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Sample records for animal virus replication

  1. Animal models for the study of hepatitis C virus infection and replication.

    Science.gov (United States)

    MacArthur, Kristin L; Wu, Catherine H; Wu, George Y

    2012-06-21

    Hepatitis C virus (HCV) hepatitis, initially termed non-A, non-B hepatitis, has become one of the leading causes of cirrhosis and hepatocellular carcinoma worldwide. With the help of animal models, our understanding of the virus has grown substantially from the time of initial discovery. There is a paucity of available animal models for the study of HCV, mainly because of the selective susceptibility limited to humans and primates. Recent work has focused modification of animals to permit HCV entry, replication and transmission. In this review, we highlight the currently available models for the study of HCV including chimpanzees, tupaia, mouse and rat models. Discussion will include methods of model design as well as the advantages and disadvantages of each model. Particular focus is dedicated to knowledge of pathophysiologic mechanisms of HCV infection that have been elucidated through animal studies. Research within animal models is critically important to establish a complete understanding of HCV infection, which will ultimately form the basis for future treatments and prevention of disease.

  2. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  3. DNA Virus Replication Compartments

    Science.gov (United States)

    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

  4. Animal Mitochondrial DNA Replication

    Science.gov (United States)

    Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

    2016-01-01

    Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

  5. The molecular biology of Bluetongue virus replication.

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    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Ultrastructural Characterization of Zika Virus Replication Factories

    Directory of Open Access Journals (Sweden)

    Mirko Cortese

    2017-02-01

    Full Text Available Summary: 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. : Cortese et al. show that ZIKV infection in both human hepatoma and neuronal progenitor cells induces drastic structural modification of the cellular architecture. Microtubules and intermediate filaments surround the viral replication factory composed of vesicles corresponding to ER membrane invagination toward the ER lumen. Importantly, alteration of microtubule flexibility impairs ZIKV replication. Keywords: Zika virus, flavivirus, human neural progenitor cells, replication factories, replication organelles, microtubules, intermediate filaments, electron microscopy, electron tomography, live-cell imaging

  7. Methadone enhances human influenza A virus replication.

    Science.gov (United States)

    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.

  8. Replicating viruses for gynecologic cancer therapy.

    Science.gov (United States)

    Park, J W; Kim, M

    2016-01-01

    Despite advanced therapeutic treatments, gynecologic malignancies such as cervical and ovarian cancers are still the top ten leading cause of cancer death among women in South Korea. Thus a novel and innovative approach is urgently needed. Naturally occurring viruses are live, replication-proficient viruses that specifically infect human cancer cells while sparing normal cell counterparts. Since the serendipitous discovery of the naturally oncotropic virus targeting gynecologic cancer in 1920s, various replicating viruses have shown various degrees of safety and efficacy in preclinical or clinical applications for gynecologic cancer therapy. Cellular oncogenes and tumor suppressor genes, which are frequently dysregulated in gynecologic malignancies, play an important role in determining viral oncotropism. Published articles describing replicating, oncolytic viruses for gynecologic cancers are thoroughly reviewed. This review outlines the discovery of replication-proficient virus strains for targeting gynecologic malignancies, recent progresses elucidating molecular connections between oncogene/tumor suppressor gene abnormalities and viral oncotropism, and the associated preclinical/clinical implications. The authors would also like to propose future directions in the utility of the replicating viruses for gynecologic cancer therapy.

  9. Replication-Competent Controlled Herpes Simplex Virus.

    Science.gov (United States)

    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

  10. Functional analysis of replication determinantsin classical swine fever virus

    DEFF Research Database (Denmark)

    Hadsbjerg, Johanne

    and animal pathogens should facilitate finding new approaches for efficient disease control. The principal aim of this thesis is to characterise determinants involved in the replication of classical swine fever virus (CSFV). Classical swine fever is a highly contagious virus disease of domestic pigs and wild...... in cell culture. Knowledge of these sequence variations and putative long-range interactions will provide valuable insights into mechanisms underlying virustranslation and replication. In manuscript 3, a selection marker has been inserted into a CSFV-based replicon making it suitable for screening...

  11. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

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    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.

  12. Replication of Japanese Encephalitis Virus.

    Science.gov (United States)

    1980-12-10

    several reasons: 1) All flaviviruses appear to be human pathogens and thus require special laboratory hand- ling. 2) Most flaviviruses grow more slowly...Ishii et al., 1968). In view of the growing realization of the role of persis- tent viruses in some chronic human disease (Nathanson, 1977), it...Tamm. (1970) The role of cytoplasmic membranes in poliovirus biosynthesis. Virology 42,100. Cleaves, G. R. and D. T. Dubin. (1979) Methylation status of

  13. Engineering attenuated virus vaccines by controlling replication fidelity.

    Science.gov (United States)

    Vignuzzi, Marco; Wendt, Emily; Andino, Raul

    2008-02-01

    Long-lasting protection against viral infection is best achieved by vaccination with attenuated viruses. Obtaining stably attenuated vaccine strains has traditionally been an empirical process, which greatly restricts the number of effective vaccines for viral diseases. Here we describe a rational approach for engineering stably attenuated viruses that can serve as safe and effective vaccines. Our approach exploits the observation that restricting viral population diversity by increasing replication fidelity greatly reduces viral tissue tropism and pathogenicity. We show that poliovirus variants with reduced genetic diversity elicit a protective immune response in an animal model of infection. Indeed, these novel vaccine candidates are comparable in efficacy to the currently available Sabin type 1 vaccine strain, but have the added advantage of being more stable, as their increased replication fidelity prevents reversion to the pathogenic wild-type phenotype. We propose that restricting viral quasispecies diversity provides a general approach for the rational design of stable, attenuated vaccines for a wide variety of viruses.

  14. Ultrastructural Characterization of Zika Virus Replication Factories.

    Science.gov (United States)

    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. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Quantitative estimation of Nipah virus replication kinetics in vitro

    Directory of Open Access Journals (Sweden)

    Hassan Sharifah

    2006-06-01

    Full Text Available Abstract Background Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero using the one-step SYBR® Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR assay. Results The qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/μL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every ~40 minutes and attained peak intracellular virus RNA level of ~8.4 log PFU/μL at about 32 hours post-infection (PI. Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at ~7.9 log PFU/μL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was ~0.07 log PFU/μL per hour and less than 10% of the released Nipah virus RNA was infectious. Conclusion The SYBR® Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection.

  16. Replication strategy of human hepatitis B virus

    International Nuclear Information System (INIS)

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

    1987-01-01

    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

  17. Animal Models of Zika Virus

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    Bradley, Michael P; Nagamine, Claude M

    2017-01-01

    Zika virus has garnered great attention over the last several years, as outbreaks of the disease have emerged throughout the Western Hemisphere. Until quite recently Zika virus was considered a fairly benign virus, with limited clinical severity in both people and animals. The size and scope of the outbreak in the Western Hemisphere has allowed for the identification of severe clinical disease that is associated with Zika virus infection, most notably microcephaly among newborns, and an association with Guillian–Barré syndrome in adults. This recent association with severe clinical disease, of which further analysis strongly suggested causation by Zika virus, has resulted in a massive increase in the amount of both basic and applied research of this virus. Both small and large animal models are being used to uncover the pathogenesis of this emerging disease and to develop vaccine and therapeutic strategies. Here we review the animal-model–based Zika virus research that has been performed to date. PMID:28662753

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

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

    1993-12-01

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

  19. Autophagic machinery activated by dengue virus enhances virus replication

    International Nuclear Information System (INIS)

    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.

