival compared with cows with lower OD values. Cows with OD values above 0.5 were at 40% greater risk of dying or being culled than were their uninfected herd mates. These results support the contention that the association of BLV with cow longevity, when added to other economic impacts, may warrant the control of BLV in our US dairy cow population. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Abelson murine leukemia virus: structural requirements for transforming gene function.

    PubMed Central

    Srinivasan, A; Dunn, C Y; Yuasa, Y; Devare, S G; Reddy, E P; Aaronson, S A

    1982-01-01

    The integrated Abelson murine leukemia virus (A-MuLV) genome cloned in bacteriophage lambda gtWES.lambda B was used to localize viral genetic sequences required for transformation. Comparison of the biological activity of cloned A-MuLV genomic and subgenomic fragments showed that subgenomic clones that lacked the 5' long terminal repeat and adjoining sequences (300 base pairs downstream of the repeat) were not biologically active. In contrast, subgenomic clones that lacked the 3' long terminal repeat and as much as 1.3 kilobase pairs of the A-MuLV cell-derived abl gene were as efficient as wild-type viral DNA in transformation. The A-MuLV-encoded polyprotein P120 and its associated protein kinase activity were detected in transformants obtained by transfection with Cla I, BamHI, and HindIII subgenomic clones. In contrast, individual transformants obtained with subgenomic Sal I clones expressed A-MuLV proteins ranging in size from 82,000 to 95,000 daltons. Each demonstrated an associated protein kinase activity. These results provide direct genetic evidence that only the proximal 40% of abl with its associated 5' helper viral sequences is required for fibroblast transformation. Images PMID:6291048

  13. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

    PubMed Central

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A.; Li, Jianhui; Randall, Glenn; Belov, George A.; Wang, Xiaofeng

    2016-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

  14. Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity.

    PubMed

    Derse, D; Martarano, L

    1990-01-01

    A recombinant bovine leukemia virus (BLV) was constructed in which the X region was replaced with the bacterial neomycin resistance gene controlled by the simian virus 40 early promoter. This virus, termed BLV-SVNEO, is a self-packaging, activator-dependent retroviral vector. Introduction of the plasmid pBLV-SVNEO into mammalian cells resulted in constitutive expression of the neo gene, whereas the BLV structural genes, gag, pol, and env, were expressed only in the presence of the two regulatory proteins, Tax and Rex. The production and release of recombinant virus by cells transfected with pBLV-SVNEO were proportional to the number of G418-resistant colonies that developed after susceptible cells were exposed to the filtered culture medium. BLV-SVNEO was able to infect cell lines of human, bovine, canine, feline, and murine origin. BLV-producing cell lines were resistant to superinfection with BLV-SVNEO. This cell-virus system should facilitate molecular genetic studies of BLV and will provide a rapid, quantitative measure of BLV infectivity in a variety of cell types. These studies also demonstrate the feasibility of using activator-dependent retroviral vectors such as BLV-SVNEO to deliver foreign genes into cells and eventually animals.

  15. Managing the future: the Special Virus Leukemia Program and the acceleration of biomedical research.

    PubMed

    Scheffler, Robin Wolfe

    2014-12-01

    After the end of the Second World War, cancer virus research experienced a remarkable revival, culminating in the creation in 1964 of the United States National Cancer Institute's Special Virus Leukemia Program (SVLP), an ambitious program of directed biomedical research to accelerate the development of a leukemia vaccine. Studies of cancer viruses soon became the second most highly funded area of research at the Institute, and by far the most generously funded area of biological research. Remarkably, this vast infrastructure for cancer vaccine production came into being before a human leukemia virus was shown to exist. The origins of the SVLP were rooted in as much as shifts in American society as laboratory science. The revival of cancer virus studies was a function of the success advocates and administrators achieved in associating cancer viruses with campaigns against childhood diseases such as polio and leukemia. To address the urgency borne of this new association, the SVLP's architects sought to lessen the power of peer review in favor of centralized Cold War management methods, fashioning viruses as "administrative objects" in order to accelerate the tempo of biomedical research and discovery.

  16. A review of feline leukemia virus and feline immunodeficiency virus seroprevalence in cats in Canada.

    PubMed

    Little, Susan

    2011-10-15

    Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are common and important infectious diseases of cats in Canada. Prevalence data are necessary to define prophylactic, management, and therapeutic measures for stray, feral and owned cats. Recently, comprehensive data on the seroprevalence of retrovirus infections of cats in Canada have become available and are reviewed. Further investigation into geographic variations in retrovirus seroprevalence within Canada is warranted, and may provide information to improve recommendations for testing and prevention. As well, more information is needed on FIV subtypes in Canada to improve diagnostics and vaccines, as well as to provide information on disease outcomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Conditions for Copackaging Rous Sarcoma Virus and Murine Leukemia Virus Gag Proteins during Retroviral Budding

    PubMed Central

    Bennett, Robert P.; Wills, John W.

    1999-01-01

    Rous sarcoma virus (RSV) and murine leukemia virus (MLV) are examples of distantly related retroviruses that normally do not encounter one another in nature. Their Gag proteins direct particle assembly at the plasma membrane but possess very little sequence similarity. As expected, coexpression of these two Gag proteins did not result in particles that contain both. However, when the N-terminal membrane-binding domain of each molecule was replaced with that of the Src oncoprotein, which is also targeted to the cytoplasmic face of the plasma membrane, efficient copackaging was observed in genetic complementation and coimmunoprecipitation assays. We hypothesize that the RSV and MLV Gag proteins normally use distinct locations on the plasma membrane for particle assembly but otherwise have assembly domains that are sufficiently similar in function (but not sequence) to allow heterologous interactions when these proteins are redirected to a common membrane location. PMID:9971785

  18. Acute leukemia viruses E26 and avian myeloblastosis virus have related transformation-specific RNA sequences but different genetic structures, gene products, and oncogenic properties

    PubMed Central

    Bister, Klaus; Nunn, Michael; Moscovici, Carlo; Perbal, Bernard; Baluda, Marcel A.; Duesberg, Peter H.

    1982-01-01

    Replication-defective acute leukemia viruses E26 and myeloblastosis virus (AMV) cause distinct leukemias although they belong to the same subgroup of oncogenic avian tumor viruses based on shared transformation-specific (onc) RNA sequences. E26 causes predominantly erythroblastosis in chicken and in quail, whereas AMV induces a myeloid leukemia. However, upon cultivation in vitro for >1 month, a majority of surviving hemopoietic cells of E26-infected animals bear myeloid markers similar to those of AMV-transformed cells. We have analyzed the genetic structure and gene products of E26 virus for a comparison with those of AMV. An E26/helper virus complex was found to contain two RNA species: a 5.7-kilobase (kb) RNA that hybridizes with cloned AMV-specific proviral DNA and hence is probably the E26 genome; and an 8.5-kb RNA that is unrelated to AMV and represents helper virus RNA. Thus, E26 RNA is smaller than 7.5-kb AMV RNA. Hybridization of size-selected poly(A)-terminating E26 RNA fragments with AMV-specific DNA indicated that the shared specific sequences are located in the 5′ half of the E26 genome as opposed to a 3′ location in AMV RNA. In nonproducer cells transformed in vitro by E26, a gag-related nonstructural 135,000-dalton protein (p135) was found. No gag(Pr76) or gag-pol (Pr180) precursors of essential virion proteins, which are present in AMV nonproducer cells, were observed. p135 was also found in cultured E26 virus producing cells of several leukemic chickens, and its intracellular concentration relative to that of the essential virion proteins encoded by the helper virus correlates with the ratio of E26 to helper RNA in virions released by these cells. p135 is phosphorylated but not glycosylated; antigenically it is not related to the pol or env gene products. It appears to be coded for by a partial gag gene and by E26-specific RNA sequences, presumably including those shared with AMV. Hence, AMV and E26 appear to use different strategies for the

  19. Presence of Gumprecht shadows (smudge cells) in bovine leukemia virus-positive cattle.

    PubMed

    Panei, Carlos Javier; Larsen, Alejandra; González, Ester Teresa; Echeverría, María Gabriela

    2013-11-01

    Enzootic Bovine Leukosis is a chronic disease caused by the bovine leukemia virus (BLV). Smudge cells, also known as Gumprecht shadows, are not simple artifacts of slide preparation, but ragged lymphoid cells found mainly in peripheral blood smears from human patients with chronic lymphocytic leukemia. In this study, we report the presence of Gumprecht shadows in peripheral blood from BLV-positive cattle. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Replication of avian influenza viruses in equine tracheal epithelium but not in horses.

    PubMed

    Chambers, Thomas M; Balasuriya, Udeni B R; Reedy, Stephanie E; Tiwari, Ashish

    2013-12-01

    We evaluated a hypothesis that horses are susceptible to avian influenza viruses by in vitro testing, using explanted equine tracheal epithelial cultures, and in vivo testing by aerosol inoculation of ponies. Results showed that several subtypes of avian influenza viruses detectably replicated in vitro. Three viruses with high in vitro replication competence were administered to ponies. None of the three demonstrably replicated or caused disease signs in ponies. While these results do not exhaustively test our hypothesis, they do highlight that the tracheal explant culture system is a poor predictor of in vivo infectivity.

  1. Cryptoporus volvatus extract inhibits influenza virus replication in vitro and in vivo.

    PubMed

    Gao, Li; Sun, Yipeng; Si, Jianyong; Liu, Jinhua; Sun, Guibo; Sun, Xiaobo; Cao, Li

    2014-01-01

    Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy.

  2. Tomato Mosaic Virus Replication Protein Suppresses Virus-Targeted Posttranscriptional Gene Silencing

    PubMed Central

    Kubota, Kenji; Tsuda, Shinya; Tamai, Atsushi; Meshi, Tetsuo

    2003-01-01

    Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L11 and L11A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L11A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed. PMID:14512550

  3. How the Double Spherules of Infectious Bronchitis Virus Impact Our Understanding of RNA Virus Replicative Organelles

    PubMed Central

    Neuman, Benjamin W.

    2013-01-01

    ABSTRACT Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct “replication factories” inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology. PMID:24345746

  4. Seroprevalence of Toxoplasma gondii and concurrent bartonella spp., feline immunodeficiency virus, and feline leukemia infections in cats from Grenada, West Indies

    USDA-ARS?s Scientific Manuscript database

    Toxoplasma gondii and Bartonella spp. are zoonotic pathogens of cats. Feline Immunodeficiency Virus (FIV), and Feline Leukemia Virus (FeLv) are related to Human Iimmunodeficiency Virus, and Human Leukemia Virus, respectively, and these viruses are immunosuppressive. In the present study, the prevale...

  5. Involvement of the skin during bluetongue virus infection and replication in the ruminant host.

    PubMed

    Darpel, Karin E; Monaghan, Paul; Simpson, Jennifer; Anthony, Simon J; Veronesi, Eva; Brooks, Harriet W; Elliott, Heather; Brownlie, Joe; Takamatsu, Haru-Hisa; Mellor, Philip S; Mertens, Peter Pc

    2012-04-30

    Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence".BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.

  6. Isolation and Characterization of Mink Cell Focus-Inducing Murine Leukemia Viruses with Xenotropic Host Range from Mouse Strain SL

    PubMed Central

    Adachi, Akio; Sakai, Koji; Ishimoto, Akinori

    1983-01-01

    A new type of mink cell focus-inducing virus was persistently isolated from the leukemic tissues of SL mice. In contrast to the dual tropic mink cell focus-inducing viruses reported to date, the new virus has the host range of the xenotropic murine leukemia virus. Analysis of RNase T1 fingerprints of genomic RNAs suggested that the mink cell focus-inducing virus with the xenotropic host range isolated from SL mice is a recombinant virus deriving from xenotropic murine leukemia virus. Images PMID:6296452

  7. A replication-incompetent influenza virus bearing the HN glycoprotein of human parainfluenza virus as a bivalent vaccine.

    PubMed

    Kobayashi, Hirofumi; Iwatsuki-Horimoto, Kiyoko; Kiso, Maki; Uraki, Ryuta; Ichiko, Yurie; Takimoto, Toru; Kawaoka, Yoshihiro

    2013-12-16

    Influenza virus and human parainfluenza virus (HPIV) are major etiologic agents of acute respiratory illness in young children. Inactivated and live attenuated influenza vaccines are approved in several countries, yet no vaccine is licensed for HPIV. We previously showed that a replication-incompetent PB2-knockout (PB2-KO) virus that possesses a reporter gene in the coding region of the PB2 segment can serve as a platform for a bivalent vaccine. To develop a bivalent vaccine against influenza and parainfluenza virus, here, we generated a PB2-KO virus possessing the hemagglutinin-neuraminidase (HN) glycoprotein of HPIV type 3 (HPIV3), a major surface antigen of HPIV, in its PB2 segment. We confirmed that this virus replicated only in PB2-expressing cells and expressed HN. We then examined the efficacy of this virus as a bivalent vaccine in a hamster model. High levels of virus-specific IgG antibodies in sera and IgA, IgG, and IgM antibodies in bronchoalveolar lavage fluids against both influenza virus and HPIV3 were detected from hamsters immunized with this virus. The neutralizing capability of these serum antibodies was also confirmed. Moreover, the immunized hamsters were completely protected from virus challenge with influenza virus or HPIV3. These results indicate that PB2-KO virus expressing the HN of HPIV3 has the potential to be a novel bivalent vaccine against influenza and human parainfluenza viruses.

  8. Xenotropic Murine Leukemia Virus-Related Virus in Chronic Fatigue Syndrome and Prostate Cancer

    PubMed Central

    2010-01-01

    Xenotropic murine leukemia virus-related virus (XMRV) is a γ retrovirus that has been associated with chronic fatigue syndrome (CFS) and prostate cancer. The search for viral causes of these syndromes was reignited by the finding that RNase L activity was low in hereditary prostate cancer and some CFS patients. The six strains of XMRV that have been sequenced have greater than 99% identity, indicating a new human infection rather than laboratory contamination. DNA, RNA, and proteins from XMRV have been detected in 50% to 67% of CFS patients and in about 3.7% of healthy controls. XMRV infections could be transmitted to permissive cell lines from CFS plasma, suggesting the potential for communicable and blood-borne spread of the virus and potentially CFS. This troubling concept is currently under intense evaluation. The most important steps now are to independently confirm the initial findings; develop reliable assays of biomarkers; and to move on to investigations of XMRV pathophysiology and treatment in CFS, prostate cancer, and potentially other virus-related syndromes, if they exist. PMID:20425007

  9. Ascorbic acid inhibits replication and infectivity of avian RNA tumor virus

    SciTech Connect

    BISSELL, MINA J; HATIE, CARROLL; FARSON, DEBORAH A.; SCHWARZ, RICHARD I.; SOO, WHAI-JEN

    1980-04-01

    Ascorbic acid, at nontoxic concentrations, causes a substantial reduction in the ability of avian tumor viruses to replicate in both primary avian tendon cells and chicken embryo fibroblasts. The virus-infected cultures appear to be less transformed in the presence of ascorbic acid by the criteria of morphology, reduced glucose uptake, and increased collagen synthesis. The vitamin does not act by altering the susceptibility of the cells to initial infection and transformation, but instead appears to interfere with the spread of infection through a reduction in virus replication and virus infectivity. The effect is reversible and requires the continuous presence of the vitamin in the culture medium.

  10. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  11. Potential Antivirals: Natural Products Targeting Replication Enzymes of Dengue and Chikungunya Viruses.

    PubMed

    Oliveira, Ana Flávia Costa da Silveira; Teixeira, Róbson Ricardo; Oliveira, André Silva de; Souza, Ana Paula Martins de; Silva, Milene Lopes da; Paula, Sérgio Oliveira de

    2017-03-22

    Dengue virus (DENV) and chikungunya virus (CHIKV) are reemergent arboviruses that are transmitted by mosquitoes of the Aedes genus. During the last several decades, these viruses have been responsible for millions of cases of infection and thousands of deaths worldwide. Therefore, several investigations were conducted over the past few years to find antiviral compounds for the treatment of DENV and CHIKV infections. One attractive strategy is the screening of compounds that target enzymes involved in the replication of both DENV and CHIKV. In this review, we describe advances in the evaluation of natural products targeting the enzymes involved in the replication of these viruses.

  12. Inhibition of Bim Enhances Replication of Varicella-Zoster Virus and Delays Plaque Formation in Virus-Infected Cells

    PubMed Central

    Liu, XueQiao

    2014-01-01

    Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication. PMID:24227856

  13. Inhibition of Bim enhances replication of varicella-zoster virus and delays plaque formation in virus-infected cells.

    PubMed

    Liu, Xueqiao; Cohen, Jeffrey I

    2014-01-01

    Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.

  14. Immunopathogenesis of Human T-Cell Leukemia Virus Type-1-Associated Myelopathy/Tropical Spastic Paraparesis: Recent Perspectives

    PubMed Central

    Saito, Mineki; Bangham, Charles R. M.

    2012-01-01

    Human T-cell leukemia virus type-1 (HTLV-1) is a replication-competent human retrovirus associated with two distinct types of disease only in a minority of infected individuals: the malignancy known as adult T-cell leukemia (ATL) and a chronic inflammatory central nervous system disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic progressive myelopathy characterized by spastic paraparesis, sphincter dysfunction, and mild sensory disturbance in the lower extremities. Although the factors that cause these different manifestations of HTLV-1 infection are not fully understood, accumulating evidence from host population genetics, viral genetics, DNA expression microarrays, and assays of lymphocyte function suggests that complex virus-host interactions and the host immune response play an important role in the pathogenesis of HAM/TSP. Especially, the efficiency of an individual's cytotoxic T-cell (CTL) response to HTLV-1 limits the HTLV-1 proviral load and the risk of HAM/TSP. This paper focuses on the recent advances in HAM/TSP research with the aim to identify the precise mechanisms of disease, in order to develop effective treatment and prevention. PMID:23198155

  15. Myxoma virus M063R is a host range gene essential for virus replication in rabbit cells.

    PubMed

    Barrett, John W; Shun Chang, Chew; Wang, Gen; Werden, Steven J; Shao, Zhuhong; Barrett, Catherine; Gao, Xiujuan; Belsito, Tara A; Villenevue, Danielle; McFadden, Grant

    2007-04-25

    The myxoma virus M063R gene product exhibits some sequence similarity to the poxvirus host range gene, C7L, of vaccinia virus. To address the potential host range function of the M063R gene product in rabbits, a deletion mutant of myxoma virus (vMyx63KO) was generated and characterized. vMyx63KO replicated to normal titre levels and produced foci that were indistinguishable from those produced by MV in vitro in a monkey kidney cell line (BGMK) that are permissive for wild type MV. However, vMyx63KO failed to replicate in all rabbit cell lines tested, including both primary and established cells lines, as well as cells derived from a variety of tissues. M063R expression was not required for myxoma virus binding, entry or early gene expression, whereas DNA replication was aborted and late genes were not expressed in vMyx63KO infected rabbit cells. Thus, the replication block for vMyx63KO in rabbit cells preceded the stage of late gene expression and DNA replication. Finally, an in vivo pathogenesis study indicated that vMyx63KO failed to cause any signs of classic myxomatosis in infected rabbits, but functioned as a non-replicating vaccine and provided protection for subsequent challenge by wild type myxoma virus. Altogether, these observations demonstrate that M063R plays a critical role in determining the host specificity of myxoma virus in rabbit cells.

  16. 3'-Azido-3'-deoxythymidine inhibits the replication of avian leukosis virus.