    2008-01-01

    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

  20. Replication and clearance of respiratory syncytial virus - Apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus

    DEFF Research Database (Denmark)

    Viuff, B.; Tjørnehøj, Kirsten; Larsen, Lars Erik

    2002-01-01

    and the infections with human respiratory syncytial. virus and BRSV have similar clinical, pathological, and epidemiological characteristics. In this study we used experimental BRSV infection in calves as a model of respiratory syncytial virus infection to demonstrate important aspects of viral replication...... and clearance in a natural target animal. Replication of BRSV was demonstrated in the luminal part of the respiratory epithelial cells and replication in the upper respiratory tract preceded the replication in the lower respiratory tract. Virus excreted to the lumen of the respiratory tract was cleared...

  1. Zika Virus-Induced Antibody Response Enhances Dengue Virus Serotype 2 Replication In Vitro.

    Science.gov (United States)

    Kawiecki, Anna B; Christofferson, Rebecca C

    2016-11-01

    Zika virus has emerged in the Americas, where dengue virus is endemic. Among the 4 serotypes of dengue virus, antibody-dependent enhancement is thought to enhance viral replication and disease severity. Reports suggest that anti-dengue virus antibody may enhance Zika virus replication. We investigated whether Zika virus antibodies enhance dengue virus replication, by exposing C57Bl/6 mice to Zika virus. Polyclonal serum was verified for strong Zika virus-neutralizing, dengue virus-subneutralizing capacity. Then we determined the enhancement capabilities of Zika virus-immune serum for dengue virus in vitro. We showed that Zika virus antibodies have the ability to enhance dengue virus infections, which is important, because in many Zika virus-affected areas, dengue virus is expected to remain endemic. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  2. Influenza virus replication in macrophages: balancing protection and pathogenesis.

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    Cline, Troy D; Beck, Donald; Bianchini, Elizabeth

    2017-10-01

    Macrophages are essential for protection against influenza A virus infection, but are also implicated in the morbidity and mortality associated with severe influenza disease, particularly during infection with highly pathogenic avian influenza (HPAI) H5N1 virus. While influenza virus infection of macrophages was once thought to be abortive, it is now clear that certain virus strains can replicate productively in macrophages. This may have important consequences for the antiviral functions of macrophages, the course of disease and the outcome of infection for the host. In this article, we review findings related to influenza virus replication in macrophages and the impact of productive replication on macrophage antiviral functions. A clear understanding of the interactions between influenza viruses and macrophages may lead to new antiviral therapies to relieve the burden of severe disease associated with influenza viruses.

  3. Small-Animal Models of Zika Virus.

    Science.gov (United States)

    Julander, Justin G; Siddharthan, Venkatraman

    2017-12-16

    Zika virus (ZIKV) infection can result in serious consequences, including severe congenital manifestations, persistent infection in the testes, and neurologic sequelae. After a pandemic emergence, the virus has spread to much of North and South America and has been introduced to many countries outside of ZIKV-endemic areas as infected travelers return to their home countries. Rodent models have been important in gaining a better understanding of the wide range of disease etiologies associated with ZIKV infection and for the initial phase of developing countermeasures to prevent or treat viral infections. We discuss herein the advantages and disadvantages of small-animal models that have been developed to replicate various aspects of disease associated with ZIKV infection. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

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

    Directory of Open Access Journals (Sweden)

    Takashi Irie

    2012-01-01

    Full Text Available 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. The Genomic Replication of the Crenarchaeal Virus SIRV2

    DEFF Research Database (Denmark)

    Martinez Alvarez, Laura

    Archaeal viruses have been found to be remarkably diverse in terms of their morphologies and genomes. But knowledge about their replication cycles and interactions with their hosts is still lacking. The aim of this work was to gain insight about the molecular mechanism of the genomic replication ...

  6. Host factors involved in chikungunya virus replication

    NARCIS (Netherlands)

    Scholte, Florine Elisabeth Maria

    2015-01-01

    In this thesis the interplay of CHIKV with cellular (host) factors involved in its replication is addressed. An in-depth understanding of the interactions between the viral proteins and those of their host is required for the elucidation of molecular mechanisms underlying viral replication. A

  7. Hendra virus and Nipah virus animal vaccines.

    Science.gov (United States)

    Broder, Christopher C; Weir, Dawn L; Reid, Peter A

    2016-06-24

    Hendra virus (HeV) and Nipah virus (NiV) are zoonotic viruses that emerged in the mid to late 1990s causing disease outbreaks in livestock and people. HeV appeared in Queensland, Australia in 1994 causing a severe respiratory disease in horses along with a human case fatality. NiV emerged a few years later in Malaysia and Singapore in 1998-1999 causing a large outbreak of encephalitis with high mortality in people and also respiratory disease in pigs which served as amplifying hosts. The key pathological elements of HeV and NiV infection in several species of mammals, and also in people, are a severe systemic and often fatal neurologic and/or respiratory disease. In people, both HeV and NiV are also capable of causing relapsed encephalitis following recovery from an acute infection. The known reservoir hosts of HeV and NiV are several species of pteropid fruit bats. Spillovers of HeV into horses continue to occur in Australia and NiV has caused outbreaks in people in Bangladesh and India nearly annually since 2001, making HeV and NiV important transboundary biological threats. NiV in particular possesses several features that underscore its potential as a pandemic threat, including its ability to infect humans directly from natural reservoirs or indirectly from other susceptible animals, along with a capacity of limited human-to-human transmission. Several HeV and NiV animal challenge models have been developed which have facilitated an understanding of pathogenesis and allowed for the successful development of both active and passive immunization countermeasures. Published by Elsevier Ltd.

  8. Replication of avian influenza A viruses in mammals.

    OpenAIRE

    Hinshaw, V S; Webster, R G; Easterday, B C; Bean, W J

    1981-01-01

    The recent appearance of an avian influenza A virus in seals suggests that viruses are transmitted from birds to mammals in nature. To examine this possibility, avian viruses of different antigenic subtypes were evaluated for their ability to replicate in three mammals-pigs, ferrets, and cats. In each of these mammals, avian strains replicated to high titers in the respiratory tract (10(5) to 10(7) 50% egg infective doses per ml of nasal wash), with peak titers at 2 to 4 days post-inoculation...

  9. Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

    Directory of Open Access Journals (Sweden)

    Antonio Postigo

    2017-05-01

    Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

  10. Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

    Science.gov (United States)

    Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

    2018-01-01

    Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

  11. NB protein does not affect influenza B virus replication in vitro and is not required for replication in or transmission between ferrets

    Science.gov (United States)

    Elderfield, Ruth A.; Koutsakos, Marios; Frise, Rebecca; Bradley, Konrad; Ashcroft, Jonathan; Miah, Shanhjahan; Lackenby, Angie

    2016-01-01

    The influenza B virus encodes a unique protein, NB, a membrane protein whose function in the replication cycle is not, as yet, understood. We engineered a recombinant influenza B virus lacking NB expression, with no concomitant difference in expression or activity of viral neuraminidase (NA) protein, an important caveat since NA is encoded on the same segment and initiated from a start codon just 4 nt downstream of NB. Replication of the virus lacking NB was not different to wild-type virus with full-length NB in clonal immortalized or complex primary cell cultures. In the mouse model, virus lacking NB induced slightly lower IFN-α levels in infected lungs, but this did not affect virus titres or weight loss. In ferrets infected with a mixture of viruses that did or did not express NB, there was no fitness advantage for the virus that retained NB. Moreover, virus lacking NB protein was transmitted following respiratory droplet exposure of sentinel animals. These data suggest no role for NB in supporting replication or transmission in vivo in this animal model. The role of NB and the nature of selection to retain it in all natural influenza B viruses remain unclear. PMID:26703440

  12. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication.

    Science.gov (United States)

    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.

  13. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication

    Directory of Open Access Journals (Sweden)

    Gefei Wang

    2016-01-01

    Full Text Available 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.

  14. Murine leukemia virus (MLV replication monitored with fluorescent proteins

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    Bittner Alexandra

    2004-12-01

    Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

  15. Inhibition of virus replication by RNA interference

    NARCIS (Netherlands)

    Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

    2003-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

  16. Structure and replication of hepatitis delta virus

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-29

    Dec 29, 2008 ... important feature of the virus genomic and antigenomic. RNA molecules is the 85 nucleotide ribozyme ... overall secondary structure consisting of base-paired elements, joining sequences and hairpin .... accumulation of specific HDV components in discrete foci in the nucleus is not probably relevant for ...

  17. Inhibitors of Nucleotidyltransferase Superfamily Enzymes Suppress Herpes Simplex Virus Replication

    Science.gov (United States)

    Wang, Hong; Tollefson, Ann E.; Ying, Baoling; Korom, Maria; Cheng, Xiaohong; Cao, Feng; Davis, Katie L.; Wold, William S. M.

    2014-01-01

    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. PMID:25267681

  18. Animal Models of Dengue Virus Infection

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    Eva Harris

    2012-01-01

    Full Text Available The development of animal models of dengue virus (DENV infection and disease has been challenging, as epidemic DENV does not naturally infect non-human species. Non-human primates (NHPs can sustain viral replication in relevant cell types and develop a robust immune response, but they do not develop overt disease. In contrast, certain immunodeficient mouse models infected with mouse-adapted DENV strains show signs of severe disease similar to the ‘vascular-leak’ syndrome seen in severe dengue in humans. Humanized mouse models can sustain DENV replication and show some signs of disease, but further development is needed to validate the immune response. Classically, immunocompetent mice infected with DENV do not manifest disease or else develop paralysis when inoculated intracranially; however, a new model using high doses of DENV has recently been shown to develop hemorrhagic signs after infection. Overall, each model has its advantages and disadvantages and is differentially suited for studies of dengue pathogenesis and immunopathogenesis and/or pre-clinical testing of antiviral drugs and vaccines.

  19. Inhibition of Monkeypox virus replication by RNA interference

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    Jahrling Peter B

    2009-11-01

    Full Text Available Abstract The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV, Vaccinia (VACV, monkeypox (MPV and Variola (VARV viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA. Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R or an important gene in viral entry (E8L, inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.

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

    Science.gov (United States)

    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.

  1. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    Science.gov (United States)

    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.

  2. Distinct Contributions of Autophagy Receptors in Measles Virus Replication.

    Science.gov (United States)

    Petkova, Denitsa S; Verlhac, Pauline; Rozières, Aurore; Baguet, Joël; Claviere, Mathieu; Kretz-Remy, Carole; Mahieux, Renaud; Viret, Christophe; Faure, Mathias

    2017-05-22

    Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO₂ and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.

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

    Science.gov (United States)

    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).

  4. DNA intercalator stimulates influenza transcription and virus replication

    Directory of Open Access Journals (Sweden)

    Poon Leo LM

    2011-03-01

    Full Text Available Abstract 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 RNAPIIa in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPIIa to hyperphosphorylated RNAPII (RNAPIIo.

  5. Replicative intermediates in UV-irradiated Simian virus 40

    International Nuclear Information System (INIS)

    Clark, J.M.; Hanawalt, P.C.

    1984-01-01

    The authors have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m 2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [ 3 H]thymidine. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h by which time the size of the newly-synthesized DNA exceeded the interdimer distance. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strand contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from 0V-damaged SV40 replicative intermediates. (Auth.)

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

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    Xiaozhen Guo

    2017-03-01

    Full Text Available 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.

  7. Cathepsin B & L are not required for ebola virus replication.

    Directory of Open Access Journals (Sweden)

    Andrea Marzi

    Full Text Available Ebola virus (EBOV, family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV. EBOV encodes one viral surface glycoprotein (GP, which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL, which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control, catB(-/- and catL(-/- mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

  8. Inhibition of herpes simplex virus replication by tobacco extracts.

    Science.gov (United States)

    Hirsch, J M; Svennerholm, B; Vahlne, A

    1984-05-01

    Herpes simplex virus type 1 (HSV-1) has been associated with the genesis of leukoplakias, epithelial atypia, and oral cancer. Tobacco habits, such as snuff dipping, are also definitely correlated with this type of lesion. The normal cytolytic HSV-1 infection can, after in vitro inactivation, transform cells. Extracts of snuff were prepared and assayed for their ability to inhibit HSV-1 replication. Plaque formation assays of HSV-1 in the presence of snuff extract showed that a reduced number of plaques was formed. Different batches of one brand of snuff were tested for inhibition of herpes simplex virus (HSV) production. More than 99% inhibition of 24-hr HSV production was obtained with undiluted batches. The 1:5 dilutions of snuff had an inhibitory effect of 85% and 1:25 dilutions, 39%. In agreement, the attachment of the virus to the host cell and penetration of the virus to the cell nuclei were found to be inhibited as was the synthesis of viral DNA. Nicotine had an inhibitory effect, while aromatic additions to snuff were found to have no major inhibitory effect on HSV replication. Snuff extracts were prepared from different brands of snuff reported to contain high and low quantities of tobacco-specific N-nitrosamines. Brands with reported high levels of tobacco-specific N-nitrosamines had significantly greater ability to inhibit HSV replication. In conclusion, this study has shown that extracts of snuff have inhibitory effects on the production of cytolytic HSV-1 infections. A chronic snuff dipper keeps tobacco in the mouth for the major part of the day. Thus, virus shed in the oral cavity in connection with a reactivated latent HSV-1 infection has great possibilities of being affected by snuff or derivatives of snuff. It is suggested that an interaction between tobacco products and HSV-1 might be involved in the development of dysplastic lesions in the oral cavity.

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

    Science.gov (United States)

    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.

  10. Animal models for the study of hepatitis B virus infection

    Directory of Open Access Journals (Sweden)

    Wei-Na Guo

    2018-01-01

    Full Text Available Even with an effective vaccine, an estimated 240 million people are chronically infected with hepatitis B virus (HBV worldwide. Current antiviral therapies, including interferon and nucleot(side analogues, rarely cure chronic hepatitis B. Animal models are very crucial for understanding the pathogenesis of chronic hepatitis B and developing new therapeutic drugs or strategies. HBV can only infect humans and chimpanzees, with the use of chimpanzees in HBV research strongly restricted. Thus, most advances in HBV research have been gained using mouse models with HBV replication or infection or models with HBV-related hepadnaviral infection. This review summarizes the animal models currently available for the study of HBV infection.