    PubMed Central

    Olsen, J C; Furman, P; Fyfe, J A; Swanstrom, R

    1987-01-01

    We tested the ability of the thymidine analog 3'-azido-3'-deoxythymidine (BWA509U) to inhibit the replication of the retrovirus avian leukosis virus. Inhibition was measured with two different assays: inhibition of a single round of virus replication and inhibition of virus spread through a cell culture. With both assays, we detected inhibition of virus growth, although inhibition of a single round of virus replication required a 40-fold higher drug concentration than did inhibition of virus spread. We also detected variations in the concentrations of drug needed to inhibit virus replication in different cell types. Higher concentrations of drug were needed to inhibit virus replication in chicken embryo fibroblasts than in the continuous quail cell line QT6. Viral DNA synthesis in infected cells was shown to be inhibited in the presence of the drug. The triphosphate form of the analog acted as a competitive inhibitor of purified viral reverse transcriptase, with a Ki of 0.09 +/- 0.003 microM, and was incorporated as a chain terminator during reverse transcription of the natural viral RNA substrate in vitro. Images PMID:2441079

  17. Replication of swine and human influenza viruses in juvenile and layer turkey hens.

    PubMed

    Ali, Ahmed; Yassine, Hadi; Awe, Olusegun O; Ibrahim, Mahmoud; Saif, Yehia M; Lee, Chang-Won

    2013-04-12

    Since the first reported isolation of swine influenza viruses (SIVs) in turkeys in the 1980s, transmission of SIVs to turkeys was frequently documented. Recently, the 2009 pandemic H1N1 virus, that was thought to be of swine origin, was detected in turkeys with a severe drop in egg production. In this study, we assessed the infectivity of different mammalian influenza viruses including swine, pandemic H1N1 and seasonal human influenza viruses in both juvenile and layer turkeys. In addition, we investigated the potential influenza virus dissemination in the semen of experimentally infected turkey toms. Results showed that all mammalian origin influenza viruses tested can infect turkeys. SIVs were detected in respiratory and digestive tracts of both juvenile and layer turkeys. Variations in replication efficiencies among SIVs were observed especially in the reproductive tract of layer turkeys. Compared to SIVs, limited replication of seasonal human H1N1 and no detectable replication of recent human-like swine H1N2, pandemic H1N1 and seasonal human H3N2 viruses was noticed. All birds seroconverted to all tested viruses regardless of their replication level. In turkey toms, we were able to detect swine H3N2 virus in semen and reproductive tract of infected toms by real-time RT-PCR although virus isolation was not successful. These data suggest that turkey hens could be affected by diverse influenza strains especially SIVs. Moreover, the differences in the replication efficiency we demonstrated among SIVs and between SIV and human influenza viruses in layer turkeys suggest a possible use of turkeys as an animal model to study host tropism and pathogenesis of influenza viruses. Our results also indicate a potential risk of venereal transmission of influenza viruses in turkeys.

  18. Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

    SciTech Connect

    Malkinson, Mertyn . E-mail: malkins@agri.huji.ac.il; Winocour, Ernest . E-mail: ernest.winocour@weizmann.ac.il

    2005-06-05

    The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM. AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.

  19. Evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents

    PubMed Central

    Rosenfeld, Paul; Turner, Patricia V.; MacInnes, Janet I.; Nagy, Éva; Yoo, Dongwan

    2009-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses in the swine industry. The disease is widespread worldwide, and so PRRSV-negative pigs are often difficult to find for the study of PRRSV in vivo. To determine if a small animal model could be developed for PRRSV, 3 strains of laboratory rodent were examined for their susceptibility to the virus. No virus replication was detected in BALB/c or SCID (severe combined immunodeficiency) mice after intraperitoneal inoculation. Moderate replication of PRRSV was detected in primary cotton rat lung cell cultures, but no viral replication was detected following intranasal or intraperitoneal inoculation. Following intratracheal inoculation, viral transcripts were detected in the lungs of cotton rats, but only for 1 day. This study indicates that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate. PMID:20046635

  20. Unusual roles of host metabolic enzymes and housekeeping proteins in plant virus replication.

    PubMed

    Huang, Ying-Wen; Hu, Chung-Chi; Lin, Na-Sheng; Hsu, Yau-Heiu

    2012-12-01

    Viruses have developed the ability to improvise their own replication machineries with host proteins, adapt to different environments, and overcome difficulties encountered during various stages of their infection cycles. The modular nature of protein functional motifs allows for the novel use of ordinary host factors. Recent studies have revealed that positive-sense RNA [(+)RNA] viruses may adapt regular metabolic enzymes and housekeeping proteins of host plants by exploiting unusual functions to accommodate their need for replication, mainly for recruitment and subcellular localization of RNA templates or components of replicase complexes and for controlling switches in different stages of replication. This review compares the newly discovered roles of selected metabolic enzymes and housekeeping proteins in plant (+)RNA virus replication with their original cellular functions and the different consequences when utilized by different viruses. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Fowlpox virus host range restriction: gene expression, DNA replication, and morphogenesis in nonpermissive mammalian cells.

    PubMed

    Somogyi, P; Frazier, J; Skinner, M A

    1993-11-01

    Fowlpox virus (FPV), type species of the Avipoxvirus genus, causes a slow-spreading pox disease of chickens. Following infection of mammalian cells there is no evidence of productive replication of FPV although cytopathic effects are induced and FPV recombinants have been shown to express foreign genes from vaccinia virus early/late promoters. Here we report results of a study to investigate the expression of FPV genes, the replication of FPV genomic DNA, and any ultrastructural changes in mammalian cells infected by wild-type virus, undertaken as a first step in elucidating the nature of the block (or blocks) to productive replication of FPV in mammalian cells. Early and late gene expression as well as genomic DNA replication was observed in fibroblast-like cell lines of monkey and human origin. Furthermore, viral morphogenesis was observed in monkey cells, with the production mainly of immature particles though smaller numbers of apparently mature virus particles were observed.

  2. Studies on the replication of Mayaro virus grown in interferon treated cells.

    PubMed

    Rebello, M C; Fonseca, M E; Marinho, J O; Rebello, M A

    1994-01-01

    Mayaro virus grown in interferon treated infected cells has been characterized with regard to its ability to replicate in vertebrate (TC7) and invertebrate (Aedes albopictus) cells. Virus purified from interferon treated TC7 cells adsorbs and penetrates to the same extent as the control virus. During infection, these virus particles caused inhibition of host protein synthesis and synthesized the same spectrum of viral proteins as normal virus. This population however, was apparently more sensitive to interferon treatment. Electron microscopy of TC7 cells showed the presence of numerous aberrant virus particles budding from the plasma membrane.

  3. Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets

    PubMed Central

    Nachbagauer, Raffael; Miller, Matthew S.; Hai, Rong; Ryder, Alex B.; Rose, John K.; Palese, Peter; García-Sastre, Adolfo

    2015-01-01

    We assessed whether influenza virus hemagglutinin stalk-based immunity protects ferrets against aerosol-transmitted H1N1 influenza virus infection. Immunization of ferrets by a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were cohoused with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers and delayed or no virus replication at all following natural exposure to influenza virus. PMID:26719251

  4. Neuroimmunological aspects of human T cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis.

    PubMed

    Saito, Mineki

    2014-04-01

    Human T cell leukemia virus type 1 (HTLV-1) is a human retrovirus etiologically associated with adult T cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Only approximately 0.25-4 % of infected individuals develop HAM/TSP; the majority of infected individuals remain lifelong asymptomatic carriers. Recent data suggest that immunological aspects of host-virus interactions might play an important role in the development and pathogenesis of HAM/TSP. This review outlines and discusses the current understanding, ongoing developments, and future perspectives of HAM/TSP research.

  5. Meningitis caused by lymphocytic choriomeningitis virus in a patient with leukemia.

    PubMed

    Al-Zein, Naser; Boyce, Thomas G; Correa, Armando G; Rodriguez, Vilmarie

    2008-10-01

    We report a case of 15-year-old girl with T-cell acute lymphoblastic leukemia who had fever, neutropenia, and severe headache while receiving maintenance chemotherapy. Cerebrospinal fluid testing revealed a lymphocytic pleocytosis and no evidence of relapsed leukemia. Meningitis caused by lymphocytic choriomeningitis virus was identified serologically. The patient's course was complicated by hydrocephalus requiring ventriculoperitoneal shunt placement and by an intracranial hemorrhage. Lymphocytic choriomeningitis virus is a rare cause of aseptic meningitis that should be considered in the symptomatic immunocompromised patient with an appropriate exposure history.

  6. Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication

    USGS Publications Warehouse

    Debing, Yannick; Winton, James; Neyts, Johan; Dallmeier, Kai

    2013-01-01

    Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2′-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17β-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.

  7. Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication.

    PubMed

    Debing, Yannick; Winton, James; Neyts, Johan; Dallmeier, Kai

    2013-10-01

    Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2'-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17β-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.

  8. Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication

    PubMed Central

    van Gennip, René G. P.; van de Water, Sandra G. P.; van Rijn, Piet A.

    2014-01-01

    Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUGMet→GCCAla mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector. PMID:24465709

  9. Host–Pathogen Interactions in Measles Virus Replication and Anti-Viral Immunity

    PubMed Central

    Jiang, Yanliang; Qin, Yali; Chen, Mingzhou

    2016-01-01

    The measles virus (MeV) is a contagious pathogenic RNA virus of the family Paramyxoviridae, genus Morbillivirus, that can cause serious symptoms and even fetal complications. Here, we summarize current molecular advances in MeV research, and emphasize the connection between host cells and MeV replication. Although measles has reemerged recently, the potential for its eradication is promising with significant progress in our understanding of the molecular mechanisms of its replication and host-pathogen interactions. PMID:27854326

  10. Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease

    SciTech Connect

    Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander

    2012-10-23

    Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K{sub i}, depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.

  11. Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

    PubMed

    Hand, Erin S; Haller, Sherry L; Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R

    2015-01-01

    As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.

  12. Ultrastructure of the replication sites of positive-strand RNA viruses

    SciTech Connect

    Harak, Christian; Lohmann, Volker

    2015-05-15

    Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.

  13. How viruses use the endoplasmic reticulum for entry, replication, and assembly.

    PubMed

    Inoue, Takamasa; Tsai, Billy

    2013-01-01

    To cause infection, a virus enters a host cell, replicates, and assembles, with the resulting new viral progeny typically released into the extracellular environment to initiate a new infection round. Virus entry, replication, and assembly are dynamic and coordinated processes that require precise interactions with host components, often within and surrounding a defined subcellular compartment. Accumulating evidence pinpoints the endoplasmic reticulum (ER) as a crucial organelle supporting viral entry, replication, and assembly. This review focuses on the molecular mechanism by which different viruses co-opt the ER to accomplish these crucial infection steps. Certain bacterial toxins also hijack the ER for entry. An interdisciplinary approach, using rigorous biochemical and cell biological assays coupled with advanced microscopy strategies, will push to the next level our understanding of the virus-ER interaction during infection.

  14. The Unexpected Roles of Eukaryotic Translation Elongation Factors in RNA Virus Replication and Pathogenesis

    PubMed Central

    Li, Dongsheng; Wei, Ting; Abbott, Catherine M.

    2013-01-01

    SUMMARY The prokaryotic translation elongation factors were identified as essential cofactors for RNA-dependent RNA polymerase activity of the bacteriophage Qβ more than 40 years ago. A growing body of evidence now shows that eukaryotic translation elongation factors (eEFs), predominantly eEF1A, acting in partially characterized complexes sometimes involving additional eEFs, facilitate virus replication. The functions of eEF1A as a protein chaperone and an RNA- and actin-binding protein enable its “moonlighting” roles as a virus replication cofactor. A diverse group of viruses, from human immunodeficiency type 1 and West Nile virus to tomato bushy stunt virus, have adapted to use eEFs as cofactors for viral transcription, translation, assembly, and pathogenesis. Here we review the mechanisms used by viral pathogens to usurp these abundant cellular proteins for their replication. PMID:23699257

  15. Enhanced replication of UV-damaged Simian virus 40 DNA in carcinogen-treated mammalian cells

    SciTech Connect

    Maga, J.A.

    1983-01-01

    The replication of UV-damaged Simian virus 40 (SV40) in carcinogen-treated monkey cells has been studied to elucidate the mechanism of carcinogen-enhanced reactivation. Carcinogen enhanced reactivation is the observed increase in UV-irradiated virus survival in host cells treated with low doses of carcinogen compared to UV-irradiated virus survival in untreated hosts. Carcinogen treatment of monkey kidney cells with either N-acetoxy-2-acetylaminofluorene (AAAF) or UV radiation leads to an enhanced capacity to replicate UV-damaged virus during the first round of infection. To further define the mechanism leading to enhanced replication, a detailed biochemical analysis of replication intermediates in carcinogen-treated cells was performed. Several conclusions can be drawn. First enhanced replication can be observed in the first four rounds of replication after UV irradiation of viral templates. The second major finding is that the relaxed circular intermediate model proposed for the replication of UV-damaged templates in untreated cells appears valid for replication of UV-damaged templates in carcinogen-treated cells. Possible mechanisms and the supporting evidence are discussed and future experiments outlined.

  16. Are viruses alive? The replicator paradigm sheds decisive light on an old but misguided question.

    PubMed

    Koonin, Eugene V; Starokadomskyy, Petro

    2016-10-01

    The question whether or not "viruses are alive" has caused considerable debate over many years. Yet, the question is effectively without substance because the answer depends entirely on the definition of life or the state of "being alive" that is bound to be arbitrary. In contrast, the status of viruses among biological entities is readily defined within the replicator paradigm. All biological replicators form a continuum along the selfishness-cooperativity axis, from the completely selfish to fully cooperative forms. Within this range, typical, lytic viruses represent the selfish extreme whereas temperate viruses and various mobile elements occupy positions closer to the middle of the range. Selfish replicators not only belong to the biological realm but are intrinsic to any evolving system of replicators. No such system can evolve without the emergence of parasites, and moreover, parasites drive the evolution of biological complexity at multiple levels. The history of life is a story of parasite-host coevolution that includes both the incessant arms race and various forms of cooperation. All organisms are communities of interacting, coevolving replicators of different classes. A complete theory of replicator coevolution remains to be developed, but it appears likely that not only the differentiation between selfish and cooperative replicators but the emergence of the entire range of replication strategies, from selfish to cooperative, is intrinsic to biological evolution. Published by Elsevier Ltd.

  17. Are viruses alive? The replicator paradigm sheds decisive light on an old but misguided question

    PubMed Central

    Koonin, Eugene V.; Starokadomskyy, Petro

    2016-01-01

    The question whether or not “viruses are alive” has caused considerable debate over many years. Yet, the question is effectively without substance because the answer depends entirely on the definition of life or the state of “being alive” that is bound to be arbitrary. In contrast, the status of viruses among biological entities is readily defined within the replicator paradigm. All biological replicators form a continuum along the selfishness-cooperativity axis, from the completely selfish to fully cooperative forms. Within this range, typical, lytic viruses represent the selfish extreme whereas temperate viruses and various mobile elements occupy positions closer to the middle of the range. Selfish replicators not only belong to the biological realm but are intrinsic to any evolving system of replicators. No such system can evolve without the emergence of parasites, and moreover, parasites drive the evolution of biological complexity at multiple levels. The history of life is a story of parasite-host coevolution that includes both the incessant arms race and various forms of cooperation. All organisms are communities of interacting, coevolving replicators of different classes. A complete theory of replicator coevolution remains to be developed, but it appears likely that not only the differentiation between selfish and cooperative replicators but the emergence of the entire range of replication strategies, from selfish to cooperative, is intrinsic to biological evolution. PMID:26965225

  18. Tissue selectivity of murine leukemia virus infection is determined by long terminal repeat sequences.

    PubMed Central

    Rosen, C A; Haseltine, W A; Lenz, J; Ruprecht, R; Cloyd, M W

    1985-01-01

    Here we show that the tissue specificity of murine retrovirus infections is determined by the long terminal repeat (LTR) of an otherwise isogenic set of viruses. The isogenic viruses used for this study contain the coding gag, pol, and env genes of the avirulent Akv virus. Recombinant viruses that contain the LTR of a virus that induces T-cell leukemia lymphoma preferentially infect T lymphocytes. Viruses that carry the LTR of a virus that induces erythroleukemia preferentially infect non-T lymphoblastoid cell lines in the marrow and spleen. The Akv virus itself displays no tissue preference for hematopoietic cells. These experiments suggest that retroviruses that carry appropriate enhancer-promoters can be used to infect selectively specific target cells in animals. PMID:2991605

  19. Recognition of Computer Viruses by Detecting Their Gene of Self Replication

    DTIC Science & Technology

    2006-03-01

    addresses that are shared with write access. It uses entry-point obscuring ( EPO ) and an encryption method that is both very simple to implement and very...and code-optimized at a lower level. "* Replication includes a split-inject- regenerate mechanism for the virus body "* Replication includes a correct...sections and a file alignment: ghost° = Sheader + Sseci + Ssec2 + +. SseN Fa Viral code regeneration mechanism is indicative of cavity replication. Unless

  20. DNA tumor viruses: Control of gene expression and replication

    SciTech Connect

    Botchan, M.; Grodzicker, T.; Sharp, P.A.

    1986-01-01

    This book contains eight sections, each consisting of several papers. The sections are: Introduction, Transcription; Regulation of Transcription; RNA Processing and Translation; Transformation; Transforming Proteins; Replication; and Papillomaviruses.

  1. Surface glycoproteins of influenza A H3N2 virus modulate virus replication in the respiratory tract of ferrets.

    PubMed

    Cheng, Xing; Zengel, James R; Xu, Qi; Jin, Hong

    2012-10-10

    The hemagglutinin (HA) genes of the influenza A H3N2 subtype viruses isolated from 1968 to 2010 have evolved substantially but their neuraminidase (NA) genes have been relatively less divergent. The H3N2 viruses isolated since 1995 were found to replicate in the lower respiratory tract of ferrets less efficiently than the earlier isolates. To evaluate whether the HA or/and NA or the internal protein gene segments of the H3N2 virus affected viral replication in the respiratory tract of ferrets, recombinant A/California/07/2004 (CA04) (H3N2) virus and its reassortants that contained the same CA04 internal protein gene segments and the HA and/or NA of A/Udorn/309/1972 (UD72) or A/Wuhan/359/1995 (WH95) H3N2 viruses were generated and evaluated for their replication in the respiratory tract of ferrets. All the reassortant viruses replicated efficiently in the upper respiratory tract of ferrets, but their replication in the lower respiratory tract of ferrets varied. In contrast to the UD72-HA reassortant virus that replicated efficiently in the lungs of ferrets, the virus with the WH95-HA or the CA04-HA either replicated modestly or did not replicate in the lungs of ferrets. The reassortants with the WH95-HA and UD72-NA or CA04-NA had the tendency to lose a N-linked glycosylation site at residue 246 in the HA, resulting in viral lung titer of 100-fold higher than the virus with the HA and NA from WH95. The UD72-NA had the highest neuraminidase activity and increased viral replication by up to 100-fold in tissue culture cells during early infection. Thus, our data indicate that both the HA and NA glycoproteins play important roles in viral replication of the H3N2 influenza virus in ferrets. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Dissociation between lymphoproliferative responses and virus replication in mice with different sensitivities to retrovirus-induced immunodeficiency.

    PubMed Central

    Pozsgay, J M; Reid, S; Pitha, P M

    1993-01-01

    Murine AIDS (MAIDS) is induced by a replication-defective virus (BM5d). In susceptible mice (C57BL/6J), inoculation with LP-BM5 murine leukemia virus, which consists of the BM5d virus and replication-competent B-tropic ecotropic (BM5e) and milk cell focus-inducing (BM5-MCF) helper viruses results in the polyclonal proliferation of T and B cells, immunodeficiency, and the expansion of B cells containing the BM5d provirus followed by the development of B-cell lymphomas. Several strains of mice that are resistant to LP-BM5-induced murine AIDS have been identified, and major histocompatibility complex genes as well as non-major histocompatibility complex genes were shown to play a role in this resistance. In the present study, we have examined and compared the replication of the BM5d and BM5e viruses after inoculation of LP-BM5 into sensitive (C57BL/6J) and resistant (C57BL/KSJ) mice. Using a specific polymerase chain reaction, we could detect the BM5d and BM5e proviruses as early as 1 week postinfection in the sensitive mice, and the levels of both viruses increased significantly with the progression of the disease. In contrast, in the resistant C57BL/KSJ mice, replication of BM5d and BM5e was restricted and no BM5d and only very low levels of the BM5e provirus could be detected either at early or late times postinoculation with the LP-BM5 virus mixture. Inoculation with LP-BM5 did not lead to the production of antibodies that could recognize the BM5d-encoded Pr60gag in either the sensitive or resistant mice; however, production of antibodies recognizing the env-related proteins of the helper virus was detected in the resistant but not in the sensitive mice at late times postinfection. Interestingly, inoculation with LP-BM5 increased polyclonal stimulation of spleen cells and decreased mitogen stimulation in both strains of mice. This stimulation of splenocytes persisted in the sensitive mice but decreased after a few weeks in the resistant mice. These results show an

  3. The hemagglutinin protein of highly pathogenic H5N1 influenza viruses overcomes an early block in the replication cycle to promote productive replication in macrophages.