  11. Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors

    Directory of Open Access Journals (Sweden)

    Moss Bernard

    2005-03-01

    Full Text Available Abstract Background Replication of the vaccinia virus genome occurs in cytoplasmic factory areas and is dependent on the virus-encoded DNA polymerase and at least four additional viral proteins. DNA synthesis appears to start near the ends of the genome, but specific origin sequences have not been defined. Surprisingly, transfected circular DNA lacking specific viral sequences is also replicated in poxvirus-infected cells. Origin-independent plasmid replication depends on the viral DNA polymerase, but neither the number of additional viral proteins nor the site of replication has been determined. Results Using a novel real-time polymerase chain reaction assay, we detected a >400-fold increase in newly replicated plasmid in cells infected with vaccinia virus. Studies with conditional lethal mutants of vaccinia virus indicated that each of the five proteins known to be required for viral genome replication was also required for plasmid replication. The intracellular site of replication was determined using a plasmid containing 256 repeats of the Escherichia coli lac operator and staining with an E. coli lac repressor-maltose binding fusion protein followed by an antibody to the maltose binding protein. The lac operator plasmid was localized in cytoplasmic viral factories delineated by DNA staining and binding of antibody to the viral uracil DNA glycosylase, an essential replication protein. In addition, replication of the lac operator plasmid was visualized continuously in living cells infected with a recombinant vaccinia virus that expresses the lac repressor fused to enhanced green fluorescent protein. Discrete cytoplasmic fluorescence was detected in cytoplasmic juxtanuclear sites at 6 h after infection and the area and intensity of fluorescence increased over the next several hours. Conclusion Replication of a circular plasmid lacking specific poxvirus DNA sequences mimics viral genome replication by occurring in cytoplasmic viral factories

  12. Quinic acid derivatives inhibit dengue virus replication in vitro.

    Science.gov (United States)

    Zanello, Paula Rodrigues; Koishi, Andrea Cristine; Rezende Júnior, Celso de Oliveira; Oliveira, Larissa Albuquerque; Pereira, Adriane Antonia; de Almeida, Mauro Vieira; Duarte dos Santos, Claudia Nunes; Bordignon, Juliano

    2015-12-22

    Dengue is the most prevalent arboviral disease in tropical and sub-tropical areas of the world. The incidence of infection is estimated to be 390 million cases and 25,000 deaths per year. Despite these numbers, neither a specific treatment nor a preventive vaccine is available to protect people living in areas of high risk. With the aim of seeking a treatment that can mitigate dengue infection, we demonstrated that the quinic acid derivatives known as compound 2 and compound 10 were effective against all four dengue virus serotypes and safe for use in a human hepatoma cell line (Huh7.5). Both compounds were non-virucidal to dengue virus particles and did not interfere with early steps of the dengue virus life cycle, including binding and internalization. Experiments using a replicon system demonstrated that compounds 2 and 10 impaired dengue virus replication in Huh7.5 cells. Additionally, the anti-dengue virus effects of the quinic acid derivatives were preserved in human peripheral blood mononuclear cells. Taken together, these data suggest that quinic acid derivatives represent a novel chemical class of active compounds that could be used to combat dengue virus infection.

  13. Companion Animals as a Source of Viruses for Human Beings and Food Production Animals.

    Science.gov (United States)

    Reperant, L A; Brown, I H; Haenen, O L; de Jong, M D; Osterhaus, A D M E; Papa, A; Rimstad, E; Valarcher, J-F; Kuiken, T

    2016-07-01

    Companion animals comprise a wide variety of species, including dogs, cats, horses, ferrets, guinea pigs, reptiles, birds and ornamental fish, as well as food production animal species, such as domestic pigs, kept as companion animals. Despite their prominent place in human society, little is known about the role of companion animals as sources of viruses for people and food production animals. Therefore, we reviewed the literature for accounts of infections of companion animals by zoonotic viruses and viruses of food production animals, and prioritized these viruses in terms of human health and economic importance. In total, 138 virus species reportedly capable of infecting companion animals were of concern for human and food production animal health: 59 of these viruses were infectious for human beings, 135 were infectious for food production mammals and birds, and 22 were infectious for food production fishes. Viruses of highest concern for human health included hantaviruses, Tahyna virus, rabies virus, West Nile virus, tick-borne encephalitis virus, Crimean-Congo haemorrhagic fever virus, Aichi virus, European bat lyssavirus, hepatitis E virus, cowpox virus, G5 rotavirus, influenza A virus and lymphocytic choriomeningitis virus. Viruses of highest concern for food production mammals and birds included bluetongue virus, African swine fever virus, foot-and-mouth disease virus, lumpy skin disease virus, Rift Valley fever virus, porcine circovirus, classical swine fever virus, equine herpesvirus 9, peste des petits ruminants virus and equine infectious anaemia virus. Viruses of highest concern for food production fishes included cyprinid herpesvirus 3 (koi herpesvirus), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus. Of particular concern as sources of zoonotic or food production animal viruses were domestic carnivores, rodents and food production animals kept as companion animals. The current list of viruses provides an objective

  14. Advances in Animal Models of Hepatitis B Virus Infection

    Directory of Open Access Journals (Sweden)

    Zhang Hang

    2015-12-01

    Full Text Available Hepatitis B virus (HBV infection seriously affects human health. Stable and reliable animal models of HBV infection bear significance in studying pathogenesis of this health condition and development of intervention measures. HBV exhibits high specificity for hosts, and chimpanzee is long used as sole animal model of HBV infection. However, use of chimpanzees is strictly constrained because of ethical reasons. Many methods were used to establish small-animal models of HBV infection. Tupaia is the only nonprimate animal that can be infected by HBV. Use of HBV-related duck hepatitis virus and marmot hepatitis virus infection model contributed to evaluation of mechanism of HBV replication and HBV treatment methods. In recent years, development of human–mouse chimeric model provided possibility of using common experimental animals to carry out HBV research. These models feature their own advantages and disadvantages and can be complementary in some ways. This study provides an overview of current and commonly used animal models of HBV infection.

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

    Directory of Open Access Journals (Sweden)

    Bin Zhou

    2014-10-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  17. Promotion of Hendra virus replication by microRNA 146a.

    Science.gov (United States)

    Stewart, Cameron R; 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

    2013-04-01

    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.

  18. Ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells.

    Science.gov (United States)

    Junjhon, Jiraphan; Pennington, Janice G; Edwards, Thomas J; Perera, Rushika; Lanman, Jason; Kuhn, Richard J

    2014-05-01

    During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells. Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.

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

    Science.gov (United States)

    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).

  20. RNA Editing and its Control in Hepatitis Delta Virus Replication

    Directory of Open Access Journals (Sweden)

    John L. Casey

    2010-01-01

    Full Text Available The hepatitis delta virus genome is a small circular RNA, similar to viroids. Although HDV contains a gene, the protein produced (HDAg is encoded by less than half the genome and possesses no RNA polymerase activity. Because of this limited coding capacity, HDV relies heavily on host functions and on structural features of the viral RNA—very much like viroids. The virus’ use of host RNA editing activity to produce two functionally distinct forms of HDAg is a particularly good example of this reliance. This review covers the mechanisms and control of RNA editing in the HDV replication cycle.

  1. Trigocherrierin A, a Potent Inhibitor of Chikungunya Virus Replication

    Directory of Open Access Journals (Sweden)

    Mélanie Bourjot

    2014-03-01

    Full Text Available 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.

  2. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species.

    Science.gov (United States)

    Di Giallonardo, Francesca; Schlub, Timothy E; Shi, Mang; Holmes, Edward C

    2017-04-15

    Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies and

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

    Energy Technology Data Exchange (ETDEWEB)

    Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Hampton-Smith, Rachel J.; Aloia, Amanda L. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Eddes, James S. [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Simpson, Kaylene J. [Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville (Australia); Hoffmann, Peter [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide (Australia); Beard, Michael R. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia)

    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.

  4. Increased adiposity in animals due to a human virus.

    Science.gov (United States)

    Dhurandhar, N V; Israel, B A; Kolesar, J M; Mayhew, G F; Cook, M E; Atkinson, R L

    2000-08-01

    Four animal models of virus-induced obesity including adiposity induced by an avian adenovirus have been described previously. This is the first report of adiposity induced in animals by a human virus. We investigated the adiposity promoting effect of a human adenovirus (Ad-36) in two different animal models. Due to the novel nature of the findings we replicated the experiments using a chicken model three times and a mammal model once. In four separate experiments, chickens and mice were inoculated with human adenovirus Ad-36. Weight matched groups inoculated with tissue culture media were used as non-infected controls in each experiment. Ad-36 inoculated and uninfected control groups were housed in separate rooms under biosafety level 2 or better containment. The first experiment included an additional weight matched group of chickens that was inoculated with CELO (chick embryo lethal orphan virus), an avian adenovirus. Food intakes and body weights were measured weekly. At the time of sacrifice blood was drawn and visceral fat was carefully separated and weighed. Total body fat was determined by chemical extraction of carcass fat. Animals inoculated with Ad-36 developed a syndrome of increased adipose tissue and paradoxically low levels of serum cholesterol and triglycerides. This syndrome was not seen in chickens inoculated with CELO virus. Sections of the brain and hypothalamus of Ad-36 inoculated animals did not show any overt histopathological changes. Ad-36 DNA could be detected in adipose tissue, but not skeletal muscles of randomly selected animals for as long as 16 weeks after Ad-36 inoculation. Data from these animal models suggest that the role of viral disease in the etiology of human obesity must be considered.

  5. A dimeric Rep protein initiates replication of a linear archaeal virus genome: implications for the Rep mechanism and viral replication

    DEFF Research Database (Denmark)

    Oke, Muse; Kerou, Melina; Liu, Huanting

    2011-01-01

    that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between...... positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral......The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report...

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

    OpenAIRE

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

    2013-01-01

    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 exh...

  7. Suppressing active replication of a live attenuated simian immunodeficiency virus vaccine does not abrogate protection from challenge

    International Nuclear Information System (INIS)

    Gabriel, Benjamin; Fiebig, Uwe; Hohn, Oliver; Plesker, Roland; Coulibaly, Cheick; Cichutek, Klaus; Mühlebach, Michael D.; Bannert, Norbert; Kurth, Reinhard; Norley, Stephen

    2016-01-01

    Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequent challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection. - Highlights: • A Nef-deleted RT-SHIV was used as a live attenuated vaccine in macaques. • Vaccine virus replication was shut down to investigate its role in protection. • Ongoing vaccine virus replication did not appear to be necessary for protection. • An analysis of T- and B-cell responses failed to identify a correlate of protection.

  8. Suppressing active replication of a live attenuated simian immunodeficiency virus vaccine does not abrogate protection from challenge

    Energy Technology Data Exchange (ETDEWEB)

    Gabriel, Benjamin; Fiebig, Uwe; Hohn, Oliver [Robert Koch-Institut, Berlin (Germany); Plesker, Roland; Coulibaly, Cheick; Cichutek, Klaus; Mühlebach, Michael D. [Paul-Ehrlich-Institut, Langen (Germany); Bannert, Norbert; Kurth, Reinhard [Robert Koch-Institut, Berlin (Germany); Norley, Stephen, E-mail: NorleyS@rki.de [Robert Koch-Institut, Berlin (Germany)

    2016-02-15

    Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequent challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection. - Highlights: • A Nef-deleted RT-SHIV was used as a live attenuated vaccine in macaques. • Vaccine virus replication was shut down to investigate its role in protection. • Ongoing vaccine virus replication did not appear to be necessary for protection. • An analysis of T- and B-cell responses failed to identify a correlate of protection.

  9. Hepatitis C virus translation preferentially depends on active RNA replication.

    Directory of Open Access Journals (Sweden)

    Helene Minyi Liu

    Full Text Available Hepatitis C virus (HCV RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.

  10. Dendritic cells in dengue virus infection: Targets of virus replication and mediators of immunity

    Directory of Open Access Journals (Sweden)

    Michael A. Schmid

    2014-12-01

    Full Text Available Dendritic cells (DCs are sentinels of the immune system and detect pathogens at sites of entry, such as the skin. In addition to the ability of DCs to control infections directly via their innate immune functions, DCs help to prime adaptive B and T cell responses via antigen presentation in lymphoid tissues. Infected Aedes aegypti or Ae. albopictus mosquitoes transmit the four dengue virus (DENV serotypes to humans while probing for small blood vessels in the skin. DENV causes the most prevalent arthropod-borne viral disease in humans, yet no vaccine or specific therapeutic is currently approved. Although primary DENV infection confers life-long protective immunity against re-infection with the same DENV serotype, secondary infection with a different DENV serotype can lead to increased disease severity via cross-reactive T cells or enhancing antibodies. This review summarizes recent findings in humans and animal models about DENV infection of DCs, monocytes and macrophages. We discuss the dual role of DCs as both targets of DENV replication and mediators of innate and adaptive immunity, and summarize immune evasion strategies whereby DENV impairs the function of infected DCs. We suggest that DCs play a key role in priming DENV-specific neutralizing or potentially harmful memory B and T cell responses, and that future DC-directed therapies may help induce protective memory responses and reduce dengue pathogenesis.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Silva, Laurie A.; Dermody, Terence S.

    2017-01-01

    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. PMID:28248203

  13. Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

    OpenAIRE

    Ermak, G; Paszkowski, U; Wohlmuth, M; Scheid, O M; Paszkowski, J

    1993-01-01

    Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibi...

  14. Interference of hepatitis C virus RNA replication by short interfering RNAs

    Science.gov (United States)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  15. Emetine inhibits replication of RNA and DNA viruses without generating drug-resistant virus variants.

    Science.gov (United States)

    Khandelwal, Nitin; Chander, Yogesh; Rawat, Krishan Dutt; Riyesh, Thachamvally; Nishanth, Chikkahonnaiah; Sharma, Shalini; Jindal, Naresh; Tripathi, Bhupendra N; Barua, Sanjay; Kumar, Naveen

    2017-08-01

    At a noncytotoxic concentration, emetine was found to inhibit replication of DNA viruses [buffalopoxvirus (BPXV) and bovine herpesvirus 1 (BHV-1)] as well as RNA viruses [peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)]. Using the time-of-addition and virus step-specific assays, we showed that emetine treatment resulted in reduced synthesis of viral RNA (PPRV and NDV) and DNA (BPXV and BHV-1) as well as inhibiting viral entry (NDV and BHV-1). In addition, emetine treatment also resulted in decreased synthesis of viral proteins. In a cell free endogenous viral polymerase assay, emetine was found to significantly inhibit replication of NDV, but not BPXV genome, suggesting that besides directly inhibiting specific viral polymerases, emetine may also target other factors essentially required for efficient replication of the viral genome. Moreover, emetine was found to significantly inhibit BPXV-induced pock lesions on chorioallantoic membrane (CAM) along with associated mortality of embryonated chicken eggs. At a lethal dose 50 (LD 50 ) of 126.49 ng/egg and at an effective concentration 50 (EC 50 ) of 3.03 ng/egg, the therapeutic index of the emetine against BPXV was determined to be 41.74. Emetine was also found to significantly delay NDV-induced mortality in chicken embryos associated with reduced viral titers. Further, emetine-resistant mutants were not observed upon long-term (P = 25) sequential passage of BPXV and NDV in cell culture. Collectively, we have extended the effective antiviral activity of emetine against diverse groups of DNA and RNA viruses and propose that emetine could provide significant therapeutic value against some of these viruses without inducing an antiviral drug-resistant phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Syrian Hamster as an Animal Model for the Study of Human Influenza Virus Infection.