    PubMed

    Cline, Troy D; Karlsson, Erik A; Seufzer, Bradley J; Schultz-Cherry, Stacey

    2013-02-01

    Macrophages are known to be one of the first lines of defense against influenza virus infection. However, they may also contribute to severe disease caused by the highly pathogenic avian (HPAI) H5N1 influenza viruses. One reason for this may be the ability of certain influenza virus strains to productively replicate in macrophages. However, studies investigating the productive replication of influenza viruses in macrophages have been contradictory, and the results may depend on both the type of macrophages used and the specific viral strain. In this work, we investigated the ability of H1 to H16 viruses to productively replicate in primary murine alveolar macrophages and RAW264.7 macrophages. We show that only a subset of HPAI H5N1 viruses, those that cause high morbidity and mortality in mammals, can productively replicate in macrophages, as measured by the release of newly synthesized virus particles into the cell supernatant. Mechanistically, we found that these H5 strains can overcome a block early in the viral life cycle leading to efficient nuclear entry, viral transcription, translation, and ultimately replication. Studies with reassortant viruses demonstrated that expression of the hemagglutinin gene from an H5N1 virus rescued replication of H1N1 influenza virus in macrophages. This study is the first to characterize H5N1 influenza viruses as the only subtype of influenza virus capable of productive replication in macrophages and establishes the viral gene that is required for this characteristic. The ability to productively replicate in macrophages is unique to H5N1 influenza viruses and may contribute to their increased pathogenesis.

  4. In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences.

    PubMed

    Irie, Takashi; Carnero, Elena; García-Sastre, Adolfo; Harty, Ronald N

    2012-11-19

    The M40 VSV recombinant was engineered to contain overlapping PTAP and PPxY L-domain motifs and flanking residues from the VP40 protein of Ebola virus. Replication of M40 in cell culture is virtually indistinguishable from that of control viruses. However, the presence of the Ebola PTAP motif in the M40 recombinant enabled this virus to interact with and recruit host Tsg101, which was packaged into M40 virions. In this brief report, we compared replication and the pathogenic profiles of M40 and the parental virus M51R in mice to determine whether the presence of the Ebola L-domains and flanking residues altered in vivo characteristics of the virus. Overall, the in vivo characteristics of M40 were similar to those of the parental M51R virus, indicating that the Ebola sequences did not alter pathogenesis of VSV in this small animal model of infection.

  5. Epidemiologic survey of feline leukemia virus in domestic cats on Tsushima Island, Japan: Management strategy for Tsushima leopard cats.

    PubMed

    Makundi, Isaac; Koshida, Yushi; Kuse, Kyohei; Hiratsuka, Takahiro; Ito, Jumpei; Baba, Takuya; Watanabe, Shinya; Kawamura, Maki; Odahara, Yuka; Miyake, Ariko; Yamamoto, Hanae; Kuniyoshi, Sawako; Onuma, Manabu; Nishigaki, Kazuo

    2017-08-01

    The Tsushima leopard cat (TLC) Prionailurus bengalensis euptilurus, a subspecies of P. bengalensis, is designated a National Natural Monument of Japan, and lives only on Tsushima Island, Nagasaki Prefecture, Japan. TLCs are threatened by various infectious diseases. Feline leukemia virus (FeLV) causes a serious infectious disease with a poor prognosis in cats. Therefore, the transmission of FeLV from Tsushima domestic cats (TDCs) to TLCs may threaten the TLC population. We investigated the FeLV infection status of both TDCs and TLCs on Tsushima Island by screening blood samples for FeLV p27 antigen and using PCR to amplify the full-length FeLV env gene. The prevalence of FeLV was 6.4% in TDCs and 0% in TLCs. We also demonstrated that the virus can replicate in the cells of TLCs, suggesting its potential cross-species transmission. The viruses in TDCs were classified as genotype I/clade 3, which is prevalent on a nearby island, based on previous studies of FeLV genotypes and FeLV epidemiology. The FeLV viruses identified on Tsushima Island can be further divided into 2 lineages within genotype I/clade 3, which are geographically separated in Kamijima and Shimojima, indicating that FeLV may have been transmitted to Tsushima Island at least twice. Monitoring FeLV infection in the TDC and TLC populations is highly recommended as part of the TLC surveillance and management strategy.

  6. An upstream open reading frame modulates ebola virus polymerase translation and virus replication.

    PubMed

    Shabman, Reed S; Hoenen, Thomas; Groseth, Allison; Jabado, Omar; Binning, Jennifer M; Amarasinghe, Gaya K; Feldmann, Heinz; Basler, Christopher F

    2013-01-01

    Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5' and 3' untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5'-UTRs lack internal ribosomal entry site function. However, the 5'-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5'-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5'-UTR. The L 5'-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5'-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a "weak" Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10-100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target.

  7. Identification of critical elements within the JC virus DNA replication origin.

    PubMed Central

    Lynch, K J; Frisque, R J

    1990-01-01

    The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins. Images PMID:2173768

  8. Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase

    PubMed Central

    Ndongwe, Tanyaradzwa P.; Adedeji, Adeyemi O.; Michailidis, Eleftherios; Ong, Yee Tsuey; Hachiya, Atsuko; Marchand, Bruno; Ryan, Emily M.; Rai, Devendra K.; Kirby, Karen A.; Whatley, Angela S.; Burke, Donald H.; Johnson, Marc; Ding, Shilei; Zheng, Yi-Min; Liu, Shan-Lu; Kodama, Ei-Ichi; Delviks-Frankenberry, Krista A.; Pathak, Vinay K.; Mitsuya, Hiroaki; Parniak, Michael A.; Singh, Kamalendra; Sarafianos, Stefan G.

    2012-01-01

    We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (koff) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1. PMID:21908397

  9. Feline immunodeficiency virus and feline leukemia virus: frequency and associated factors in cats in northeastern Brazil.

    PubMed

    Lacerda, L C; Silva, A N; Freitas, J S; Cruz, R D S; Said, R A; Munhoz, A D

    2017-05-10

    Our aims were to determine the frequencies of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in owned and stray cats in the northeastern region of Brazil, ascertain the status of FeLV infection, and investigate potential associated factors among the owned cats. Blood samples from 200 asymptomatic owned cats and 30 stray cats were processed using nested PCR and commercial immunochromatographic tests to diagnose infections. To evaluate the factors associated with FIV and/or FeLV in owned cats, a semi-structured interview was conducted with each owner about the animal's environment, and these data were subjected to unconditional logistic regression. The frequencies for owned cats were 6% (12/200) and 3% (6/200) for FIV and FeLV, respectively. No owned cat was positive for both viruses. Stray cats showed frequencies of 6.66% (2/30) and 0% (0/30) for FIV and FeLV, respectively. Contact with other cats and living in peri-urban areas were considered to be risk factors (P < 0.05) for FIV. We did not identify any factors associated with infections with FeLV. Our results confirm the presence of these two retroviruses in the region under study. Our use of different diagnostic techniques allowed us to determine the frequency of retroviruses in the feline population more accurately, particularly with regard to infections by FeLV, which have complex pathogenesis.

  10. Tumorigenic Potential of a Recombinant Retrovirus Containing Sequences from Moloney Murine Leukemia Virus and Feline Leukemia Virus

    PubMed Central

    Starkey, C. R.; Lobelle-Rich, P. A.; Granger, S.; Brightman, B. K.; Fan, H.; Levy, L. S.

    1998-01-01

    A recombinant retrovirus, termed MoFe2-MuLV, was constructed in which the U3 region of T-lymphomagenic Moloney murine leukemia virus (Mo-MuLV) was replaced by that of FeLV-945, a provirus of unique long terminal repeat (LTR) structure identified only in non-T-cell, non-B-cell lymphomas of the domestic cat. The LTR of FeLV-945 is unusual in that it contains only a single copy of the transcriptional enhancer followed 25 bp downstream by a 21-bp sequence in triplicate in tandem. Infectivity of MoFe2-MuLV was demonstrated in vitro in SC-1 cells and in vivo in neonatal NIH-Swiss mice. Tumors occurred in MoFe2-MuLV-infected animals following a latency period of 4 to 10 months (average, 6 months). The results of Southern blot analysis of the T-cell receptor beta locus demonstrated that all tumors were lymphomas of T-cell origin. MoFe2-MuLV LTRs were amplified by PCR from tumor DNA and were characterized by nucleotide sequence analysis. LTRs from the tumors that occurred with relatively shorter latency predominantly retained the original MoFe2-MuLV sequence intact and unaltered. Tumors that occurred with relatively longer latency contained LTRs that also retained the 21-bp sequence triplication characteristic of the original virus but had acquired various duplications of enhancer sequences. The repeated identification of enhancer duplications in late-appearing tumors suggests that the duplication affords a selective advantage, although apparently not in the efficient induction of T-cell lymphoma. Proto-oncogenes known to be targets of insertional mutagenesis in the majority of Mo-MuLV-induced tumors or in feline non-T-cell, non-B-cell lymphomas were shown not to be rearranged in any tumor examined. Mink cell focus-inducing (MCF) proviral DNA was readily detectable in some, but not all, tumors. The presence or absence of MCF did not correlate with the kinetics of tumor induction. These studies indicate that the single-enhancer, triplication-containing FeLV LTR, typical of

  11. IRES-dependent replication of Venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells

    PubMed Central

    Volkova, Eugenia; Frolova, Elena; Darwin, Justin; Forrester, Naomi L.; Weaver, Scott C.; Frolov, Ilya

    2008-01-01

    The development of infectious cDNA for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. In the present study, we developed a recombinant VEEV that is more attenuated than TC-83 and capable of replicating only in vertebrate cells. This phenotype was achieved by rendering the translation of the viral structural proteins, and ultimately viral replication, dependent on the internal ribosome entry site of encephalomyocarditis virus (EMCV IRES). This recombinant virus was viable, but required additional, adaptive mutations in nsP2 that strongly increased its replication rates. In spite of efficient replication in cultured vertebrate cells, the genetically modified VEEV demonstrated a highly attenuated phenotype in newborn mice, and yet induced protective immunity against VEEV infection. PMID:18501401

  12. Cryptoporic acid E from Cryptoporus volvatus inhibits influenza virus replication in vitro.

    PubMed

    Gao, Li; Han, Jiayuan; Si, Jianyong; Wang, Junchi; Wang, Hexiang; Sun, Yipeng; Bi, Yuhai; Liu, Jinhua; Cao, Li

    2017-07-01

    Influenza virus infection is a global public health issue. The efficacy of antiviral agents for influenza virus has been limited by the emergence of drug-resistant virus strains. Thus, there is an urgent need to identify novel antiviral therapies. Our previous studies have found that Cryptoporus volvatus extract can potently inhibit influenza virus replication in vitro and in vivo. However, the effective component of Cryptoporus volvatus, which mediates the antiviral activity, hasn't been identified. Here, we identified a novel anti-influenza virus molecule, Cryptoporic acid E (CAE), from Cryptoporus volvatus. Our results showed that CAE had broad-spectrum anti-influenza activity against 2009 pandemic strain A/Beijing/07/2009 (H1N1/09pdm), seasonal strain A/Beijing/CAS0001/2007(H3N2), mouse adapted strains A/WSN/33 (H1N1), and A/PR8/34 (H1N1). We further investigated the mode of CAE action. Time-course-analysis indicated that CAE exerted its inhibition mainly at the middle stages of the replication cycle of influenza virus. Subsequently, we confirmed that CAE inhibited influenza virus RNA polymerase activity and blocked virus RNA replication and transcription in MDCK cells. In addition, we found that CAE also impaired influenza virus infectivity by directly targeting virus particles. Our data suggest that CAE is a major effective component of Cryptoporus volvatus. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Influenza virus neuraminidase contributes to the dextran sulfate-dependent suppressive replication of some influenza A virus strains.

    PubMed

    Yamada, Hiroshi; Moriishi, Eiko; Haredy, Ahmad M; Takenaka, Nobuyuki; Mori, Yasuko; Yamanishi, Koichi; Okamoto, Shigefumi

    2012-12-01

    Dextran sulfate (DS), a negatively charged, sulfated polysaccharide, suppresses the replication of an influenza A virus strain, and this suppression is associated with inhibition of the hemagglutinin (HA)-dependent fusion activity. However, it remains unknown whether the replication of all or just some influenza A virus strains is suppressed by DS, or whether HA is the only target for the replication suppression. In the present study, we found that DS inhibited the replication of some, but not all influenza A virus strains. The suppression in the DS-sensitive strains was dose-dependent and neutralized by diethylaminoethyl-dextran (DD), which has a positive charge. The suppression by DS was observed not only at the initial stage of viral infection, which includes viral attachment and entry, but also at the late stage, which includes virus assembly and release from infected cells. Electron microscopy revealed that the DS induced viral aggregation at the cell surface. The neuraminidase (NA) activity of the strains whose viral replication was inhibited at the late stage was also more suppressed by DS than that of the strains whose replication was not inhibited, and this inhibition of NA activity was also neutralized by adding positively charged DD. Furthermore, we found that replacing the NA gene of a strain in which viral replication was inhibited by DS at the late stage with the NA gene from a strain in which viral replication was not inhibited, eliminated the DS-dependent suppression. These results suggest that the influenza virus NA contributes to the DS-suppressible virus release from infected cells at the late stage, and the suppression may involve the inhibition of NA activity by DS's negative charge.

  14. Reply to conclusive evidence of replication of a plant virus in honeybees is lacking

    USDA-ARS?s Scientific Manuscript database

    This reply provides clarifications regarding comment “Conclusive Evidence of Replication of a Plant Virus in Honeybees Is Lacking” about our paper “Systemic Spread and Propagation of a Plant-Pathogenic Virus in European Honeybees, Apis mellifera” [mBio 2014, 5(1):e00898-13]. The strand-specific rea...

  15. Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

    PubMed Central

    Panavas, Tadas; Serviene, Elena; Brasher, Jeremy; Nagy, Peter D.

    2005-01-01

    Viruses are devastating pathogens of humans, animals, and plants. To further our understanding of how viruses use the resources of infected cells, we systematically tested the yeast single-gene-knockout library for the effect of each host gene on the replication of tomato bushy stunt virus (TBSV), a positive-strand RNA virus of plants. The genome-wide screen identified 96 host genes whose absence either reduced or increased the accumulation of the TBSV replicon. The identified genes are involved in the metabolism of nucleic acids, lipids, proteins, and other compounds and in protein targeting/transport. Comparison with published genome-wide screens reveals that the replication of TBSV and brome mosaic virus (BMV), which belongs to a different supergroup among plus-strand RNA viruses, is affected by vastly different yeast genes. Moreover, a set of yeast genes involved in vacuolar targeting of proteins and vesicle-mediated transport both affected replication of the TBSV replicon and enhanced the cytotoxicity of the Parkinson's disease-related α-synuclein when this protein was expressed in yeast. In addition, a set of host genes involved in ubiquitin-dependent protein catabolism affected both TBSV replication and the cytotoxicity of a mutant huntingtin protein, a candidate agent in Huntington's disease. This finding suggests that virus infection and disease-causing proteins might use or alter similar host pathways and may suggest connections between chronic diseases and prior virus infection. PMID:15883361

  16. Characterization of the replication cycle of the Lymantria dispar nuclear polyhedrosis virus

    Treesearch

    Christopher I. Riegel; James M. Slavicek

    1997-01-01

    The life cycle of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) was characterized through analysis of budded virus (BV) release, the temporal formation of polyhedra, the temporal transcription pattern of representative early, late, and hyper-expressed late genes, and the onset of DNA replication in the Ld652Y cell line. Transcripts from...

  17. Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses.

    PubMed

    Cheng, Li-Lin; Luk, Yan-Yeung; Murphy, Christopher J; Israel, Barbara A; Abbott, Nicholas L

    2005-12-01

    We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using

  18. Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle.

    PubMed Central

    Yuan, B; Li, X; Goff, S P

    1999-01-01

    The p12 Gag protein of Moloney murine leukemia virus is a small polypeptide of unknown function, containing two proline-rich motifs. To determine its role in replication, we introduced a series of deletion and alanine-scanning substitution mutations throughout the p12 coding region of a proviral DNA, and characterized the phenotypes of the resulting mutant viruses. Complete deletion of p12 and mutations affecting the PPPY motif caused substantial reduction in the yield of virions and a modest reduction in Gag processing. Proteolytic cleavage of the R-peptide from the cytoplasmic tail of the envelope protein TM was abolished in these mutants, suggesting that the PPPY motif is crucial for the viral protease to access the TM tail. The resulting virions were non-infectious, and unable to initiate DNA synthesis in infected cells. Mutants with alterations in both the N- and C-terminal portions of p12 exhibited a distinct phenotype. The production of virions and processing of Gag, Pol and Env precursors were normal. The viruses were able to direct synthesis of linear viral DNA, but there was almost no detectable circular DNAs or LTR-LTR junction. These data suggest that p12 plays a critical role in the early events of the virus life cycle. PMID:10469649

  19. Quasispecies spatial models for RNA viruses with different replication modes and infection strategies.

    PubMed

    Sardanyés, Josep; Elena, Santiago F

    2011-01-01

    Empirical observations and theoretical studies suggest that viruses may use different replication strategies to amplify their genomes, which impact the dynamics of mutation accumulation in viral populations and therefore, their fitness and virulence. Similarly, during natural infections, viruses replicate and infect cells that are rarely in suspension but spatially organized. Surprisingly, most quasispecies models of virus replication have ignored these two phenomena. In order to study these two viral characteristics, we have developed stochastic cellular automata models that simulate two different modes of replication (geometric vs stamping machine) for quasispecies replicating and spreading on a two-dimensional space. Furthermore, we explored these two replication models considering epistatic fitness landscapes (antagonistic vs synergistic) and different scenarios for cell-to-cell spread, one with free superinfection and another with superinfection inhibition. We found that the master sequences for populations replicating geometrically and with antagonistic fitness effects vanished at low critical mutation rates. By contrast, the highest critical mutation rate was observed for populations replicating geometrically but with a synergistic fitness landscape. Our simulations also showed that for stamping machine replication and antagonistic epistasis, a combination that appears to be common among plant viruses, populations further increased their robustness by inhibiting superinfection. We have also shown that the mode of replication strongly influenced the linkage between viral loci, which rapidly reached linkage equilibrium at increasing mutations for geometric replication. We also found that the strategy that minimized the time required to spread over the whole space was the stamping machine with antagonistic epistasis among mutations. Finally, our simulations revealed that the multiplicity of infection fluctuated but generically increased along time.

  20. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    SciTech Connect

    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-10-25

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  1. Aurintricarboxylic Acid Inhibits the Early Stage of Vaccinia Virus Replication by Targeting both Cellular and Viral Factors▿

    PubMed Central

    Myskiw, Chad; Deschambault, Yvon; Jefferies, Kristel; He, Runtao; Cao, Jingxin

    2007-01-01

    Aurintricarboxylic acid (ATA) has been shown to inhibit the replication of viruses from several different families, including human immunodeficiency virus, vesicular stomatitis virus, and the coronavirus causing severe acute respiratory syndrome. This study characterizes the inhibitory effect of ATA on vaccinia virus replication in HeLa, Huh7, and AD293 cells. Vaccinia virus replication is significantly abrogated upon ATA treatment, which is associated with the inhibition of early viral gene transcription. This inhibitory effect may be attributed to two findings. First, ATA blocks the phosphorylation of extracellular signal-regulated kinase 1/2, an event shown to be essential for vaccinia virus replication. Second, ATA inhibits the phosphatase activity of the viral enzyme H1L, which is required to initiate viral transcription. Thus, ATA inhibits vaccinia virus replication by targeting both cellular and viral factors essential for the early stage of replication. PMID:17192307

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

    PubMed

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

    2014-11-01

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

  3. Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication

    PubMed Central

    DiNapoli, Sarah R.; Greene, Justin M.; Lehrer-Brey, Gabrielle; Gieger, Samantha M.; Buechler, Connor R.; Crosno, Kristin A.; Peterson, Eric J.; Wiseman, Roger W.; Estes, Jacob D.; Sacha, Jonah B.; Brenchley, Jason M.; O’Connor, David H.

    2016-01-01

    Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute “pre-peak” phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque ‘elite controllers’ with a short-course of dextran sulfate sodium (DSS)–stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host’s capacity to contain infection. PMID:27926931

  4. Prostaglandin A1 inhibits replication of Mayaro virus in Aedes albopictus cells.

    PubMed

    Barbosa, J A; Rebello, M A

    1995-01-01

    Prostaglandin A1 (PGA1) reduced Mayaro virus replication in Aedes albopictus (mosquito) cells in culture. The highest nontoxic dose of PGA1, 7.5 microM, decreased virus production by 90%. In Mayaro virus-infected cells, PGA1 inhibited virus-specific protein synthesis. However, in mock-infected cells the presence of PGA1 stimulated the synthesis of several proteins with molecular masses of 70, 57 and 23 kDa, respectively. The data obtained from this study show that PGA1 plays a role in the metabolic regulation of Aedes albopictus cells, blocking the synthesis of Mayaro virus and inducing the synthesis of cellular polypeptides.