    Science.gov (United States)

    Iwatsuki-Horimoto, Kiyoko; Nakajima, Noriko; Ichiko, Yurie; Sakai-Tagawa, Yuko; Noda, Takeshi; Hasegawa, Hideki; Kawaoka, Yoshihiro

    2018-02-15

    Ferrets and mice are frequently used as animal models for influenza research. However, ferrets are demanding in terms of housing space and handling, whereas mice are not naturally susceptible to infection with human influenza A or B viruses. Therefore, prior adaptation of human viruses is required for their use in mice. In addition, there are no mouse-adapted variants of the recent H3N2 viruses, because these viruses do not replicate well in mice. In this study, we investigated the susceptibility of Syrian hamsters to influenza viruses with a view to using the hamster model as an alternative to the mouse model. We found that hamsters are sensitive to influenza viruses, including the recent H3N2 viruses, without adaptation. Although the hamsters did not show weight loss or clinical signs of H3N2 virus infection, we observed pathogenic effects in the respiratory tracts of the infected animals. All of the H3N2 viruses tested replicated in the respiratory organs of the hamsters, and some of them were detected in the nasal washes of infected animals. Moreover, a 2009 pandemic (pdm09) virus and a seasonal H1N1 virus, as well as one of the two H3N2 viruses, but not a type B virus, were transmissible by the airborne route in these hamsters. Hamsters thus have the potential to be a small-animal model for the study of influenza virus infection, including studies of the pathogenicity of H3N2 viruses and other strains, as well as for use in H1N1 virus transmission studies. IMPORTANCE We found that Syrian hamsters are susceptible to human influenza viruses, including the recent H3N2 viruses, without adaptation. We also found that a pdm09 virus and a seasonal H1N1 virus, as well as one of the H3N2 viruses, but not a type B virus tested, are transmitted by the airborne route in these hamsters. Syrian hamsters thus have the potential to be used as a small-animal model for the study of human influenza viruses. Copyright © 2018 American Society for Microbiology.

  17. Gamma ray inactivation of some animal viruses.

    Science.gov (United States)

    Thomas, F C; Davies, A G; Dulac, G C; Willis, N G; Papp-Vid, G; Girard, A

    1981-10-01

    Twenty samples of animal viruses comprising 14 different viruses in 12 families were subjected to varying doses of gamma irradiation from a 60Co source in a Gamma Cell 220 (Atomic Energy of Canada Limited) to determine lethal dose levels. The dose responses appeared linear throughout inactivation. The D10 values, that is the dose necessary to reduce infectivity by one log10, ranged from less than 0.20 Megarads to approximately 0.55 Megarads. There was not a complete inverse correlation between the target size (virion core) and the D10 value.

  18. Gamma ray inactivation of some animal viruses.

    OpenAIRE

    Thomas, F C; Davies, A G; Dulac, G C; Willis, N G; Papp-Vid, G; Girard, A

    1981-01-01

    Twenty samples of animal viruses comprising 14 different viruses in 12 families were subjected to varying doses of gamma irradiation from a 60Co source in a Gamma Cell 220 (Atomic Energy of Canada Limited) to determine lethal dose levels. The dose responses appeared linear throughout inactivation. The D10 values, that is the dose necessary to reduce infectivity by one log10, ranged from less than 0.20 Megarads to approximately 0.55 Megarads. There was not a complete inverse correlation betwee...

  19. Short hairpin-looped oligodeoxynucleotides reduce hepatitis C virus replication

    Directory of Open Access Journals (Sweden)

    Broecker Felix

    2012-07-01

    Full Text Available Abstract Background Persistent infection with hepatitis C virus (HCV is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Standard therapy consists of a combination of interferon-alpha and ribavirin, but many patients respond poorly, especially those infected with HCV genotypes 1 and 4. Furthermore, standard therapy is associated with severe side-effects. Thus, alternative therapeutic approaches against HCV are needed. Findings Here, we studied the effect of a new class of antiviral agents against HCV, short, partially double-stranded oligodeoxynucleotides (ODNs, on viral replication. We targeted the 5’ nontranslated region (5’ NTR of the HCV genome that has previously been shown as effective target for small interfering RNAs (siRNAs in vitro. One of the investigated ODNs, ODN 320, significantly and efficiently reduced replication of HCV replicons in a sequence-, time- and dose-dependent manner. ODN 320 targets a genomic region highly conserved among different HCV genotypes and might thus be able to inhibit a broad range of genotypes and subtypes. Conclusions ODNs provide an additional approach for inhibition of HCV, might be superior to siRNAs in terms of stability and cellular delivery, and suitable against HCV resistant to standard therapy. This study underlines the potential of partially double-stranded ODNs as antiviral agents.

  20. Enhancement of animal viruses growth on RK13 cells pretreated with 6-azauridine.

    Science.gov (United States)

    Tozzini, F

    1976-09-30

    In these experiments a technique for enhancing the virus replication in tissue culture (RK13 cells) has been used. The method consisted in growing the cells in presence of mug 0.4-0.8 of 6-Azauridine/ml of cellular suspension until the monolayers were formed. This pretreatment enhances the replication of several animal viruses with increased infectious titers (vesicular stomatitis, equine arteritis, equine rhinopneumonitis, Aujeszky disease and myxomatosis virus) and with increased yield of total virus in the culture (myxomatosis virus). In other experiment the 6-Azauridine pretreatment of the cells has shown to render the cells more susceptible to interferon preparation action with subsequent high rate of vesicular stomatitis virus plaques reduction.

  1. Dengue Virus Type 2: Protein Binding and Active Replication in Human Central Nervous System Cells

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    Ma Isabel Salazar

    2013-01-01

    Full Text Available An increased number of dengue cases with neurological complications have been reported in recent years. The lack of reliable animal models for dengue has hindered studies on dengue virus (DENV pathogenesis and cellular tropism in vivo. We further investigate the tropism of DENV for the human central nervous system (CNS, characterizing DENV interactions with cell surface proteins in human CNS cells by virus overlay protein binding assays (VOPBA and coimmunoprecipitations. In VOPBA, three membrane proteins (60, 70, and 130 kDa from the gray matter bound the entire virus particle, whereas only a 70 kDa protein bound in white matter. The coimmunoprecipitation assays revealed three proteins from gray matter consistently binding virus particles, one clearly distinguishable protein (~32 kDa and two less apparent proteins (100 and 130 kDa. Monoclonal anti-NS3 targeted the virus protein in primary cell cultures of human CNS treated with DENV-2, which also stained positive for NeuH, a neuron-specific marker. Thus, our results indicate (1 that DENV-2 exhibited a direct tropism for human neurons and (2 that human neurons sustain an active DENV replication as was demonstrated by the presence of the NS3 viral antigen in primary cultures of these cells treated with DENV-2.