  5. Identification and characterization of the role of c-terminal Src kinase in dengue virus replication

    PubMed Central

    Kumar, Rinki; Agrawal, Tanvi; Khan, Naseem Ahmed; Nakayama, Yuji; Medigeshi, Guruprasad R.

    2016-01-01

    We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication. PMID:27457684

  6. The leukemia-associated Rho guanine nucleotide exchange factor LARG is required for efficient replication stress signaling

    PubMed Central

    Beveridge, Ryan D; Staples, Christopher J; Patil, Abhijit A; Myers, Katie N; Maslen, Sarah; Skehel, J Mark; Boulton, Simon J; Collis, Spencer J

    2014-01-01

    We previously identified and characterized TELO2 as a human protein that facilitates efficient DNA damage response (DDR) signaling. A subsequent yeast 2-hybrid screen identified LARG; Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (also known as Arhgef12), as a potential novel TELO2 interactor. LARG was previously shown to interact with Pericentrin (PCNT), which, like TELO2, is required for efficient replication stress signaling. Here we confirm interactions between LARG, TELO2 and PCNT and show that a sub-set of LARG co-localizes with PCNT at the centrosome. LARG-deficient cells exhibit replication stress signaling defects as evidenced by; supernumerary centrosomes, reduced replication stress-induced γH2AX and RPA nuclear foci formation, and reduced activation of the replication stress signaling effector kinase Chk1 in response to hydroxyurea. As such, LARG-deficient cells are sensitive to replication stress-inducing agents such as hydroxyurea and mitomycin C. Conversely we also show that depletion of TELO2 and the replication stress signaling kinase ATR leads to RhoA signaling defects. These data therefore reveal a level of crosstalk between the RhoA and DDR signaling pathways. Given that mutations in both ATR and PCNT can give rise to the related primordial dwarfism disorders of Seckel Syndrome and Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) respectively, which both exhibit defects in ATR-dependent checkpoint signaling, these data also raise the possibility that mutations in LARG or disruption to RhoA signaling may be contributory factors to the etiology of a sub-set of primordial dwarfism disorders. PMID:25485589

  7. The leukemia-associated Rho guanine nucleotide exchange factor LARG is required for efficient replication stress signaling.

    PubMed

    Beveridge, Ryan D; Staples, Christopher J; Patil, Abhijit A; Myers, Katie N; Maslen, Sarah; Skehel, J Mark; Boulton, Simon J; Collis, Spencer J

    2014-01-01

    We previously identified and characterized TELO2 as a human protein that facilitates efficient DNA damage response (DDR) signaling. A subsequent yeast 2-hybrid screen identified LARG; Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (also known as Arhgef12), as a potential novel TELO2 interactor. LARG was previously shown to interact with Pericentrin (PCNT), which, like TELO2, is required for efficient replication stress signaling. Here we confirm interactions between LARG, TELO2 and PCNT and show that a sub-set of LARG co-localizes with PCNT at the centrosome. LARG-deficient cells exhibit replication stress signaling defects as evidenced by; supernumerary centrosomes, reduced replication stress-induced γH2AX and RPA nuclear foci formation, and reduced activation of the replication stress signaling effector kinase Chk1 in response to hydroxyurea. As such, LARG-deficient cells are sensitive to replication stress-inducing agents such as hydroxyurea and mitomycin C. Conversely we also show that depletion of TELO2 and the replication stress signaling kinase ATR leads to RhoA signaling defects. These data therefore reveal a level of crosstalk between the RhoA and DDR signaling pathways. Given that mutations in both ATR and PCNT can give rise to the related primordial dwarfism disorders of Seckel Syndrome and Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) respectively, which both exhibit defects in ATR-dependent checkpoint signaling, these data also raise the possibility that mutations in LARG or disruption to RhoA signaling may be contributory factors to the etiology of a sub-set of primordial dwarfism disorders.

  8. Accommodation of pyrimidine dimers during replication of UV-damaged simian virus 40 DNA.

    PubMed Central

    Stacks, P C; White, J H; Dixon, K

    1983-01-01

    UV irradiation of simian virus 40-infected cells at fluences between 20 and 60 J/m2, which yield one to three pyrimidine dimers per simian virus 40 genome, leads to a fluence-dependent progressive decrease in simian virus 40 DNA replication as assayed by incorporation of [3H]deoxyribosylthymine into viral DNA. We used a variety of biochemical and biophysical techniques to show that this decrease is due to a block in the progression of replicative-intermediate molecules to completed form I molecules, with a concomitant decrease in the entry of molecules into the replicating pool. Despite this UV-induced inhibition of replication, some pyrimidine dimer-containing molecules become fully replicated after UV irradiation. The fraction of completed molecules containing dimers goes up with time such that by 3 h after a UV fluence of 40 J/m2, more than 50% of completed molecules contain pyrimidine dimers. We postulate that the cellular replication machinery can accommodate limited amounts of UV-induced damage and that the progressive decrease in simian virus 40 DNA synthesis after UV irradiation is due to the accumulation in the replication pool of blocked molecules containing levels of damage greater than that which can be tolerated. PMID:6621531

  9. Accommodation of pyrimidine dimers during replication of UV-damaged simian virus 40 DNA

    SciTech Connect

    Stacks, P.C.; White, J.H.; Dixon, K.

    1983-08-01

    UV irradiation of simian virus 40-infected cells at fluences between 20 and 60 J/m2, which yield one to three pyrimidine dimers per simian virus 40 genome, leads to a fluence-dependent progressive decrease in simian virus 40 DNA replication as assayed by incorporation of (3H)deoxyribosylthymine into viral DNA. We used a variety of biochemical and biophysical techniques to show that this decrease is due to a block in the progression of replicative-intermediate molecules to completed form I molecules, with a concomitant decrease in the entry of molecules into the replicating pool. Despite this UV-induced inhibition of replication, some pyrimidine dimer-containing molecules become fully replicated after UV irradiation. The fraction of completed molecules containing dimers goes up with time such that by 3 h after a UV fluence of 40 J/m2, more than 50% of completed molecules contain pyrimidine dimers. We postulate that the cellular replication machinery can accommodate limited amounts of UV-induced damage and that the progressive decrease in simian virus 40 DNA synthesis after UV irradiation is due to the accumulation in the replication pool of blocked molecules containing levels of damage greater than that which can be tolerated.

  10. Tomato yellow leaf curl virus: No evidence for replication in the insect vector Bemisia tabaci

    PubMed Central

    Sánchez-Campos, Sonia; Rodríguez-Negrete, Edgar A.; Cruzado, Lucía; Grande-Pérez, Ana; Bejarano, Eduardo R.; Navas-Castillo, Jesús; Moriones, Enrique

    2016-01-01

    Begomovirus ssDNA plant virus (family Geminiviridae) replication within the Bemisia tabaci vector is controversial. Transovarial transmission, alteration to whitefly biology, or detection of viral transcripts in the vector are proposed as indirect evidence of replication of tomato yellow leaf curl virus (TYLCV). Recently, contrasting direct evidence has been reported regarding the capacity of TYLCV to replicate within individuals of B. tabaci based on quantitave PCR approaches. Time-course experiments to quantify complementary and virion sense viral nucleic acid accumulation within B. tabaci using a recently implemented two step qPCR procedure revealed that viral DNA quantities did not increase for time points up to 96 hours after acquisition of the virus. Our findings do not support a recent report claiming TYLCV replication in individuals of B. tabaci. PMID:27476582

  11. Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

    PubMed Central

    Zhao, Yongqian; Soh, Tingjin Sherryl; Chan, Kitti Wing Ki; Fung, Sarah Suet Yin; Swaminathan, Kunchithapadam; Lim, Siew Pheng; Shi, Pei-Yong; Huber, Thomas; Lescar, Julien

    2015-01-01

    We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication. PMID:26269182

  12. Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells.

    PubMed

    Ishimaru, D; Marcicano, F G; Rebello, M A

    1998-09-01

    The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 micrograms/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.

  13. Bovine leukemia virus seroprevalence among cattle presented for slaughter in the United States

    USDA-ARS?s Scientific Manuscript database

    Infection with bovine leukemia virus (BLV) results in economic loss due reduced productivity, especially the reduction of milk production and early culling. In the USA.,USA, previous studies in 1996, 1999 and 2007 showed BLV infections widespread, especially in the dairy herds. The goal of this stud...

  14. Bovine leukemia virus infection in a juvenile alpaca with multicentric lymphoma

    PubMed Central

    Lee, Laura C.; Scarratt, William K.; Buehring, Gertrude C.; Saunders, Geoffrey K.

    2012-01-01

    A 13-month-old alpaca (Vicugna pacos) was presented for mandibular masses and weight loss. Histopathology of biopsy tissue was consistent with lymphoma. The alpaca was euthanized and necropsy revealed lymphoma masses in multiple organs. Immunohistochemistry for T- and B-cell typing was inconclusive. Serology and in-situ polymerase chain reaction hybridization were positive for bovine leukemia virus. PMID:22942445

  15. Milk and fat production in dairy cattle influenced by advanced subclinical bovine leukemia virus infection.

    PubMed

    Wu, M C; Shanks, R D; Lewin, H A

    1989-02-01

    Genetic potentials (pedigree-estimated breeding value) for milk and for fat were compared in cows grouped according to subclinical stage of bovine leukemia virus infection. Genetic potential for milk production was significantly greater in seropositive cows with persistent lymphocytosis (622 +/- 72 kg) and in seropositive hematologically normal cows (554 +/- 34 34 kg) than in seronegative herdmates (418 +/- 53 kg). When 305-day twice-daily-milking mature equivalent milk production records for the current lactation were adjusted for genetic potential, bovine leukemia virus-infected cows that were hematologically normal had significantly greater milk production than did seronegative herdmates, suggesting that early bovine leukemia virus infection was positively associated with milk yield. Genetic potential for fat production was significantly greater for cows with persistent lymphocytosis (21 +/- 2 kg) than for other seropositive (16 +/- 1 kg) and seronegative herdmates (13 +/- 2 kg); however, 305-day twice-daily-milking mature equivalent fat production for the current lactation was not significantly different between the groups. Thus, cows with persistent lymphocytosis did not produce fat according to their genetic potential. As an apparent consequence of tendencies for greater milk yield and less fat production, milk fat percentage was significantly reduced in cows with persistent lymphocytosis (3.33 +/- 0.09%) and other seropositive cows (3.48 +/- 0.05%) relative to seronegative herdmates (3.67 +/- 0.07%). These results suggest a need to reevaluate the economic impact of bovine leukemia virus infection on the dairy industry.

  16. Effect of phenylhydrazine pretreatment on splenectomized Rauscher leukemia virus-infected mice.

    PubMed

    Bergson, A; Lobue, J; Gordon, A S; Fredickson, T N

    1978-01-01

    The protective effect of phenylhydrazine pretreatment seen in Rauscher leukemia virus-infected intact mice is not observed when splenectomized mice are used. Such mice succumb to infection even earlier than viral potency controls. Since phenylhydrazine is known to increase both splenic erythropoiesis and hematopoietic stem cell numbers, the results suggest that these two events may be involved in phenylhydrazine prophylaxis.

  17. T-cell lymphoma of the tympanic bulla in a feline leukemia virus-negative cat.

    PubMed

    de Lorimier, Louis-Philippe; Alexander, Suzanne D; Fan, Timothy M

    2003-12-01

    This report constitutes the first description of a T-cell lymphoma of the tympanic bulla in a cat. This feline leukemia virus (FeLV)-negative cat originally presented with signs referable to middle ear disease; it deteriorated rapidly after definitive diagnosis. Lymphoma of the middle ear is extremely rare in all species.

  18. Milk and fat production in dairy cattle influenced by advanced subclinical bovine leukemia virus infection.

    PubMed Central

    Wu, M C; Shanks, R D; Lewin, H A

    1989-01-01

    Genetic potentials (pedigree-estimated breeding value) for milk and for fat were compared in cows grouped according to subclinical stage of bovine leukemia virus infection. Genetic potential for milk production was significantly greater in seropositive cows with persistent lymphocytosis (622 +/- 72 kg) and in seropositive hematologically normal cows (554 +/- 34 34 kg) than in seronegative herdmates (418 +/- 53 kg). When 305-day twice-daily-milking mature equivalent milk production records for the current lactation were adjusted for genetic potential, bovine leukemia virus-infected cows that were hematologically normal had significantly greater milk production than did seronegative herdmates, suggesting that early bovine leukemia virus infection was positively associated with milk yield. Genetic potential for fat production was significantly greater for cows with persistent lymphocytosis (21 +/- 2 kg) than for other seropositive (16 +/- 1 kg) and seronegative herdmates (13 +/- 2 kg); however, 305-day twice-daily-milking mature equivalent fat production for the current lactation was not significantly different between the groups. Thus, cows with persistent lymphocytosis did not produce fat according to their genetic potential. As an apparent consequence of tendencies for greater milk yield and less fat production, milk fat percentage was significantly reduced in cows with persistent lymphocytosis (3.33 +/- 0.09%) and other seropositive cows (3.48 +/- 0.05%) relative to seronegative herdmates (3.67 +/- 0.07%). These results suggest a need to reevaluate the economic impact of bovine leukemia virus infection on the dairy industry. PMID:2536940

  19. Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice.

    PubMed Central

    Massey, A C; Coppola, M A; Thomas, C Y

    1990-01-01

    The acquisition of U3 region sequences derived from the endogenous xenotropic provirus Bxv-1 appears to be an important step in the generation of leukemogenic recombinant viruses in AKR, HRS, C58, and some CWD mice. We report here that each of three CWD lymphomas produced infectious xenotropic murine leukemia virus related to Bxv-1. In Southern blot experiments, these proviruses hybridized to probes that were specific for the xenotropic envelope and Bxv-1 U3 region sequences. Nucleotide sequence analysis of a cloned CWD xenotropic provirus, CWM-S-5X, revealed that the envelope gene was closely related to but distinct from those of other known xenotropic viruses. In addition, the U3 region of CWM-S-5X contained a viral enhancer sequence that was identical to that found in MCF 247, a recombinant AKR virus that is thought to contain the Bxv-1 enhancer. Finally, restriction enzyme sites in the CWM-S-5X provirus were analogous to those reported within Bxv-1. These results establish that the virus progeny of Bxv-1 have the potential to donate pathogenic enhancer sequences to recombinant polytropic murine leukemia viruses. Interestingly, the three CWD polytropic viruses that were isolated from the same tumor cells that produced the Bxv-1-like viruses had not incorporated Bxv-1 sequences into the U3 region. Images PMID:2170683

  20. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.

    PubMed

    Martins, Laura J; Bonczkowski, Pawel; Spivak, Adam M; De Spiegelaere, Ward; Novis, Camille L; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Typsteen, Wim; Bosque, Alberto; Vanderkerckhove, Linos; Planelles, Vicente

    2016-02-01

    HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput.

  1. Experimental African swine fever: apoptosis of lymphocytes and virus replication in other cells.

    PubMed

    Gómez-Villamandos, J C; Hervás, J; Méndez, A; Carrasco, L; Martín de las Mulas, J; Villeda, C J; Wilkinson, P J; Sierra, M A

    1995-09-01

    In order to determine the cause of cellular death of lymphocytes in pigs with acute African swine fever and the relationships between African swine fever virus (ASFV) and interstitial cells, ten pigs were inoculated with a highly virulent strain of ASFV (Malawi '83) and samples taken for ultrastructural study of hepatic and renal interstitial tissues. We demonstrated death by apoptosis of lymphocytes and virus replication in fibroblasts, smooth muscle cells and endothelial cells in the interstitial tissues of pigs inoculated with ASFV. From day 5 onwards, apoptotic lymphocyte and intense virus replication in hepatic interstitial macrophages and fibroblasts were observed. By day 7, apoptotic lymphocytes and virus replication in macrophages, interstitial capillary endothelial cells and fibroblasts in the kidney were observed. Virus replication was also seen in smooth muscle cells of hepatic and renal arterioles and venules. Our results suggest that mononuclear phagocyte system (MPS) cell activation, and the resulting release of cytokines, could induce apoptosis of lymphocytes and virus replication in non-MPS cells.

  2. Enhanced replication of herpes simplex virus type 1 in human cells

    SciTech Connect

    Miller, C.S.; Smith, K.O. )

    1991-02-01

    The effects of DNA-damaging agents on the replication of herpes simplex virus type 1 (HSV-1) were assessed in vitro. Monolayers of human lung fibroblast cell lines were exposed to DNA-damaging agents (methyl methanesulfonate (MMS), methyl methanethiosulfonate (MMTS), ultraviolet light (UV), or gamma radiation (GR)) at specific intervals, before or after inoculation with low levels of HSV-1. The ability of cell monolayers to support HSV-1 replication was measured by direct plaque assay and was compared with that of untreated control samples. In this system, monolayers of different cell lines infected with identical HSV-1 strains demonstrated dissimilar levels of recovery of the infectious virus. Exposure of DNA-repair-competent cell cultures to DNA-damaging agents produced time-dependent enhanced virus replication. Treatment with agent before virus inoculation significantly (p less than 0.025) increased the number of plaques by 10 to 68%, compared with untreated control cultures, while treatment with agent after virus adsorption significantly increased (p less than 0.025) the number of plaques by 7 to 15%. In a parallel series of experiments, cells deficient in DNA repair (xeroderma pigmentosum) failed to support enhanced virus replication. These results suggest that after exposure to DNA-damaging agents, fibroblasts competent in DNA repair amplify the replication of HSV-1, and that DNA-repair mechanisms that act on a variety of chromosomal lesions may be involved in the repair and biological activation of HSV-1 genomes.

  3. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus

    PubMed Central

    Martins, Laura J.; Bonczkowski, Pawel; Spivak, Adam M.; De Spiegelaere, Ward; Novis, Camille L.; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Typsteen, Wim; Vanderkerckhove, Linos

    2016-01-01

    Abstract HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4+ T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput. PMID:26171776

  4. Interactions between tobamovirus replication proteins and cellular factors: their impacts on virus multiplication.

    PubMed

    Ishibashi, Kazuhiro; Nishikiori, Masaki; Ishikawa, Masayuki

    2010-11-01

    Most viral gene products function inside cells in the presence of various host proteins, nucleic acids, and lipids. Thus, viral gene products come into direct contact with these molecules. The replication proteins of tobamovirus participate not only in viral genome replication but also in counterdefense mechanisms against RNA silencing and other plant defense systems. Accumulating evidence indicates that these functions are carried out through interactions with specific host components. Interactions with some cellular factors, however, are inhibitory to virus multiplication and contribute to host range restriction of tobamovirus. The interactions that have positive and negative impacts on virus multiplication should have been maintained and lost, respectively, during adaptation of the viruses to their respective natural hosts. This review lists the host factors that interact with the replication proteins of tobamovirus and discusses how they influence multiplication of the virus.

  5. Hepatitis B virus replication in steroid-treated severe HBsAg-positive chronic active hepatitis.

    PubMed

    Davis, G L; Czaja, A J; Taswell, H F; Ludwig, J; Go, V L

    1985-02-01

    To determine the effect of corticosteroids on the replication of hepatitis B virus and to assess the relationship between virus replication and prognosis, the behavior of serum and tissue HBcAg was evaluated in 16 patients with severe HBsAg-positive chronic active hepatitis who were treated with prednisone and followed for up to 10 years (mean +/- SEM, 66 +/- 9 months). Hepatitis B virus replication was assessed in serum by a solid-phase radioimmunoassay of Dane particle-associated HBcAg and in liver tissue by indirect immunoperoxidase staining for HBcAg. Despite the presence of severe inflammatory activity, only low levels of hepatitis B virus replication were demonstrated. Mean serum HBcAg levels were low at accession and remained essentially unchanged or gradually decreased during corticosteroid therapy. Serum HBcAg appeared in only one patient in whom no virus replication was detected prior to therapy. HBeAg was frequently detected at low titers by radioimmunoassay when serum HBcAg was undetectable. Loss of HBcAg preceded loss of HBeAg by radioimmunoassay, and disappearance of both markers was a prerequisite for sustained histologic remission. In eight patients, inflammation was present despite absence of serum or tissue HBcAg; in three of these, disease activity continued after loss of HBeAg. We conclude that low levels of hepatitis B virus replication may be associated with severe inflammatory activity, and these levels are not increased by long-term corticosteroid therapy. Inflammation can continue despite loss of HBeAg and absence of detectable virus replication.

  6. Replication and persistence of measles virus in defined subpopulations of human leukocytes.

    PubMed Central

    Joseph, B S; Lampert, P W; Oldstone, M B

    1975-01-01

    Replication of Edmonston strain measles virus was studied in several human lymphoblast lines, as well as in defined subpopulations of circulating human leukocytes. It was found that measles virus can productively infect T cells, B cells, and monocytes from human blood. These conclusions were derived from infectious center studies on segregated cell populations, as well as from ultrastructural analyses on cells labeled with specific markers. In contrast, mature polymorphonuclear cells failed to synthesize measles virus nucleocapsids even after infection at a relatively high multiplicity of infection. Measles virus replicated more efficiently in lymphocytes stimulated with mitogens than in unstimulated cells. However, both phytohemagglutinin and pokeweed mitogen had a negligible stimulatory effect on viral synthesis in purified populations of monocytes. In all instances the efficiency of measles virus replication by monocytes was appreciably less than that of mitogenically stimulated lymphocytes or of continuously culture lymphoblasts. Under standard conditions of infection, all of the surveyed lymphoblast lines produced equivalent amounts of measles virus regardless of the major histocompatibility (HL-A) haplotype. Hence, no evidence was found that the HL-A3,7 haplotype conferred either an advantage or disadvantage with respect to measles virus synthesis in an immunologically neutral environment. A persistent infection with measles virus could be established in both T and B lymphoblasts. The release of infectious virus from such persistently infected cells was stable over a period of several weeks and was approximately 100-fold less than peak viral titers obtained in each respective line after acute infection. Images PMID:1081602

  7. Virus replication in engineered human cells that do not respond to interferons.

    PubMed

    Young, D F; Andrejeva, L; Livingstone, A; Goodbourn, S; Lamb, R A; Collins, P L; Elliott, R M; Randall, R E

    2003-02-01

    The V protein of the paramyxovirus simian virus 5 blocks interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. Here we report on the isolation of human cell lines that express the V protein and can no longer respond to IFN. A variety of viruses, particularly slow-growing wild-type viruses and vaccine candidate viruses (which are attenuated due to mutations that affect virus replication, virus spread, or ability to circumvent the IFN response), form bigger plaques and grow to titers that are increased as much as 10- to 4,000-fold in these IFN-nonresponsive cells. We discuss the practical applications of using such cells in vaccine development and manufacture, virus diagnostics and isolation of newly emerging viruses, and studies on host cell tropism and pathogenesis.

  8. Replication and immunogenicity of swine, equine, and avian h3 subtype influenza viruses in mice and ferrets.

    PubMed

    Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Jin, Hong; Subbarao, Kanta

    2013-06-01

    Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines.

  9. Replication and Immunogenicity of Swine, Equine, and Avian H3 Subtype Influenza Viruses in Mice and Ferrets

    PubMed Central

    Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Jin, Hong

    2013-01-01

    Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines. PMID:23576512

  10. Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and pro-inflammatory dysregulation.

    PubMed

    Chang, Pengxiang; Kuchipudi, Suresh V; Mellits, Kenneth H; Sebastian, Sujith; James, Joe; Liu, Jinhua; Shelton, Holly; Chang, Kin-Chow

    2015-12-08

    Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) influenza viruses in PAM. We found that PAM were readily susceptible to initial infection with all five avian and mammalian influenza viruses but only avian viruses caused early and extensive apoptosis (by 6 h of infection) resulting in reduced virus progeny and moderated pro-inflammation. Full length viral PB1-F2 present only in avian influenza viruses is a virulence factor that targets AM for mitochondrial-associated apoptotic cell death. With the use of reverse genetics on an avian H5N1 virus, we found that full length PB1-F2 contributed to increased apoptosis and pro-inflammation but not to reduced virus replication. Taken together, we propose that early apoptosis of PAM limits the spread of avian influenza viruses and that PB1-F2 could play a contributory role in the process.

  11. Replication of chicken anemia virus (CAV) requires apoptin and is complemented by VP3 of human torque teno virus (TTV).

    PubMed

    Prasetyo, Afiono Agung; Kamahora, Toshio; Kuroishi, Ayumu; Murakami, Kyoko; Hino, Shigeo

    2009-03-01

    To test requirement for apoptin in the replication of chicken anemia virus (CAV), an apoptin-knockout clone, pCAV/Ap(-), was constructed. DNA replication was completely abolished in cells transfected with replicative form of CAV/Ap(-). A reverse mutant competent in apoptin production regained the full level of DNA replication. DNA replication and virus-like particle (VLP) production of CAV/Ap(-) was fully complemented by supplementation of the wild-type apoptin. The virus yield of a point mutant, CAV/ApT(108)I, was 1/40 that of the wild type, even though its DNA replication level was full. The infectious titer of CAV was fully complemented by supplementing apoptin. Progeny virus was free from reverse mutation for T(108)I. To localize the domain within apoptin molecule inevitable for CAV replication, apoptin-mutant expressing plasmids, pAp1, pAp2, pAp3, and pAp4, were constructed by deleting amino acids 10-36, 31-59, 59-88 and 80-112, respectively. While Ap1 and Ap2 were preferentially localized in nuclei, Ap3 and Ap4 were mainly present in cytoplasm. Although complementation capacity of Ap3 and Ap4 was 1/10 of the wild type, neither of them completely lost its activity. VP3 of TTV did fully complement the DNA replication and VLP of CAV/Ap(-). These data suggest that apoptin is inevitable not only for DNA replication but also VLP of CAV. The common feature of apoptin and TTV-VP3 presented another evidence for close relatedness of CAV and TTV.

  12. Differential replication properties among H9N2 avian influenza viruses of Eurasian origin.

    PubMed

    Parvin, Rokshana; Shehata, Awad A; Heenemann, Kristin; Gac, Malgorzata; Rueckner, Antje; Halami, Mohammad Y; Vahlenkamp, Thomas W

    2015-07-06

    Avian influenza H9N2 viruses have become panzootic in Eurasia causing respiratory manifestations, great economic losses and occasionally being transmitted to humans. To evaluate the replication properties and compare the different virus quantification methods, four Eurasian H9N2 viruses from different geographical origins were propagated in embryonated chicken egg (ECE) and Madin-Darby canine kidney epithelial cell systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID50), Hemagglutination (HA) test and quantitative real time RT-PCR (qRT-PCR). The cellular morphology was analyzed using immunofluorescence (IF) and cellular ELISA was used to screen the sensitivity of the viruses to amantadine. The Eurasian wild type-H9N2 virus produced lower titers compared to the three G1-H9N2 viruses at respective time points. Detectable titers were observed earliest at 16 h post inoculation (hpi), significant morphological changes on cells were first observed at 32 hpi. Few nucleotide and amino acid substitutions were noticed in the HA, NA and NS gene sequences but none of them are related to the known conserved region that can alter pathogenesis or virulence following a single passage in cell culture. All studied H9N2 viruses were sensitive to amantadine. The G1-H9N2 viruses have higher replication capabilities compared to the European wild bird-H9N2 probably due to their specific genetic constitutions which is prerequisite for a successful vaccine candidate. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period.

  13. Xenotropic Murine Leukemia Virus-Related Virus in Monozygotic Twins Discordant for Chronic Fatigue Syndrome

    PubMed Central

    Jerome, Keith R.; Diem, Kurt; Huang, Meei-Li; Selke, Stacy; Corey, Lawrence; Buchwald, Dedra

    2011-01-01

    A recent report suggested an association between xenotropic murine leukemia virus-related virus (XMRV) and chronic fatigue syndrome (CFS). If confirmed, this would suggest that antiretroviral therapy might benefit patients suffering from CFS. We validated a set of assays for XMRV, and evaluated the prevalence of XMRV in a cohort of monozygotic twins discordant for CFS. Stored PBMC were tested with 3 separate PCR assays (one of which was nested) for XMRV DNA, and serum/plasma was tested for XMRV RNA by reverse transcription (RT)-PCR. None of the PBMC samples from the twins with CFS or their unaffected co-twins were positive for XMRV, by any of the assays. One plasma sample, from an unaffected co-twin, was reproducibly positive by RT-PCR. However, serum from the same day was negative, as was a followup plasma sample obtained 2 days after the positive specimen. These data do not support an association of XMRV with CFS. PMID:21795004

  14. Evaluation of feline immunodeficiency virus and feline leukemia virus transmembrane peptides for serological diagnosis.

    PubMed Central

    Fontenot, J D; Hoover, E A; Elder, J H; Montelaro, R C

    1992-01-01

    The general model for retrovirus transmembrane (TM) proteins proposed by Gallaher et al. (W. R. Gallaher, J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro, AIDS Res. Hum. Retroviruses 5:431-440, 1989) suggests that all retrovirus TM proteins may contain an immunodominant domain (Imd-TM peptide) located at the apex of the TM polypeptide. Although this Imd-TM peptide has been shown to be immunodominant in a variety of lentivirus infections, there has not been a detailed serological analysis of an oncovirus Imd-TM peptide as a diagnostic agent. We describe here an analysis of the antigenic properties and diagnostic potentials of the predicted Imd-TM peptides of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in serological assays of sera from infected cats. The results of these studies demonstrate that antibodies specific to the FIV Imd-TM peptide are detected within 2 weeks postinfection, are maintained at high levels for extended periods, and are not detectable in uninfected or FeLV-infected cats. In marked contrast, the FeLV Imd-TM peptide displayed only negligible levels of serological reactivity in FeLV-infected cats. These studies indicate that the peptide is a useful reagent for the detection of antibodies to FIV. PMID:1629349

  15. Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells

    PubMed Central

    Mänz, Benjamin; de Graaf, Miranda; Mögling, Ramona; Richard, Mathilde; Bestebroer, Theo M.; Rimmelzwaan, Guus F.

    2016-01-01

    ABSTRACT A strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified using in vitro assays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts. IMPORTANCE Influenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions—most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2—have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans. PMID:27076644

  16. Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication

    PubMed Central

    Bateman, Allen C.; Karasin, Alexander I.; Olsen, Christopher W.

    2013-01-01

    Please cite this paper as: Bateman et al. (2013) Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication. Influenza and Other Respiratory Viruses 7(2) 139–150. Background  Differentiated human airway epithelial cell cultures have been utilized to investigate cystic fibrosis, wound healing, and characteristics of viral infections. These cultures, grown at an air–liquid interface (ALI) in media with defined hormones and growth factors, recapitulate many aspects of the in vivo respiratory tract and allow for experimental studies at the cellular level. Objectives  To optimize growth conditions for differentiated swine airway epithelial cultures and to use these cultures to examine influenza virus infection and replication. Methods  Primary swine respiratory epithelial cells were grown at an air–liquid interface with varying amounts of retinoic acid and epidermal growth factor. Cells grown with optimized concentrations of these factors for 4 weeks differentiated into multilayer epithelial cell cultures resembling the lining of the swine respiratory tract. Influenza virus infection and replication were examined in these cultures. Results/Conclusions  Retinoic acid promoted ciliogenesis, whereas epidermal growth factor controlled the thickness of the pseudoepithelium. The optimal concentrations for differentiated swine cell cultures were 1·5 ng/ml epidermal growth factor and 100 nm retinoic acid. Influenza A viruses infected and productively replicated in these cultures in the absence of exogenous trypsin, suggesting that the cultures express a protease capable of activating influenza virus hemagglutinin. Differences in virus infection and replication characteristics found previously in pigs in vivo were recapitulated in the swine cultures. This system could be a useful tool for a range of applications, including investigating influenza virus species specificity, defining cell tropism

  17. Autophagy induction regulates influenza virus replication in a time-dependent manner.

    PubMed

    Feizi, Neda; Mehrbod, Parvaneh; Romani, Bizhan; Soleimanjahi, Hoorieh; Bamdad, Taravat; Feizi, Amir; Jazaeri, Ehsan Ollah; Targhi, Hadiseh Shokouhi; Saleh, Maryam; Jamali, Abbas; Fotouhi, Fatemeh; Nargesabad, Reza Nasrollahi; Abdoli, Asghar

    2017-04-01

    Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research. In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin-1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation.Results/Key findings. Beclin-1 overexpression in the cells infected by virus induced autophagy to 26 %. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50 % infection) of replicating virus were measured at 24 and 48 h post-infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post-infection (P≤0.01), but it was not significantly different from the control at 48 h post-infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post-infection. Additionally, we showed that inhibition of autophagy using 3-methyladenine reduced viral replication. This study revealed that the virus (H1N1) titre was controlled in a time-dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production.

  18. Replication Capacity of Viruses from Acute Infection Drives HIV-1 Disease Progression.

    PubMed

    Selhorst, Philippe; Combrinck, Carina; Ndabambi, Nonkululeko; Ismail, Sherazaan D; Abrahams, Melissa-Rose; Lacerda, Miguel; Samsunder, Natasha; Garrett, Nigel; Abdool Karim, Quarraisha; Abdool Karim, Salim S; Williamson, Carolyn

    2017-04-15

    The viral genotype has been shown to play an important role in HIV pathogenesis following transmission. However, the viral phenotypic properties that contribute to disease progression remain unclear. Most studies have been limited to the evaluation of Gag function in the context of a recombinant virus backbone. Using this approach, important biological information may be lost, making the evaluation of viruses obtained during acute infection, representing the transmitted virus, a more biologically relevant model. Here, we evaluate the roles of viral infectivity and the replication capacity of viruses from acute infection in disease progression in women who seroconverted in the CAPRISA 004 tenofovir microbicide trial. We show that viral replication capacity, but not viral infectivity, correlates with the set point viral load (Spearman r = 0.346; P = 0.045) and that replication capacity (hazard ratio [HR] = 4.52; P = 0.01) can predict CD4 decline independently of the viral load (HR = 2.9; P = 0.004) or protective HLA alleles (HR = 0.61; P = 0.36). We further demonstrate that Gag-Pro is not the main driver of this association, suggesting that additional properties of the transmitted virus play a role in disease progression. Finally, we find that although viruses from the tenofovir arm were 2-fold less infectious, they replicated at rates similar to those of viruses from the placebo arm. This indicates that the use of tenofovir gel did not select for viral variants with higher replication capacity. Overall, this study supports a strong influence of the replication capacity in acute infection on disease progression, potentially driven by interaction of multiple genes rather than a dominant role of the major structural gene gagIMPORTANCE HIV disease progression is known to differ between individuals, and defining which fraction of this variation can be attributed to the virus is important both clinically and epidemiologically. In this study, we show that the replication

  19. Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

    PubMed

    Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

    2013-07-01

    Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

  20. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication.

    PubMed

    Wang, Gefei; Li, Rui; Jiang, Zhiwu; Gu, Liming; Chen, Yanxia; Dai, Jianping; Li, Kangsheng

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy.

  1. Large-scale purification of gp70 from Moloney murine leukemia virus.

    PubMed

    Pyle, S W; Chabot, D J; Miller, T L; Serabyn, S A; Bess, J W; Arthur, L O

    1991-05-01

    The external envelope glycoprotein, gp70, of the Moloney murine leukemia virus was extracted from NIH 3T3 cells utilizing the detergent n-octyl-beta-D-glycopyranoside. The extracted gp70 was sequentially purified utilizing lectin-affinity, anion-exchange, and molecular-exclusion chromatography techniques. Approximately 10 mg of gp70 was purified by this method and shown to be 95% homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of purified gp70 from Moloney murine leukemia virus was confirmed by amino acid analysis, amino-terminal sequencing, and immunoreactivity with a monoclonal antibody raised against gp70. The procedure is rapid, utilizes commercially available media, and can be used to purify large amounts of retroviral envelope glycoprotein from virus.

  2. An autophagy-independent role for LC3 in equine arteritis virus replication

    PubMed Central

    Monastyrska, Iryna; Ulasli, Mustafa; Rottier, Peter J.M.; Guan, Jun-Lin; Reggiori, Fulvio; de Haan, Cornelis A.M.

    2013-01-01

    Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus. Genome replication of EAV has been associated with modified intracellular membranes that are shaped into double-membrane vesicles (DMVs). We showed by immuno-electron microscopy that the DMVs induced in EAV-infected cells contain double-strand (ds)RNA molecules, presumed RNA replication intermediates, and are decorated with the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3). Replication of EAV, however, was not affected in autophagy-deficient cells lacking autophagy-related protein 7 (ATG7). Nevertheless, colocalization of DMVs and LC3 was still observed in these knockout cells, which only contain the nonlipidated form of LC3. Although autophagy is not required, depletion of LC3 markedly reduced the replication of EAV. EAV replication could be fully restored in these cells by expression of a nonlipidated form of LC3. These findings demonstrate an autophagy-independent role for LC3 in EAV replication. Together with the observation that EAV-induced DMVs are also positive for ER degradation-enhancing α-mannosidase-like 1 (EDEM1), our data suggested that this virus, similarly to the distantly-related mouse hepatitis coronavirus, hijacks the ER-derived membranes of EDEMosomes to ensure its efficient replication. PMID:23182945

  3. Grape Seed Extract Attenuates Hepatitis C Virus Replication and Virus-Induced Inflammation

    PubMed Central

    Chen, Wei-Chun; Tseng, Chin-Kai; Chen, Bing-Hung; Lin, Chun-Kuang; Lee, Jin-Ching

    2016-01-01

    Hepatitis C virus (HCV) infection is a causative factor leading to hepatocellular carcinoma due to chronic inflammation and cirrhosis. The aim of the study was first to explore the effects of grape seed extract (GSE) in HCV replication, and then to study mechanisms. The results indicated that a GSE treatment showed significant anti-HCV activity and suppressed HCV-elevated cyclooxygenase-2 (COX-2) expression. In contrast, exogenous COX-2 expression gradually attenuated antiviral effects of GSE, suggesting that GSE inhibited HCV replication by suppressing an aberrant COX-2 expression caused by HCV, which was correlated with the inactivation of IKK-regulated NF-κB and MAPK/ERK/JNK signaling pathways. In addition, GSE also attenuated HCV-induced inflammatory cytokine gene expression. Notably, a combined administration of GSE with interferon or other FDA-approved antiviral drugs revealed a synergistic anti-HCV effect. Collectively, these findings demonstrate the possibility of developing GSE as a dietary supplement to treat patients with a chronic HCV infection. PMID:28066241

  4. Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus.

    PubMed

    Hyodo, Kiwamu; Hashimoto, Kenji; Kuchitsu, Kazuyuki; Suzuki, Nobuhiro; Okuno, Tetsuro

    2017-02-14

    As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

  5. Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

    PubMed Central

    Hashimoto, Kenji; Kuchitsu, Kazuyuki; Suzuki, Nobuhiro; Okuno, Tetsuro

    2017-01-01

    As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant–virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication. PMID:28154139

  6. Bovine immunodeficiency virus and bovine leukemia virus and their mixed infection in Iranian Holstein cattle.

    PubMed

    Brujeni, Gholamreza Nikbakht; Poorbazargani, Taghi Taghi; Nadin-Davis, Susan; Tolooie, Mohammad; Barjesteh, Neda

    2010-10-04

    Bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) have worldwide distributions, but their prevalences in Iran are unknown. We investigated the presence of infections in Iranian Holstein cattle and determined changes in hematological values for infected animals. Nested PCR was used on blood samples from 143 animals Holstein cattle to detect proviral BIV and BLV gag sequences. Flow cytometric analysis was performed using monoclonal antibodies (mAbs) against CD4, CD8, and CD21 bovine T lymphocyte subsets. Proviral BIV and BLV gag sequences were detected in 20.3% and 17% of the animals, respectively. BIV-BLV confection was also detected in 4.2% of the study population but this was not statistically significant. Flow cytometric analysis showed that both BIV-infected cows and non-infected ones had CD4/CD8 ratios of 2.45 and 1.43, respectively, and this difference was significant. BLV infected and non-infected animals had no significant differences in their CD4/CD8 ratio. In comparison to non-infected cattle, those with both BIV and BLV had a significant decrease in their CD4/CD8 ratios (1.5 % vs. 2.3; P = 0.01). This is the first report of BIV and BLV infections in Iran. We found no evidence that infection with one agent predisposed an animal to infection with the other. BIV infection may have a role in decreasing T CD8 counts, but this may depend on the genetics of the cattle and virus strains involved.