  2. The virus-receptor interaction in the replication of feline immunodeficiency virus (FIV).

    Science.gov (United States)

    Willett, Brian J; Hosie, Margaret J

    2013-12-01

    The feline and human immunodeficiency viruses (FIV and HIV) target helper T cells selectively, and in doing so they induce a profound immune dysfunction. The primary determinant of HIV cell tropism is the expression pattern of the primary viral receptor CD4 and co-receptor(s), such as CXCR4 and CCR5. FIV employs a distinct strategy to target helper T cells; a high affinity interaction with CD134 (OX40) is followed by binding of the virus to its sole co-receptor, CXCR4. Recent studies have demonstrated that the way in which FIV interacts with its primary receptor, CD134, alters as infection progresses, changing the cell tropism of the virus. This review examines the contribution of the virus-receptor interaction to replication in vivo as well as the significance of these findings to the development of vaccines and therapeutics. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  4. COS-7-based model: methodological approach to study John Cunningham virus replication cycle.

    Science.gov (United States)

    Prezioso, C; Scribano, D; Rodio, D M; Ambrosi, C; Trancassini, M; Palamara, A T; Pietropaolo, V

    2018-02-05

    John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection.Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved.Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.

  5. Crosstalk between animal and human influenza viruses

    Science.gov (United States)

    Ozawa, Makoto; Kawaoka, Yoshihiro

    2017-01-01

    Although outbreaks of highly pathogenic avian influenza in wild and domestic birds have been posing the threat of a new influenza pandemic for the last decade, the first pandemic of the 21st century came from swine viruses. This fact emphasizes the complexity of influenza viral ecology and the difficulty of predicting influenza viral dynamics. Complete control of influenza viruses seems impossible. However, we must minimize the impact of animal and human influenza outbreaks by learning lessons from past experiences and recognizing the current status. Here, we review the most recent influenza virology data in the veterinary field, including aspects of zoonotic agents and recent studies that assessed the pandemic potential of H5N1 highly pathogenic avian influenza viruses. PMID:25387011

  6. Experimental Hendra virus infection of dogs: virus replication, shedding and potential for transmission.

    Science.gov (United States)

    Middleton, D J; Riddell, S; Klein, R; Arkinstall, R; Haining, J; Frazer, L; Mottley, C; Evans, R; Johnson, D; Pallister, J

    2017-01-01

    Characterisation of experimental Hendra virus (HeV) infection in dogs and assessment of associated transmission risk. Beagle dogs were exposed oronasally to Hendra virus/Australia/Horse/2008/Redlands or to blood collected from HeV-infected ferrets. Ferrets were exposed to oral fluids collected from dogs after canine exposure to HeV. Observations made and samples tested post-exposure were used to assess the clinical course and replication sites of HeV in dogs, the infectivity for ferrets of canine oral fluids and features of HeV infection in dogs following contact with infective blood. Dogs were reliably infected with HeV and were generally asymptomatic. HeV was re-isolated from the oral cavity and virus clearance was associated with development of virus neutralising antibody. Major sites of HeV replication in dogs were the tonsils, lower respiratory tract and associated lymph nodes. Virus replication was documented in canine kidney and spleen, confirming a viraemic phase for canine HeV infection and suggesting that urine may be a source of infectious virus. Infection was transmitted to ferrets via canine oral secretions, with copy numbers for the HeV N gene in canine oral swabs comparable to those reported for nasal swabs of experimentally infected horses. HeV is not highly pathogenic for dogs, but their oral secretions pose a potential transmission risk to people. The time-window for transmission risk is circumscribed and corresponds to the period of acute infection before establishment of an adaptive immune response. The likelihood of central nervous system involvement in canine HeV infection is unclear, as is any long-term consequence. © 2017 Australian Veterinary Association.

  7. Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.

    OpenAIRE

    Gabuzda, D H; Lawrence, K; Langhoff, E; Terwilliger, E; Dorfman, T; Haseltine, W A; Sodroski, J

    1992-01-01

    The viral infectivity factor gene vif of human immunodeficiency virus type 1 has been shown to affect the infectivity but not the production of virus particles. In this study, the effect of vif in the context of the HXB2 virus on virus replication in several CD4+ T-cell lines was investigated. vif was found to be required for replication in the CD4+ T-cell lines CEM and H9 as well as in peripheral blood T lymphocytes. vif was not required for replication in the SupT1, C8166, and Jurkat T-cell...

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

    Directory of Open Access Journals (Sweden)

    Noriyuki Otsuki

    Full Text Available 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.

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

    Science.gov (United States)

    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

  10. Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

    Science.gov (United States)

    Celma, Cristina C; Stewart, Meredith; Wernike, Kerstin; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; De Clercq, Kris; Zientara, Stephan; van Rijn, Piet A; Beer, Martin; Roy, Polly

    2017-01-01

    Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce

  11. Effect of prohibitin on hepatitis C virus replication

    Directory of Open Access Journals (Sweden)

    SHI Juanjuan

    2016-09-01

    Full Text Available Objective To investigate the effect of prohibitin (PHB on hepatitis C virus (HCV replication. Methods Human hepatocarcinoma Huh-7.5 cells were transfected with the full-length genome HCV RNA in vitro to establish the full-length genome cell model of HCV, and the supernatants were collected 24, 48, 72, and 96 hours later to measure HCV copies. The indirect immunofluorescence assay was used to measure the expression of HCV core proteins, and a transmission electron microscope was used to observe the changes in the ultrastructure of HCV-infected Huh-7.5 cells and identify the full-length genome cell model of HCV. Quantitative real-time PCR and Western Blot were used to measure the expression of PHB in HCV-infected Huh-7.5 cells, and the RNA interference technique was used to measure the replication of HCV RNA. The t-test was used for comparison between groups. Results The results of identification showed that the HCV full-length genome cell model was successfully established. At 24 and 48 hours after Huh-7.5 cells were transfected with HCV RNA, the mRNA expression level of PHB was 13.41±1.35 and 16.45±1.76, respectively, which differed significantly from that in the control group (1.01±0.57 and 1.01±0.87,t=29.540 and 31.361,both P<0.01, and the 24- and 48-hour transfection groups showed a significantly higher expression level of PHB than the control group (both P<0.01. At 24 and 48 hours after Huh-7.5 cells were transfected with the RNA interference plasmids of PHB, the level of HCV RNA was 64.32±5.49 and 84.45±7.06, respectively, significantly higher than that in the control group (shRNA-control group; 10.52±1.57 and 16.34±2.97, t=29.538 and 25.908,all P<0.01. Conclusion In HCV-infected Huh-7.5 cells, the increased expression of PHB is closely related to HCV RNA infection, and to a certain extent, PHB can inhibit the replication of HCV RNA.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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 (RVΔG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVΔG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVΔG-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 RVΔG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

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

    Energy Technology Data Exchange (ETDEWEB)

    Papaneri, Amy B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Wirblich, Christoph [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Cann, Jennifer A.; Cooper, Kurt [Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Jahrling, Peter B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Schnell, Matthias J., E-mail: matthias.schnell@jefferson.edu [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Jefferson Vaccine Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Blaney, Joseph E., E-mail: jblaney@niaid.nih.gov [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States)

    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.

  14. Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

    International Nuclear Information System (INIS)

    Eckerle, Lance D.; Albarino, Cesar G.; Ball, L. Andrew.

    2003-01-01

    The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3'-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2

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

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Cuesta-Geijo

    2017-06-01

    Full Text Available 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.

  16. siRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus.