  7. Human immunodeficiency virus evolution towards reduced replicative fitness in vivo and the development of AIDS

    PubMed Central

    Wodarz, Dominik; Levy, David N

    2007-01-01

    Human immunodeficiency virus (HIV) infection progresses to AIDS following an asymptomatic period during which the virus is thought to evolve towards increased fitness and pathogenicity. We show mathematically that progression to the strongest HIV-induced pathology requires evolution of the virus towards reduced replicative fitness in vivo. This counter-intuitive outcome can happen if multiple viruses co-infect the same cell frequently, which has been shown to occur in recent experiments. According to our model, in the absence of frequent co-infection, the less fit AIDS-inducing strains might never emerge. The frequency of co-infection can correlate with virus load, which in turn is determined by immune responses. Thus, at the beginning of infection when immunity is strong and virus load is low, co-infection is rare and pathogenic virus variants with reduced replicative fitness go extinct. At later stages of infection when immunity is less efficient and virus load is higher, co-infection occurs more frequently and pathogenic virus variants with reduced replicative fitness can emerge, resulting in T-cell depletion. In support of these notions, recent data indicate that pathogenic simian immunodeficiency virus (SIV) strains occurring late in the infection are less fit in specific in vitro experiments than those isolated at earlier stages. If co-infection is blocked, the model predicts the absence of any disease even if virus loads are high. We hypothesize that non-pathogenic SIV infection within its natural hosts, which is characterized by the absence of disease even in the presence of high virus loads, could be explained by a reduced occurrence of co-infection in this system. PMID:17666377

  8. An NS-segment exonic splicing enhancer regulates influenza A virus replication in mammalian cells

    PubMed Central

    Huang, Xiaofeng; Zheng, Min; Wang, Pui; Mok, Bobo Wing-Yee; Liu, Siwen; Lau, Siu-Ying; Chen, Pin; Liu, Yen-Chin; Liu, Honglian; Chen, Yixin; Song, Wenjun; Yuen, Kwok-Yung; Chen, Honglin

    2017-01-01

    Influenza virus utilizes host splicing machinery to process viral mRNAs expressed from both M and NS segments. Through genetic analysis and functional characterization, we here show that the NS segment of H7N9 virus contains a unique G540A substitution, located within a previously undefined exonic splicing enhancer (ESE) motif present in the NEP mRNA of influenza A viruses. G540A supports virus replication in mammalian cells while retaining replication ability in avian cells. Host splicing regulator, SF2, interacts with this ESE to regulate splicing of NEP/NS1 mRNA and G540A substitution affects SF2–ESE interaction. The NS1 protein directly interacts with SF2 in the nucleus and modulates splicing of NS mRNAs during virus replication. We demonstrate that splicing of NEP/NS1 mRNA is regulated through a cis NEP-ESE motif and suggest a unique NEP-ESE may contribute to provide H7N9 virus with the ability to both circulate efficiently in avian hosts and replicate in mammalian cells. PMID:28323816

  9. No evidence of African swine fever virus replication in hard ticks.

    PubMed

    de Carvalho Ferreira, Helena C; Tudela Zúquete, Sara; Wijnveld, Michiel; Weesendorp, Eefke; Jongejan, Frans; Stegeman, Arjan; Loeffen, Willie L A

    2014-09-01

    African swine fever (ASF) is caused by African swine fever virus (ASFV), a tick-borne DNA virus. Soft ticks of the genus Ornithodoros are the only biological vectors of ASFV recognized so far. Although other hard ticks have been tested for vector competence, two commonly found tick species in Europe, Ixodes ricinus and Dermacentor reticulatus, have not been assessed for their vector competence for ASFV. In this study, we aimed to determine whether virus replication can occur in any of these two hard tick species (I. ricinus and/or D. reticulatus), in comparison with O. moubata (the confirmed vector), after feeding them blood containing different ASFV isolates using an improved in vitro system. DNA quantities of ASFV in these infected hard ticks were measured systematically, for 6 weeks in I. ricinus, and up to 8 weeks in D. reticulatus, and the results were compared to those obtained from O. moubata. There was evidence of virus replication in the O. moubata ticks. However, there was no evidence of virus replication in I. ricinus or D. reticulatus, even though viral DNA could be detected for up to 8 weeks after feeding in some cases. This study presents the first results on the possible vector competence of European hard (ixodid) ticks for ASFV, in a validated in vitro feeding setup. In conclusion, given the lack of evidence for virus replication under in vitro conditions, D. reticulatus and I. ricinus are unlikely to be relevant biological vectors of ASFV.

  10. Nigericin is a potent inhibitor of the early stage of vaccinia virus replication.

    PubMed

    Myskiw, Chad; Piper, Jessica; Huzarewich, Rhiannon; Booth, Tim F; Cao, Jingxin; He, Runtao

    2010-12-01

    Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

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

    PubMed Central

    Villarreal, Luis P.; DeFilippis, Victor R.

    2000-01-01

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

  12. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication.

    PubMed Central

    Engelman, A; Englund, G; Orenstein, J M; Martin, M A; Craigie, R

    1995-01-01

    The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes. PMID:7535863

  13. Effects of chemical carcinogens on the susceptibility of C57BL/10 and (SJL/J x C57BL/10) F/sub 1/ hybrids to Friend leukemia virus

    SciTech Connect

    Raikow, R.B.; OKunewick, J.P.; Magliere, K.C.; Brozovich, B.J.; Seeman, P.R.

    1980-01-01

    Under normal circumstances cells of C57BL/10 mice are resistant to infection by Friend leukemia virus (FLV). Pre-treatment by chemical carcinogens does not affect the susceptibility of C57BL/10 mice to FLV leukemogenesis. However, immunosuppression by cyclophosphamide or a congenitally athymic condition allows the replication of the LLV component of FLV to take place in these mice. F1 hybrids between C57BL/10 and SJL/J mice are also resistant to virus, although about twenty percent of these hybrids develop leukemia after massive doses of FLV. Unexpectedly, the F1 hybrid with the virus-sensitive SJL/J mother was more resistant than the F1 hybrid from the reciprocal cross. Pre-treatment of the F1 hybrid or SJL/J mice with chemical carcinogens, such as methyl methane sulfonate and benzo(a)pyrene, but not cyclophosphamide, increased the incidence of leukemia with a peak of increased susceptibility developing at a specific time after treatment. A chemical carcinogen-caused depression of the viability of hematopoietic cells in the spleen, which cells are the major target for FLV oncogenesis, was also temporally related with the increase in susceptibility to the virus. Our data correlated with information on the alleles known to affect resistance to murine leukemia viruses.

  14. VP40 Octamers Are Essential for Ebola Virus Replication

    PubMed Central

    Hoenen, Thomas; Volchkov, Viktor; Kolesnikova, Larissa; Mittler, Eva; Timmins, Joanna; Ottmann, Michelle; Reynard, Olivier; Becker, Stephan; Weissenhorn, Winfried

    2005-01-01

    Matrix protein VP40 of Ebola virus is essential for virus assembly and budding. Monomeric VP40 can oligomerize in vitro into RNA binding octamers, and the crystal structure of octameric VP40 has revealed that residues Phe125 and Arg134 are the most important residues for the coordination of a short single-stranded RNA. Here we show that full-length wild-type VP40 octamers bind RNA upon HEK 293 cell expression. While the Phe125-to-Ala mutation resulted in reduced RNA binding, the Arg134-to-Ala mutation completely abolished RNA binding and thus octamer formation. The absence of octamer formation, however, does not affect virus-like particle (VLP) formation, as the VLPs generated from the expression of wild-type VP40 and mutated VP40 in HEK 293 cells showed similar morphology and abundance and no significant difference in size. These results strongly indicate that octameric VP40 is dispensable for VLP formation. The cellular localization of mutant VP40 was different from that of wild-type VP40. While wild-type VP40 was present in small patches predominantly at the plasma membrane, the octamer-negative mutants were found in larger aggregates at the periphery of the cell and in the perinuclear region. We next introduced the Arg134-to-Ala and/or the Phe125-to-Ala mutation into the Ebola virus genome. Recombinant wild-type virus and virus expressing the VP40 Phe125-to-Ala mutation were both rescued. In contrast, no recombinant virus expressing the VP40 Arg134-to-Ala mutation could be recovered. These results suggest that RNA binding of VP40 and therefore octamer formation are essential for the Ebola virus life cycle. PMID:15650213

  15. The human immunodeficiency virus type 1 envelope confers higher rates of replicative fitness to perinatally transmitted viruses than to nontransmitted viruses.

    PubMed

    Kong, Xiaohong; West, John T; Zhang, Hong; Shea, Danielle M; M'soka, Tendai J; Wood, Charles

    2008-12-01

    Selection of a minor viral genotype during perinatal transmission of human Immunodeficiency virus type 1 (HIV-1) has been observed, but there is a lack of information on the correlation of the restrictive transmission with biological properties of the virus, such as replicative fitness. Recombinant viruses expressing the enhanced green fluorescent protein or the Discosoma sp. red fluorescent (DsRed2) protein carrying the V1 to V5 regions of env from seven mother-infant pairs (MIPs) infected by subtype C HIV-1 were constructed, and competition assays were carried out to compare the fitness between the transmitted and nontransmitted viruses. Flow cytometry was used to quantify the frequency of infected cells, and the replicative fitness was determined based on a calculation that takes into account replication of competing viruses in a single infection versus dual infections. Transmitted viruses from five MIPs with the mothers chronically infected showed a restrictive env genotype, and all the recombinant viruses carrying the infants' Env had higher replicative fitness than those carrying the Env from the mothers. This growth fitness is lineage specific and can be observed only within the same MIP. In contrast, in two MIPs where the mothers had undergone recent acute infection, the viral Env sequences were similar between the mothers and infants and showed no further restriction in quasispecies during perinatal transmission. The recombinant viruses carrying the Env from the infants' viruses also showed replication fitness similar to those carrying the mothers' Env proteins. Our results suggest that newly transmitted viruses from chronically infected mothers have been selected to have higher replicative fitness to favor transmission, and this advantage is conferred by the V1 to V5 region of Env of the transmitted viruses. This finding has important implications for vaccine design or development of strategies to prevent HIV-1 transmission.

  16. High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo.

    PubMed

    Lloyd, Sarah B; Lichtfuss, Marit; Amarasena, Thakshila H; Alcantara, Sheilajen; De Rose, Robert; Tachedjian, Gilda; Alinejad-Rokny, Hamid; Venturi, Vanessa; Davenport, Miles P; Winnall, Wendy R; Kent, Stephen J

    2016-05-01

    The low fidelity of HIV replication facilitates immune and drug escape. Some reverse transcriptase (RT) inhibitor drug-resistance mutations increase RT fidelity in biochemical assays but their effect during viral replication is unclear. We investigated the effect of RT mutations K65R, Q151N and V148I on SIV replication and fidelity in vitro, along with SIV replication in pigtailed macaques. SIVmac239-K65R and SIVmac239-V148I viruses had reduced replication capacity compared to wild-type SIVmac239. Direct virus competition assays demonstrated a rank order of wild-type>K65R>V148I mutants in terms of viral fitness. In single round in vitro-replication assays, SIVmac239-K65R demonstrated significantly higher fidelity than wild-type, and rapidly reverted to wild-type following infection of macaques. In contrast, SIVmac239-Q151N was replication incompetent in vitro and in pigtailed macaques. Thus, we showed that RT mutants, and specifically the common K65R drug-resistance mutation, had impaired replication capacity and higher fidelity. These results have implications for the pathogenesis of drug-resistant HIV.

  17. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    PubMed Central

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-01-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus. PMID:8627709

  18. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    PubMed

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-03-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus.

  19. Feline Leukemia Virus Infection Requires a Post-Receptor Binding Envelope-Dependent Cellular Component▿

    PubMed Central

    Hussain, Naveen; Thickett, Kelly R.; Na, Hong; Leung, Cherry; Tailor, Chetankumar S.

    2011-01-01

    Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (101 to 102 CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (104 to 106 CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells. PMID:21917946

  20. Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

    PubMed

    Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

    2011-12-01

    Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

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

    SciTech Connect

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

    2014-11-15

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

  2. Delayed IFN response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells.

    PubMed

    Takamatsu, Yuki; Uchida, Leo; Morita, Kouichi

    2015-08-01

    West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important causes of human encephalitis cases, which result in a high mortality ratio and neurological sequelae after recovery. Understanding the mechanism of neuropathogenicity in these viral infections is important for the development of specific antiviral therapy. Here, we focused on human-derived neuronal and glial cells to understand the cellular responses against WNV and JEV infection. It was demonstrated that early IFN-β induction regulated virus replication in glioblastoma tbl98G cells, whereas delayed IFN-β induction resulted in efficient virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of viral dsRNA in the intracellular membrane resulted in the delayed IFN response in SK-N-SH cells. These results, which showed different IFN responses between human neuronal and glial cells after WNV or JEV infection, are expected to contribute to our understanding of the molecular mechanisms for neuropathology in these viral infections.

  3. Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference

    PubMed Central

    Coburn, Glen A.; Cullen, Bryan R.

    2002-01-01

    Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle. PMID:12186906

  4. Evaluation of the minimal replication time of Cauliflower mosaic virus in different hosts

    SciTech Connect

    Khelifa, Mounia; Masse, Delphine; Blanc, Stephane; Drucker, Martin

    2010-01-20

    Though the duration of a single round of replication is an important biological parameter, it has been determined for only few viruses. Here, this parameter was determined for Cauliflower mosaic virus (CaMV) in transfected protoplasts from different hosts: the highly susceptible Arabidopsis and turnip, and Nicotiana benthamiana, where CaMV accumulates only slowly. Four methods of differing sensitivity were employed: labelling of (1) progeny DNA and (2) capsid protein, (3) immunocapture PCR,, and (4) progeny-specific PCR. The first progeny virus was detected about 21 h after transfection. This value was confirmed by all methods, indicating that our estimate was not biased by the sensitivity of the detection method, and approximated the actual time required for one round of CaMV replication. Unexpectedly, the replication kinetics were similar in the three hosts; suggesting that slow accumulation of CaMV in Nicotiana plants is determined by non-optimal interactions in other steps of the infection cycle.

  5. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    PubMed Central

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K.; James, John; Prescott, Alan; Haga, Ismar R.; Beard, Philippa M.

    2015-01-01

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. PMID:25462347

  6. Replication of Chinese hamster embryo cells transformed by temperature-sensitive T-antigen mutants of simian virus 40.

    PubMed Central

    Robinson, C C; Swartzendruber, D E; Lehman, J M

    1980-01-01

    Chinese hamster embryo cells transformed by simian virus 40 temperature-sensitive T-antigen mutants replicated when confluent at 40.5 degrees C, regardless of the selection method, selection temperature, or virus strain used. Images PMID:6251272

  7. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication

    PubMed Central

    Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo; Yamada, Kenneth M.; Dhawan, Subhash

    2017-01-01

    We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of > 90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retarding ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection. PMID:28068513

  8. Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes.

    PubMed

    Yao, Fangke; Chen, Yun; Shi, Jintong; Ming, Ke; Liu, Jiaguo; Xiong, Wen; Song, Meiyun; Du, Hongxu; Wang, Yixuan; Zhang, Shuaibin; Wu, Yi; Wang, Deyun; Hu, Yuanliang

    2016-04-01

    Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection, and the virus particles with size of about 50 nm including more than 20 nm of vacuum drying gold were observed on the infected cells surface. What's more, the replication lasted around 13 h after the early protein synthesis for about 5h, and the release of DHAV-1 was in steady state after 32 h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies.

  9. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication.

    PubMed

    Huang, Hanxia; Falgout, Barry; Takeda, Kazuyo; Yamada, Kenneth M; Dhawan, Subhash

    2017-03-01

    We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of >90% in ZIKV replication, with little toxicity to infected cells. Silencing expression of HO-1 or its upstream regulatory gene, nuclear factor erythroid-related factor 2 (Nrf2), attenuated hemin-induced suppression of ZIKV infection, suggesting an important role for induction of these intracellular mediators in retarding ZIKV replication. The inverse correlation between hemin-induced HO-1 levels and ZIKV replication provides a potentially useful therapeutic modality based on stimulation of an innate cellular response against Zika virus infection.

  10. Zika Virus RNA Replication and Persistence in Brain and Placental Tissue

    PubMed Central

    Rabeneck, Demi B.; Martines, Roosecelis B.; Reagan-Steiner, Sarah; Ermias, Yokabed; Estetter, Lindsey B.C.; Suzuki, Tadaki; Ritter, Jana; Keating, M. Kelly; Hale, Gillian; Gary, Joy; Muehlenbachs, Atis; Lambert, Amy; Lanciotti, Robert; Oduyebo, Titilope; Meaney-Delman, Dana; Bolaños, Fernando; Saad, Edgar Alberto Parra; Shieh, Wun-Ju; Zaki, Sherif R.

    2017-01-01

    Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections. PMID:27959260

  11. Involvement of the skin during bluetongue virus infection and replication in the ruminant host

    PubMed Central

    2012-01-01

    Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne (“arbovirus”) and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between “virus replication” and “virus presence”.BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed. PMID:22546071

  12. Chronic hepatitis E virus infection in a patient with leukemia and elevated transaminases: a case report

    PubMed Central

    2012-01-01

    Introduction Acute hepatitis E virus infection may cause mild, self-limiting hepatitis, either as epidemic outbreaks or sporadic cases, the latter of which have been reported in industrialized countries. Chronic infections are uncommon and have been reported in immunosuppressed patients, patients with human immunodeficiency virus infection, and patients with hematological malignancies. Case presentation A 46-year-old Caucasian man was admitted to the gastroenterology clinic with a history of increasing transaminases, persistent exhaustion, and occasional right-side abdominal pain over the course of a 6-month period. B-cell chronic lymphocytic leukemia had been diagnosed several years earlier, and the patient was treated with rituximab, pentostatin, and cyclophosphamide. A diagnostic workup ruled out autoimmune and metabolic liver disease, hepatitis A-C, and herpes virus infection. A physical examination revealed enlarged axillary lymph nodes. The results of an abdominal ultrasound examination were otherwise unremarkable. Hepatitis E virus infection was diagnosed by detection of hepatitis E virus-specific antibodies. Blood samples were positive for hepatitis E virus ribonucleic acid with high viral loads for at least 8 months, demonstrating a rare chronic hepatitis E virus infection. Sequencing and phylogenetic analysis revealed hepatitis E virus genotype 3c with homologies to other European isolates from humans and swine, indicating an autochthonous infection. Conclusions Usually, hepatitis E virus infection appears as an acute infection; rare chronic infections have been reported for transplant patients, patients with human immunodeficiency virus, and patients with hematological malignancies. The chronic nature of hepatitis E infection in our patient was most likely induced by the immunosuppressive B-cell chronic lymphocytic leukemia treatment. The differential diagnosis in patients with unexplained hepatitis should include hepatitis E virus infection, and

  13. The citrus flavanone naringenin impairs dengue virus replication in human cells.

    PubMed

    Frabasile, Sandra; Koishi, Andrea Cristine; Kuczera, Diogo; Silveira, Guilherme Ferreira; Verri, Waldiceu Aparecido; Duarte Dos Santos, Claudia Nunes; Bordignon, Juliano

    2017-02-03

    Dengue is one of the most significant health problems in tropical and sub-tropical regions throughout the world. Nearly 390 million cases are reported each year. Although a vaccine was recently approved in certain countries, an anti-dengue virus drug is still needed. Fruits and vegetables may be sources of compounds with medicinal properties, such as flavonoids. This study demonstrates the anti-dengue virus activity of the citrus flavanone naringenin, a class of flavonoid. Naringenin prevented infection with four dengue virus serotypes in Huh7.5 cells. Additionally, experiments employing subgenomic RepDV-1 and RepDV-3 replicon systems confirmed the ability of naringenin to inhibit dengue virus replication. Antiviral activity was observed even when naringenin was used to treat Huh7.5 cells 24 h after dengue virus exposure. Finally, naringenin anti-dengue virus activity was demonstrated in primary human monocytes infected with dengue virus sertoype-4, supporting the potential use of naringenin to control dengue virus replication. In conclusion, naringenin is a suitable candidate molecule for the development of specific dengue virus treatments.

  14. The citrus flavanone naringenin impairs dengue virus replication in human cells

    PubMed Central

    Frabasile, Sandra; Koishi, Andrea Cristine; Kuczera, Diogo; Silveira, Guilherme Ferreira; Verri, Waldiceu Aparecido; Duarte dos Santos, Claudia Nunes; Bordignon, Juliano

    2017-01-01

    Dengue is one of the most significant health problems in tropical and sub-tropical regions throughout the world. Nearly 390 million cases are reported each year. Although a vaccine was recently approved in certain countries, an anti-dengue virus drug is still needed. Fruits and vegetables may be sources of compounds with medicinal properties, such as flavonoids. This study demonstrates the anti-dengue virus activity of the citrus flavanone naringenin, a class of flavonoid. Naringenin prevented infection with four dengue virus serotypes in Huh7.5 cells. Additionally, experiments employing subgenomic RepDV-1 and RepDV-3 replicon systems confirmed the ability of naringenin to inhibit dengue virus replication. Antiviral activity was observed even when naringenin was used to treat Huh7.5 cells 24 h after dengue virus exposure. Finally, naringenin anti-dengue virus activity was demonstrated in primary human monocytes infected with dengue virus sertoype-4, supporting the potential use of naringenin to control dengue virus replication. In conclusion, naringenin is a suitable candidate molecule for the development of specific dengue virus treatments. PMID:28157234

  15. Weak bases affect late stages of Mayaro virus replication cycle in vertebrate cells.

    PubMed

    Ferreira, D F; Santo, M P; Rebello, M A; Rebello, M C

    2000-04-01

    This paper describes the effect of two weak bases (ammonium chloride and chloroquine) on the morphogenesis of Mayaro virus. When Mayaro virus-infected TC7 (monkey kidney) cells were treated with these agents it was observed that weak bases caused a significant reduction in virus yield. Also, cellular protein synthesis, which is inhibited by Mayaro virus infection, recovered to nearly normal levels. However, the synthesis of Mayaro virus proteins was affected. These phenomena were dose-dependent. The process of Mayaro virus infection in vertebrate cells is very rapid. Virus precursors are not observed in cell cytoplasm and budding through the plasma membrane seems to be the only way of virus release. Electron microscopy of cells infected with Mayaro virus and treated with weak bases revealed an accumulation of virus structures in cell cytoplasm. The study also noted an inhibition of budding through the plasma membrane and the appearance of virus particles inside intracytoplasmic vacuoles. These observations indicate an impairment at the final stages of the virus replication cycle.

  16. Increased Replicative Fitness Can Lead to Decreased Drug Sensitivity of Hepatitis C Virus

    PubMed Central

    Sheldon, Julie; Beach, Nathan M.; Moreno, Elena; Gallego, Isabel; Piñeiro, David; Martínez-Salas, Encarnación; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.

    2014-01-01

    ABSTRACT Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-α), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV. IMPORTANCE Viral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness. PMID:25122776

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

    PubMed Central

    Yamaguchi, M; DePamphilis, M L

    1986-01-01

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

  18. Swine alveolar macrophage cell model allows optimal replication of influenza A viruses regardless of their origin.

    PubMed

    Kasloff, Samantha B; Weingartl, Hana M

    2016-03-01

    The importance of pigs in interspecies transmission of influenza A viruses has been repeatedly demonstrated over the last century. Eleven influenza A viruses from avian, human and swine hosts were evaluated for replication phenotypes at three physiologically relevant temperatures (41°C, 37°C, 33°C) in an immortalized swine pulmonary alveolar macrophage cell line (IPAM 3D4/31) to determine whether this system would allow for their efficient replication. All isolates replicated well in IPAMs at 37°C while clear distinctions were observed at 41°C and 33°C, correlating to species of origin of the PB2, reflected in distinct amino acid residue profiles rather than in one particular PB2 residue. A strong TNF-α response was induced by some mammalian but not avian IAVs, while other selected cytokines remained below detection levels. Porcine IPAMs represent a natural host cell model for influenza virus replication where the only condition requiring modification for optimal IAV replication, regardless of virus origin.

  19. Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

    PubMed

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-10-21

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

  20. Hsp90 inhibitors reduce influenza virus replication in cell culture

    SciTech Connect

    Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin Brownlee, George

    2008-08-01

    The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.

  1. Restricted Semliki Forest virus replication in perforin and Fas-ligand double-deficient mice.

    PubMed

    Alsharifi, Mohammed; Lobigs, Mario; Bettadapura, Jayaram; Koskinen, Aulikki; Müllbacher, Arno

    2008-08-01

    Previously, we have shown that mice defective in granule exocytosis and/or Fas.L/Fas-mediated cytolytic pathways are significantly more resistant to alphavirus, Semliki Forest virus (SFV), infection compared with wild-type mice. Here, we evaluated SFV replication in different tissues of mice defective in both cytolytic pathways (perf(-/-)xgld) relative to that in wild-type counterparts and found that viral replication in perf(-/-)xgld mice is remarkably restricted. Although the mechanism responsible for this observation is yet to be established, the lower virus titres found in these mice indicate that the role of cytolytic effector molecules in antiviral immunity needs to be re-evaluated.

  2. Definition of herpes simplex virus type 1 helper activities for adeno-associated virus early replication events.

    PubMed

    Alazard-Dany, Nathalie; Nicolas, Armel; Ploquin, Aurélie; Strasser, Regina; Greco, Anna; Epstein, Alberto L; Fraefel, Cornel; Salvetti, Anna

    2009-03-01

    The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1-induced AAV replication.

  3. Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

    PubMed

    Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

    2015-07-01

    The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication.

  4. Effect of Temperature on Replication of Epizootic Hemorrhagic Disease Viruses in Culicoides sonorensis (Diptera: Ceratopogonidae).

    PubMed

    Ruder, Mark G; Stallknecht, David E; Howerth, Elizabeth W; Carter, Deborah L; Pfannenstiel, Robert S; Allison, Andrew B; Mead, Daniel G

    2015-09-01

    Replication of arboviruses, including orbiviruses, within the vector has been shown to be temperature dependent. Cooler ambient temperatures slow virus replication in arthropod vectors, whereas viruses replicate faster and to higher titers at warmer ambient temperatures. Previous research with epizootic hemorrhagic disease virus (EHDV) serotype 1 demonstrated that higher temperatures were associated with shorter extrinsic incubation periods in Culicoides sonorensis Wirth & Jones, a confirmed vector of EHDV in North America. To further our understanding of the effect of temperature on replication of EHDV within the vector, C. sonorensis were experimentally infected with one of three EHDV strains representing three serotypes (1, 2, and 7). Midges were fed defibrinated white-tailed deer (Odocoileus virginianus) blood spiked with EHDV (≥10(6.5) TCID(50)/ml) through a parafilm membrane using an artificial feeding device and were then held at 20, 25, or 30°C. In addition to this in vitro method, a white-tailed deer experimentally infected with EHDV-7 was used to provide an infectious bloodmeal to determine if the results were comparable with those from the in vitro feeding method. Whole midges were processed for virus isolation and titration at regular intervals following feeding; midges with ≥10(2.7) TCID(50) were considered potentially competent to transmit virus. The virus recovery rates were high throughout the study and all three viruses replicated within C. sonorensis to high titer (≥ 10(2.7) TCID(50)/midge). Across all virus strains, the time to detection of potentially competent midges decreased with increasing temperature: 12-16 d postfeeding (dpf) at 20°C, 4-6 dpf at 25°C, and 2-4 dpf at 30°C. Significant differences in replication of the three viruses in C. sonorensis were observed, with EHDV-2 replicating to a high titer in a smaller proportion of midges and with lower peak titers. The findings are consistent with previous studies of related

  5. A new look at the origins of gibbon ape leukemia virus.

    PubMed

    McKee, J; Clark, N; Shapter, F; Simmons, G

    2017-04-01

    Is the origin of gibbon ape leukemia virus (GALV) human after all? When GALV was discovered and found to cause neoplastic disease in gibbons, it stimulated a great deal of research including investigations into the origins of this virus. A number of publications have suggested that the GALV progenitor was a retrovirus present in one of several species of South East Asian rodents that had close contact with captive gibbons. However, there are no published retroviral sequences from any South East Asian species to support this view. Here we present an alternative hypothesis that the origin of GALV is a virus closely related to Melomys burtoni retrovirus, and that this virus infected human patients in Papua New Guinea from whom biological material was obtained or in some way contaminated these samples. This material we propose contained infectious MbRV-related virus that was then unwittingly introduced into gibbons which subsequently developed GALV infections.

  6. Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA.

    PubMed Central

    Brandsma, J L; Xiao, W

    1993-01-01

    The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication. Images PMID:8380092

  7. Protection Against Dengue Virus by Non-Replicating and Live Attenuated Vaccines Used Together in a Prime Boost Vaccination Strategy

    DTIC Science & Technology

    2010-01-01

    Protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy Monika Simmons a...Dengue DNA Punfied inacdvared virus Uvc artenuatcd virus Jlnmc boost A new vaccination strategy for dengue virus (DENV) was eval uated in rhesus...region (TDNA) then boosting 2 months l,ltcr with a tetravalent live aucnuated virus (TLAV) vaccine . Both vaccine combinations elicited virus

  8. Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus.

    PubMed

    Dorobantu, Cristina M; Albulescu, Lucian; Harak, Christian; Feng, Qian; van Kampen, Mirjam; Strating, Jeroen R P M; Gorbalenya, Alexander E; Lohmann, Volker; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2015-09-01

    Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid

  9. Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus

    PubMed Central

    Dorobantu, Cristina M.; Albulescu, Lucian; Harak, Christian; Feng, Qian; van Kampen, Mirjam; Strating, Jeroen R. P. M.; Gorbalenya, Alexander E.; Lohmann, Volker

    2015-01-01

    Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid

  10. Ring Expanded Nucleoside Analogues Inhibit RNA Helicase and Intracellular Human Immunodeficiency virus type 1 Replication

    PubMed Central

    Yedavalli, Venkat S.R.K; Zhang, Ning; Cai, Hongyi; Zhang, Peng; Starost, Matthew F.; Hosmane, Ramachandra S.; Jeang, Kuan-Teh

    2008-01-01

    A series of ring expanded nucleoside (REN) analogues were synthesized and screened for inhibition of cellular RNA helicase activity and human immunodeficiency virus type 1 (HIV-1) replication. We identified two compounds 1 and 2 that inhibited the ATP dependent activity of human RNA helicase DDX3. Compounds 1 and 2 also suppressed HIV-1 replication in T cells and monocyte-derived macrophages. Neither compound at therapeutic doses was significantly toxic in ex vivo cell culture or in vivo in mice. Our findings provide proof-of-concept that a cellular factor, an RNA helicase, could be targeted for inhibiting HIV-1 replication. PMID:18680273

  11. Hepatitis B virus: pathogenesis, viral intermediates, and viral replication.

    PubMed

    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).

  12. Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes

    PubMed Central

    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

  13. Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes.

    PubMed

    Dembowski, Jill A; Dremel, Sarah E; DeLuca, Neal 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.

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

    PubMed Central

    Smale, S T; Tjian, R

    1986-01-01

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

  15. Polyamine biosynthesis and the replication of turnip yellow mosaic virus

    SciTech Connect

    Balint, R.F.

    1984-01-01

    Turnip yellow mosaic virus (TYMV) contains large amounts of nonexchangeable spermidine and induces an accumulation of spermidine in infected Chinese cabbage. By seven days after inoculation, a majority of protoplasts isolated from newly-emerging leaves stain with fluorescent antibody to the virus. These protoplasts contain 1-2 x 10/sup 6/ virions per cell and continue to produce virus in culture for at least 48 hours. (/sup 14/C)-Spermidine (10 ..mu..M) was taken up by these cells in amounts comparable to the original endogenous pool within 24 hours. However, the spermidine content of the cell was only marginally affected, implying considerable regulation of the endogenous pool(s). Putrescine and spermine were major products of the metabolism of exogenous spermidine. Radioactivity from exogenous (/sup 14/C)-spermidine was also readily incorporated into the nucleic acid-containing component of the virus, where it appeared as both spermidine and spermine. Thus, newly-formed virions contained predominantly newly-synthesized spermidine and spermine. However, inhibition of spermidine synthesis by dicyclohexylamine (DCHA) led to incorporation of pre-existing spermidine and increased amounts of spermine into newly-formed virions. The latter results were tested and confirmed in a second cellular system, consisting of health protoplasts infected with TYMC in vitro.

  16. [Phosphoramidate derivatives of acyclovir--herpes virus replication inhibitors].

    PubMed

    Zakirova, N F; Shipitsyn, A V; Ias'ko, M V; Kochetkov, S N

    2011-01-01

    A number of new phosphoramidates of acyclovir--compounds of interest as anti-virals against resistant strains of virus herpes was synthesized. Several methods of synthesis of these compounds were suggested. Optimal method appeared to be the obtaining of phosphoramidates through the phosphomonocloride with its subsequent treatment with various amines. Two compounds have shown moderate activity against HSV-1.

  17. Ludwik Gross, Sarah Stewart, and the 1950s discoveries of Gross murine leukemia virus and polyoma virus.

    PubMed

    Morgan, Gregory J

    2014-12-01

    The Polish-American scientist Ludwik Gross made two important discoveries in the early 1950s. He showed that two viruses - murine leukemia virus and parotid tumor virus - could cause cancer when they were injected into susceptible animals. At first, Gross's discoveries were greeted with skepticism: it seemed implausible that viruses could cause a disease as complex as cancer. Inspired by Gross's initial experiments, similar results were obtained by Sarah Stewart and Bernice Eddy who later renamed the parotid tumor virus SE polyoma virus after finding it could cause many different types of tumors in mice, hamsters, and rats. Eventually the "SE" was dropped and virologists adopted the name "polyoma virus." After Gross's work was published, additional viruses capable of causing solid tumors or blood-borne tumors in mice were described by Arnold Graffi, Charlotte Friend, John Moloney and others. By 1961, sufficient data had been accumulated for Gross to confidently publish an extensive monograph--Oncogenic Viruses--the first history of tumor virology, which became a standard reference work and marked the emergence of tumor virology as a distinct, legitimate field of study. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Equine infectious anemia virus replication is upregulated during differentiation of blood monocytes from acutely infected horses.

    PubMed Central

    Sellon, D C; Walker, K M; Russell, K E; Perry, S T; Covington, P; Fuller, F J

    1996-01-01

    Equine infectious anemia virus is a lentivirus that replicates in mature tissue macrophages of horses. Ponies were infected with equine infectious anemia virus. During febrile episodes, proviral DNA was detectable, but viral mRNA was not detectable. As cultured blood monocytes from these ponies differentiated into macrophages, viral expression was upregulated. In situ hybridization confirmed that viral transcription occurred in mature macrophages. PMID:8523576

  19. Calcium spirulan, an inhibitor of enveloped virus replication, from a blue-green alga Spirulina platensis.

    PubMed

    Hayashi, T; Hayashi, K; Maeda, M; Kojima, I

    1996-01-01

    Bioactivity-directed fractionation of a hot H2O extract from a blue-green alga Spirulina platensis led to the isolation of a novel sulfated polysaccharide named calcium spirulan (Ca-SP) as an antiviral principle. This polysaccharide was composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, galacturonic acid, sulfate, and calcium. Ca-SP was found to inhibit the replication of several enveloped viruses, including Herpes simplex virus type 1, human cytomegalovirus, measles virus, mumps virus, influenza A virus, and HIV-1. It was revealed that Ca-SP selectively inhibited the penetration of virus into host cells. Retention of molecular conformation by chelation of calcium ion with sulfate groups was suggested to be indispensable to its antiviral effect.

  20. Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

    PubMed Central

    Lin, Chun-Kuang; Tseng, Chin-Kai; Wu, Yu-Hsuan; Liaw, Chih-Chuang; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Lee, Jin-Ching

    2017-01-01

    Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E2 (PGE2) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection. PMID:28317866

  1. High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication.

    PubMed

    Fu, Xinping; Wang, Hua; Zhang, Xiaoliu

    2002-06-01

    Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

  2. Human cytomegalovirus renders cells non-permissive for replication of herpes simplex viruses

    SciTech Connect

    Cockley, K.D.

    1988-01-01

    The herpes simplex virus (HSV) genome during production infection in vitro may be subject to negative regulation which results in modification of the cascade of expression of herpes virus macromolecular synthesis leading to establishment of HSV latency. In the present study, human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of HSV type-1 (HSV-1). A delay in HSV replication of 15 hr as well as a consistent, almost 1000-fold inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 hr after superinfection were observed compared with controls infected with HSV alone. HSV type-2 (HSV-2) replication was similarly inhibited in HCMV-infected HEL cells. Prior ultraviolet-irradiation (UV) of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HCMV deoxyribonucleic acid (DNA) negative temperature-sensitive (ts) mutants inhibited HSV replications as efficiently as wild-type (wt) HCMV at the non-permissive temperature. Evidence for penetration and replication of superinfecting HSV into HCMV-infected cells was provided by blot hybridization of HSV DNA synthesized in HSV-superinfected cell cultures and by cesium chloride density gradient analysis of ({sup 3}H)-labeled HSV-1-superinfected cells.

  3. Analysis of Bovine Leukemia Virus Gag Membrane Targeting and Late Domain Function

    PubMed Central

    Wang, Huating; Norris, Kendra M.; Mansky, Louis M.

    2002-01-01

    Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain). PMID:12134053

  4. The E89K Mutation in the Matrix Protein of the Measles Virus Affects In Vitro Cell Death and Virus Replication Efficiency in Human PBMC

    PubMed Central

    Dong, Jianbao; Zhu, Wei; Saito, Akatsuki; Goto, Yoshitaka; Iwata, Hiroyuki; Haga, Takeshi

    2012-01-01

    Matrix protein is known to have an important role in the process of virus assembly and virion release during measles virus replication. In the present in vitro study, a single mutation of E89K in the matrix protein was shown to affect cell death and virus replication efficiency in human PBMC. One strain with this mutation caused less cell death than the parental virus, and possessed high virus replication efficiency. Moreover, by Annexin V-FITC staining, polycaspase FLICA staining, and double labeling with poly-caspase FLICA and the Hoechst stain, the cell death seen was shown to be apoptosis. PMID:22715352

  5. Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro.

    PubMed

    Ocazionez, Raquel Elvira; Meneses, Rocio; Torres, Flor Angela; Stashenko, Elena

    2010-05-01

    The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37 masculineC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 microg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 microg/mL and between 1.9-33.7 microg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

  6. Adaptive strategies of the influenza virus polymerase for replication in humans.

    PubMed

    Mehle, Andrew; Doudna, Jennifer A

    2009-12-15

    Transmission of influenza viruses into the human population requires surmounting barriers to cross-species infection. Changes in the influenza polymerase overcome one such barrier. Viruses isolated from birds generally contain polymerases with the avian-signature glutamic acid at amino acid 627 in the PB2 subunit. These polymerases display restricted activity in human cells. An adaptive change in this residue from glutamic acid to the human-signature lysine confers high levels of polymerase activity in human cells. This mutation permits escape from a species-specific restriction factor that targets polymerases from avian viruses. A 2009 swine-origin H1N1 influenza A virus recently established a pandemic infection in humans, even though the virus encodes a PB2 with the restrictive glutamic acid at amino acid 627. We show here that the 2009 H1N1 virus has acquired second-site suppressor mutations in its PB2 polymerase subunit that convey enhanced polymerase activity in human cells. Introduction of this polymorphism into the PB2 subunit of a primary avian isolate also increased polymerase activity and viral replication in human and porcine cells. An alternate adaptive strategy has also been identified, whereby introduction of a human PA subunit into an avian polymerase overcomes restriction in human cells. These data reveal a strategy used by the 2009 H1N1 influenza A virus and identify other pathways by which avian and swine-origin viruses may evolve to enhance replication, and potentially pathogenesis, in humans.

  7. Requirements for the self-directed replication of flock house virus RNA 1.

    PubMed Central

    Ball, L A

    1995-01-01

    The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account

  8. Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx

    PubMed Central

    Kouwaki, Takahisa; Okamoto, Toru; Ito, Ayano; Sugiyama, Yukari; Yamashita, Kazuo; Suzuki, Tatsuya; Kusakabe, Shinji; Hirano, Junki; Fukuhara, Takasuke; Yamashita, Atsuya; Saito, Kazunobu; Okuzaki, Daisuke; Watashi, Koichi; Sugiyama, Masaya; Yoshio, Sachiyo; Standley, Daron M.; Kanto, Tatsuya; Mizokami, Masashi; Moriishi, Kohji

    2016-01-01

    ABSTRACT Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly135 with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5. IMPORTANCE HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx-mediated HBV replication is largely dependent on JMJD5. We showed that

  9. Animal models of bovine leukemia virus and human T-lymphotrophic virus type-1: insights in transmission and pathogenesis.

    PubMed

    Lairmore, Michael D

    2014-02-01

    Bovine leukemia virus (BLV) and human T-lymphotrophic virus type-1 (HTLV-1) are related retroviruses associated with persistent and lifelong infections and a low incidence of lymphomas within their hosts. Both viruses can be spread through contact with bodily fluids containing infected cells, most often from mother to offspring through breast milk. Each of these complex retroviruses contains typical gag, pol, and env genes but also unique, nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the pathogenesis of each virus. Comparisons of BLV and HTLV-1 provide insights into mechanisms of spread and tumor formation, as well as potential approaches to therapeutic intervention against the infections.

  10. Inhibition of human immunodeficiency virus type 1 replication by Z-100, an immunomodulator extracted from human-type tubercle bacilli, in macrophages.

    PubMed

    Emori, Yutaka; Ikeda, Tamako; Ohashi, Takashi; Masuda, Takao; Kurimoto, Tadashi; Takei, Mineo; Kannagi, Mari

    2004-09-01

    Z-100 is an arabinomannan extracted from Mycobacterium tuberculosis that has various immunomodulatory activities, such as the induction of interleukin 12, interferon gamma (IFN-gamma) and beta-chemokines. The effects of Z-100 on human immunodeficiency virus type 1 (HIV-1) replication in human monocyte-derived macrophages (MDMs) are investigated in this paper. In MDMs, Z-100 markedly suppressed the replication of not only macrophage-tropic (M-tropic) HIV-1 strain (HIV-1JR-CSF), but also HIV-1 pseudotypes that possessed amphotropic Moloney murine leukemia virus or vesicular stomatitis virus G envelopes. Z-100 was found to inhibit HIV-1 expression, even when added 24 h after infection. In addition, it substantially inhibited the expression of the pNL43lucDeltaenv vector (in which the env gene is defective and the nef gene is replaced with the firefly luciferase gene) when this vector was transfected directly into MDMs. These findings suggest that Z-100 inhibits virus replication, mainly at HIV-1 transcription. However, Z-100 also downregulated expression of the cell surface receptors CD4 and CCR5 in MDMs, suggesting some inhibitory effect on HIV-1 entry. Further experiments revealed that Z-100 induced IFN-beta production in these cells, resulting in induction of the 16-kDa CCAAT/enhancer binding protein (C/EBP) beta transcription factor that represses HIV-1 long terminal repeat transcription. These effects were alleviated by SB 203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), indicating that the p38 MAPK signalling pathway was involved in Z-100-induced repression of HIV-1 replication in MDMs. These findings suggest that Z-100 might be a useful immunomodulator for control of HIV-1 infection.

  11. Immunotherapy of murine leukemia. Efficacy of passive serum therapy of Friend leukemia virus-induced disease in immunocompromised mice

    SciTech Connect

    Genovesi, E.V.; Livnat, D.; Collins, J.J.

    1983-02-01

    Previous studies have demonstrated that the passive therapy of Friend murine leukemia virus (F-MuLV)-induced disease with chimpanzee anti-F-MuLV serum is accompanied by the development of host antiviral humoral and cellular immunity, the latter measurable in adoptive transfer protocols and by the ability of serum-protected mice to resist virus rechallenge. The present study was designed to further examine the contribution of various compartments of the host immune system to serum therapy itself, as well as to the acquired antiviral immunity that develops in serum-protected mice, through the use of naturally immunocompromised animals (e.g., nude athymic mice and natural killer (NK)-deficient beige mutant mice) or mice treated with immunoabrogating agents such as sublethal irradiation, cyclophosphamide (Cytoxan (Cy)), cortisone, and /sup 89/Sr. The studies in nude mice indicate that while mature T-cells are not needed for effective serum therapy, they do appear to be necessary for the long-term resistance of serum-protected mice to virus rechallenge and for the generation of the cell population(s) responsible for adoptive transfer of antiviral immunity. Furthermore, this acquired resistance is not due to virus neutralization by serum antibodies since antibody-negative, Cy-treated, serum-protected mice still reject the secondary virus infection. Lastly, while the immunocompromise systems examined did effect various host antiviral immune responses, none of them, including the NK-deficient beige mutation, significantly diminished the efficacy of the passive serum therapy of F-MuLV-induced disease.

  12. Essential role of Rta in lytic DNA replication of Epstein-Barr virus.

    PubMed

    El-Guindy, Ayman; Ghiassi-Nejad, Maryam; Golden, Sean; Delecluse, Henri-Jacques; Miller, George

    2013-01-01

    Two transcription factors, ZEBRA and Rta, switch Epstein-Barr virus (EBV) from the latent to the lytic state. While ZEBRA also plays an obligatory role as an activator of replication, it is not known whether Rta is directly required for replication. Rta is dispensable for amplification of an oriLyt-containing plasmid in a transient-replication assay. Here, we assessed the requirement for Rta in activation of viral DNA synthesis from the endogenous viral genome, a function that has not been established. Initially, we searched for a ZEBRA mutant that supports viral replication but not transcription. We found that Z(S186A), a mutant of ZEBRA unable to activate transcription of Rta or viral genes encoding replication proteins, is competent to bind to oriLyt and to function as an origin recognition protein. Ectopic expression of the six components of the EBV lytic replication machinery failed to rescue replication by Z(S186A). However, addition of Rta to Z(S186A) and the mixture of replication factors activated viral replication and late gene expression. Deletion mutagenesis of Rta indicated that the C-terminal 10 amino acids (aa) were essential for the function of Rta in replication. In vivo DNA binding studies revealed that Rta interacted with the enhancer region of oriLyt. In addition, expression of Rta and Z(S186A) together, but not individually, activated synthesis of the BHLF1 transcript, a lytic transcript required for the process of viral DNA replication. Our findings demonstrate that Rta plays an indispensable role in the process of lytic DNA replication.

  13. A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein

    PubMed Central

    Zhang, Tong; Xu, Xiongbiao; Huang, Changjun; Qian, Yajuan; Li, Zhenghe

    2015-01-01

    ABSTRACT Rolling-circle replication of single-stranded genomes of plant geminiviruses is initiated by sequence-specific DNA binding of the viral replication-related protein (Rep) to its cognate genome at the replication origin. Monopartite begomovirus-associated betasatellites can be trans replicated by both cognate and some noncognate helper viruses, but the molecular basis of replication promiscuity of betasatellites remains uncharacterized. Earlier studies showed that when tomato yellow leaf curl China virus (TYLCCNV) or tobacco curly shoot virus (TbCSV) is coinoculated with both cognate and noncognate betasatellites, the cognate betasatellite dominates over the noncognate one at the late stages of infection. In this study, we constructed reciprocal chimeric betasatellites between tomato yellow leaf curl China betasatellite and tobacco curly shoot betasatellite and assayed their competitiveness against wild-type betasatellite when coinoculated with TYLCCNV or TbCSV onto plants. We mapped a region immediately upstream of the conserved rolling-circle cruciform structure of betasatellite origin that confers the cognate Rep-mediated replication advantage over the noncognate satellite. DNase I protection and in vitro binding assays further identified a novel sequence element termed Rep-binding motif (RBM), which specifically binds to the cognate Rep protein and to the noncognate Rep, albeit at lower affinity. Furthermore, we showed that RBM-Rep binding affinity is correlated with betasatellite replication efficiency in protoplasts. Our data suggest that although strict specificity of Rep-mediated replication does not exist, betasatellites have adapted to their cognate Reps for efficient replication during coevolution. IMPORTANCE Begomoviruses are numerous circular DNA viruses that cause devastating diseases of crops worldwide. Monopartite begomoviruses are frequently associated with betasatellites which are essential for induction of typical disease symptoms

  14. Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

    PubMed Central

    Ke, Po-Yuan; Chen, Steve S.-L.

    2013-01-01

    So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

  15. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    PubMed

    Ke, Po-Yuan; Chen, Steve S-L

    2013-01-01

    So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  16. Imaging of the alphavirus capsid protein during virus replication.

    PubMed

    Zheng, Yan; Kielian, Margaret

    2013-09-01

    Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid (NC) core contains a capsid protein lattice enclosing the plus-sense RNA genome, and it is surrounded by a lipid bilayer containing a lattice of the E1 and E2 envelope glycoproteins. Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma membrane (PM), but the traffic and assembly of viral components and the exit of virions from host cells are not well understood. To visualize the dynamics of capsid protein during infection, we developed a Sindbis virus infectious clone tagged with a tetracysteine motif. Tagged capsid protein could be fluorescently labeled with biarsenical dyes in living cells without effects on virus growth, morphology, or protein distribution. Live cell imaging and colocalization experiments defined distinct groups of capsid foci in infected cells. We observed highly motile internal puncta that colocalized with E2 protein, which may represent the transport machinery that capsid protein uses to reach the PM. Capsid was also found in larger nonmotile internal structures that colocalized with cellular G3BP and viral nsP3. Thus, capsid may play an unforeseen role in these previously observed G3BP-positive foci, such as regulation of cellular stress granules. Capsid puncta were also observed at the PM. These puncta colocalized with E2 and recruited newly synthesized capsid protein; thus, they may be sites of virus assembly and egress. Together, our studies provide the first dynamic views of the alphavirus capsid protein in living cells and a system to define detailed mechanisms during alphavirus infection.

  17. Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

    PubMed

    Beaulieu, Alexandre; Gravel, Émilie; Cloutier, Alexandre; Marois, Isabelle; Colombo, Éloïc; Désilets, Antoine; Verreault, Catherine; Leduc, Richard; Marsault, Éric; Richter, Martin V

    2013-04-01

    Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

  18. Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

    PubMed Central

    Beaulieu, Alexandre; Gravel, Émilie; Cloutier, Alexandre; Marois, Isabelle; Colombo, Éloïc; Désilets, Antoine; Verreault, Catherine; Leduc, Richard; Marsault, Éric

    2013-01-01

    Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place. PMID:23365447

  19. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil

    PubMed Central

    Guedes, Duschinka RD; Paiva, Marcelo HS; Donato, Mariana MA; Barbosa, Priscilla P; Krokovsky, Larissa; Rocha, Sura W dos S; Saraiva, Karina LA; Crespo, Mônica M; Rezende, Tatiana MT; Wallau, Gabriel L; Barbosa, Rosângela MR; Oliveira, Cláudia MF; Melo-Santos, Maria AV; Pena, Lindomar; Cordeiro, Marli T; Franca, Rafael F de O; Oliveira, André LS de; Peixoto, Christina A; Leal, Walter S; Ayres, Constância FJ

    2017-01-01

    Zika virus (ZIKV) is a flavivirus that has recently been associated with an increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide; however, there is evidence that other mosquito species, including Culex quinquefasciatus, transmit the virus. To test the potential of Cx. quinquefasciatus to transmit ZIKV, we experimentally compared the vector competence of laboratory-reared Ae. aegypti and Cx. quinquefasciatus. Interestingly, we were able to detect the presence of ZIKV in the midgut, salivary glands and saliva of artificially fed Cx. quinquefasciatus. In addition, we collected ZIKV-infected Cx. quinquefasciatus from urban areas with high microcephaly incidence in Recife, Brazil. Corroborating our experimental data from artificially fed mosquitoes, ZIKV was isolated from field-caught Cx. quinquefasciatus, and its genome was partially sequenced. Collectively, these findings indicate that there may be a wider range of ZIKV vectors than anticipated. PMID:28790458

  20. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil.

    PubMed

    Guedes, Duschinka Rd; Paiva, Marcelo Hs; Donato, Mariana Ma; Barbosa, Priscilla P; Krokovsky, Larissa; Rocha, Sura W Dos S; Saraiva, Karina LA; Crespo, Mônica M; Rezende, Tatiana Mt; Wallau, Gabriel L; Barbosa, Rosângela Mr; Oliveira, Cláudia Mf; Melo-Santos, Maria Av; Pena, Lindomar; Cordeiro, Marli T; Franca, Rafael F de O; Oliveira, André Ls de; Peixoto, Christina A; Leal, Walter S; Ayres, Constância Fj

    2017-08-09

    Zika virus (ZIKV) is a flavivirus that has recently been associated with an increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide; however, there is evidence that other mosquito species, including Culex quinquefasciatus, transmit the virus. To test the potential of Cx. quinquefasciatus to transmit ZIKV, we experimentally compared the vector competence of laboratory-reared Ae. aegypti and Cx. quinquefasciatus. Interestingly, we were able to detect the presence of ZIKV in the midgut, salivary glands and saliva of artificially fed Cx. quinquefasciatus. In addition, we collected ZIKV-infected Cx. quinquefasciatus from urban areas with high microcephaly incidence in Recife, Brazil. Corroborating our experimental data from artificially fed mosquitoes, ZIKV was isolated from field-caught Cx. quinquefasciatus, and its genome was partially sequenced. Collectively, these findings indicate that there may be a wider range of ZIKV vectors than anticipated.

  1. Favipiravir elicits antiviral mutagenesis during virus replication in vivo

    PubMed Central

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-01-01

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses. DOI: http://dx.doi.org/10.7554/eLife.03679.001 PMID:25333492

  2. Recombination between feline leukemia virus subgroup B or C and endogenous env elements alters the in vitro biological activities of the viruses.

    PubMed Central

    Pandey, R; Ghosh, A K; Kumar, D V; Bachman, B A; Shibata, D; Roy-Burman, P

    1991-01-01

    An important question in feline leukemia virus (FeLV) pathogenesis is whether, as in murine leukemia virus infection, homologous recombination between the infecting FeLV and the noninfectious endogenous FeLV-like proviruses serves as a significant base for the generation of proximal pathogens. To begin an analysis of this issue, several recombinant FeLVs were produced by using two different approaches: (i) the regions of the viral envelope (env) gene of a cloned FeLV (subgroup B virus [FeLV-B], Gardner-Arnstein strain) and those of two different endogenous proviral loci were exchanged to create specific FeLV chimeras, and (ii) vectors containing endogenous env and molecularly cloned infectious FeLV-C (Sarma strain) DNA sequences were coexpressed by transfection in nonfeline cells to facilitate recombination. The results of these combined approaches showed that up to three-fourths of the envelope glycoprotein (gp70), beginning from the N-terminal end, could be replaced by endogenous FeLV sequences to produce biologically active chimeric FeLVs. The in vitro replication efficiency or cell tropism of the recombinants appeared to be influenced by the amount of gp70 sequences replaced by the endogenous partner as well as by the locus of origin of the endogenous sequences. Additionally, a characteristic biological effect, aggregation of feline T-lymphoma cells (3201B cell line), was found to be specifically induced by replicating FeLV-C or FeLV-C-based recombinants. Multiple crossover sites in the gp70 protein selected under the conditions used for coexpression were identified. The results of induced coexpression were also supported by rapid generation of FeLV recombinants when FeLV-C was used to infect the feline 3201B cell line that constitutively expresses high levels of endogenous FeLV-specific mRNAs. Furthermore, a large, highly conserved open reading frame in the pol gene of an endogenous FeLV provirus was identified. This observation, particularly in reference to

  3. Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication.

    PubMed

    Esser-Nobis, Katharina; Harak, Christian; Schult, Philipp; Kusov, Yuri; Lohmann, Volker

    2015-08-01

    Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive-strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell-culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver-derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7-Lunet cells supported HAV- and HCV-RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA-122 and phosphatidylinositol 4-kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine-triphosphate-binding cassette transporters and FK506-binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. We established a cell-culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. © 2015 by the American Association for the

  4. The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication.

    PubMed

    Takahashi, Tadanobu; Kurebayashi, Yuuki; Ikeya, Kumiko; Mizuno, Takashi; Fukushima, Keijo; Kawamoto, Hiroko; Kawaoka, Yoshihiro; Suzuki, Yasuo; Suzuki, Takashi

    2010-12-09

    The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.

  5. Complex Virus-Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview.

    PubMed

    Li, Su; Wang, Jinghan; Yang, Qian; Naveed Anwar, Muhammad; Yu, Shaoxiong; Qiu, Hua-Ji

    2017-07-05

    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating epizootic diseases of pigs in many countries. Viruses are small intracellular parasites and thus rely on the cellular factors for replication. Fundamental aspects of CSFV-host interactions have been well described, such as factors contributing to viral attachment, modulation of genomic replication and translation, antagonism of innate immunity, and inhibition of cell apoptosis. However, those host factors that participate in the viral entry, assembly, and release largely remain to be elucidated. In this review, we summarize recent progress in the virus-host interactions involved in the life cycle of CSFV and analyze the potential mechanisms of viral entry, assembly, and release. We conclude with future perspectives and highlight areas that require further understanding.

  6. Effect of brefeldin A on Mayaro virus replication in Aedes albopictus and Vero cells.

    PubMed

    Da Costa, L J; Rebello, M A

    1999-12-01

    Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit Mayaro virus replication. At the concentration of 0.05 microgram/ml, the yield of the virus was inhibited by 94% in Aedes albopictus cells and by 99.5% in Vero cells. Treatment of A. albopictus cells with BFA did not inhibit the virus protein synthesis. However, this compound drastically reduced viral protein synthesis in Vero cells. The inhibitory effect progressively declined when BFA was added at late times post infection (p.i.). The effect of BFA on protein glycosylation is discussed.

  7. Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication.

    PubMed

    Melik, Wessam; Ellencrona, Karin; Wigerius, Michael; Hedström, Christer; Elväng, Annelie; Johansson, Magnus

    2012-10-01

    The flavivirus genus includes important human neurotropic pathogens like Tick-borne encephalitis virus (TBEV) and West-Nile virus (WNV). Flavivirus replication occurs at replication complexes, where the NS5 protein provides both RNA cap methyltransferase and RNA-dependent RNA polymerase activities. TBEVNS5 contains two PDZ binding motifs (PBMs) important for specific targeting of human PDZ proteins including Scribble, an association important for viral down regulation of cellular defense systems and neurite outgrowth. To determine whether the PBMs of TBEVNS5 affects virus replication we constructed a DNA based sub-genomic TBEV replicon expressing firefly luciferase. The PBMs within NS5 were mutated individually and in concert and the replicons were assayed in cell culture. Our results show that the replication rate was impaired in all mutants, which indicates that PDZ dependent host interactions influence TBEV replication. We also find that the C-terminal PBMs present in TBEVNS5 and WNVNS5 are targeting various human PDZ domain proteins. TBEVNS5 has affinity to Zonula occludens-2 (ZO-2), GIAP C-terminus interacting protein (GIPC), calcium/calmodulin-dependent serine protein kinase (CASK), glutamate receptor interacting protein 2, (GRIP2) and Interleukin 16 (IL-16). A different pattern was observed for WNVNS5 as it associate with a broader repertoire of putative host PDZ proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase.

    PubMed

    Choudhury, Nirupam Roy; Malik, Punjab Singh; Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Kaliappan, Kosalai; Mukherjee, Sunil Kumar

    2006-01-01

    Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).

  9. The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase

    PubMed Central

    Choudhury, Nirupam Roy; Malik, Punjab Singh; Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Kaliappan, Kosalai; Mukherjee, Sunil Kumar

    2006-01-01

    Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT↓ AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring ≥6 nt space for its efficient activity, translocates in the 3′→5′ direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation (∼24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV). PMID:17142233

  10. Imipramine Inhibits Chikungunya Virus Replication in Human Skin Fibroblasts through Interference with Intracellular Cholesterol Trafficking.

    PubMed

    Wichit, Sineewanlaya; Hamel, Rodolphe; Bernard, Eric; Talignani, Loïc; Diop, Fodé; Ferraris, Pauline; Liegeois, Florian; Ekchariyawat, Peeraya; Luplertlop, Natthanej; Surasombatpattana, Pornapat; Thomas, Frédéric; Merits, Andres; Choumet, Valérie; Roques, Pierre; Yssel, Hans; Briant, Laurence; Missé, Dorothée

    2017-06-09

    Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.

  11. Programmed Ribosomal Frameshift Alters Expression of West Nile Virus Genes and Facilitates Virus Replication in Birds and Mosquitoes

    PubMed Central

    Du, Fangyao; Owens, Nick; Bosco-Lauth, Angela M.; Nagasaki, Tomoko; Rudd, Stephen; Brault, Aaron C.; Bowen, Richard A.; Hall, Roy A.; van den Hurk, Andrew F.; Khromykh, Alexander A.

    2014-01-01

    West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed −1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1′. We have previously demonstrated that mutant