    Science.gov (United States)

    Behera, Padmanava; Nagarajan, Shanmugasundaram; Murugkar, Harshad V; Kalaiyarasu, Semmannan; Prakash, Anil; Gothalwal, Ragini; Dubey, Shiv Chandra; Kulkarni, Diwakar D; Tosh, Chakradhar

    2015-06-01

    Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58 percent and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80 percent reduction in viral plaque counts and 94 percent inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68-73 percent and 87-88 percent reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.

  17. Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential.

    Directory of Open Access Journals (Sweden)

    Hongquan Wan

    2008-08-01

    Full Text Available H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu residue at amino acid position 226 in the hemagglutinin (HA receptor-binding site (RBS, responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

  18. Comparison of Rift Valley fever virus replication in North American livestock and wildlife cell lines.

    Science.gov (United States)

    Gaudreault, Natasha N; Indran, Sabarish V; Bryant, P K; Richt, Juergen A; Wilson, William C

    2015-01-01

    Rift Valley fever virus (RVFV) causes disease outbreaks across Africa and the Arabian Peninsula, resulting in high morbidity and mortality among young domestic livestock, frequent abortions in pregnant animals, and potentially severe or fatal disease in humans. The possibility of RVFV spreading to the United States or other countries worldwide is of significant concern to animal and public health, livestock production, and trade. The mechanism for persistence of RVFV during inter-epidemic periods may be through mosquito transovarial transmission and/or by means of a wildlife reservoir. Field investigations in endemic areas and previous in vivo studies have demonstrated that RVFV can infect a wide range of animals, including indigenous wild ruminants of Africa. Yet no predominant wildlife reservoir has been identified, and gaps in our knowledge of RVFV permissive hosts still remain. In North America, domestic goats, sheep, and cattle are susceptible hosts for RVFV and several competent vectors exist. Wild ruminants such as deer might serve as a virus reservoir and given their abundance, wide distribution, and overlap with livestock farms and human populated areas could represent an important risk factor. The objective of this study was to assess a variety of cell lines derived from North American livestock and wildlife for susceptibility and permissiveness to RVFV. Results of this study suggest that RVFV could potentially replicate in native deer species such as white-tailed deer, and possibly a wide range of non-ruminant animals. This work serves to guide and support future animal model studies and risk model assessment regarding this high-consequence zoonotic pathogen.

  19. Comparison of Rift Valley fever virus replication in North American livestock and wildlife cell lines.

    Directory of Open Access Journals (Sweden)

    Natasha N Gaudreault

    2015-06-01

    Full Text Available Rift Valley fever virus (RVFV causes disease outbreaks across Africa and the Arabian Peninsula, resulting in high morbidity and mortality among young domestic livestock, frequent abortions in pregnant animals, and potentially severe or fatal disease in humans. The possibility of RVFV spreading to the US or other countries worldwide is of significant concern to animal and public health, livestock production and trade. The mechanism for persistence of RVFV during inter-epidemic periods may be through mosquito transovarial transmission and/or by means of a wildlife reservoir. Field investigations in endemic areas and previous in vivo studies have demonstrated that RVFV can infect a wide range of animals, including indigenous wild ruminants of Africa. Yet no predominant wildlife reservoir has been identified, and gaps in our knowledge of RVFV permissive hosts still remain. In North America, domestic goats, sheep and cattle are susceptible hosts for RVFV and several competent vectors exist. Wild ruminants such as deer might serve as a virus reservoir and given their abundance, wide distribution, and overlap with livestock farms and human populated areas could represent an important risk factor. The objective of this study was to assess a variety of cell lines derived from North American livestock and wildlife for susceptibility and permissiveness to RVFV. Results of this study suggest that RVFV could potentially replicate in native deer species such as white-tailed deer, and possibly a wide range of non-ruminant animals. This work serves to guide and support future animal model studies and risk model assessment regarding this high-consequence zoonotic pathogen.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Nachbagauer, Raffael; Miller, Matthew S; Hai, Rong; Ryder, Alex B; Rose, John K; Palese, Peter; García-Sastre, Adolfo; Krammer, Florian; Albrecht, Randy A

    2015-12-30

    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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Selective inhibition of hepatitis C virus replication by alpha-zam, a ...

    African Journals Online (AJOL)

    Selective inhibition of hepatitis C virus replication by alpha-zam, a Nigella sativa seed formulation. Olufunmilayo G. Oyero, Masaaki Toyama, Naoki Mitsuhiro, Abdulfatah A. Onifade, Akemi Hidaka, Mika Okamoto, Masanori Baba ...

  4. Cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis e virus replication

    NARCIS (Netherlands)

    Y. Wang (Yijin); W. Wang (Wenshi); L. Xu (Lei); X. Zhou (Xinying); E. Shokrollahi (Ehsan); K. Felczak (K.); L.J.W. van der Laan (Luc); K.W. Pankiewicz (Krzysztof W.); D. Sprengers (Dave); N.J.H. Raat (Nicolaas); H.J. Metselaar (Herold); M.P. Peppelenbosch (Maikel); Q. Pan (Qiuwei)

    2016-01-01

    textabstractiruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yanliang Jiang

    2016-11-01

    Full Text Available 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.

  7. Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Plant Virus Replication

    Directory of Open Access Journals (Sweden)

    Xuejiao Jin

    2018-01-01

    Full Text Available Positive-sense (+ RNA viruses represent the most abundant group of viruses and are dependent on the host cell machinery to replicate. One remarkable feature that occurs after (+ RNA virus entry into cells is the remodeling of host endomembranes, leading to the formation of viral replication factories. Recently, rapid progress in three-dimensional (3D imaging technologies, such as electron tomography (ET and focused ion beam-scanning electron microscopy (FIB-SEM, has enabled researchers to visualize the novel membrane structures induced by viruses at high resolution. These 3D imaging technologies provide new mechanistic insights into the viral infection cycle. In this review, we summarize the latest reports on the cellular remodeling that occurs during plant virus infection; in particular, we focus on studies that provide 3D architectural information on viral replication factories. We also outline the mechanisms underlying the formation of these membranous structures and discuss possible future research directions.

  8. Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Plant Virus Replication.

    Science.gov (United States)

    Jin, Xuejiao; Cao, Xiuling; Wang, Xueting; Jiang, Jun; Wan, Juan; Laliberté, Jean-François; Zhang, Yongliang

    2018-01-01

    Positive-sense (+) RNA viruses represent the most abundant group of viruses and are dependent on the host cell machinery to replicate. One remarkable feature that occurs after (+) RNA virus entry into cells is the remodeling of host endomembranes, leading to the formation of viral replication factories. Recently, rapid progress in three-dimensional (3D) imaging technologies, such as electron tomography (ET) and focused ion beam-scanning electron microscopy (FIB-SEM), has enabled researchers to visualize the novel membrane structures induced by viruses at high resolution. These 3D imaging technologies provide new mechanistic insights into the viral infection cycle. In this review, we summarize the latest reports on the cellular remodeling that occurs during plant virus infection; in particular, we focus on studies that provide 3D architectural information on viral replication factories. We also outline the mechanisms underlying the formation of these membranous structures and discuss possible future research directions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

    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.

  10. Detection of foot-and-mouth disease virus RNA in pharyngeal epithelium biopsy samples obtained from infected cattle: Investigation of possible sites of virus replication and persistence

    DEFF Research Database (Denmark)

    Stenfeldt, Anna Carolina; Belsham, Graham

    2012-01-01

    Foot-and-mouth disease (FMD) is a highly contagious viral infection of significant financial importance to the export and trade of agricultural products. The occurrence of persistently infected ‘‘carriers’’ of FMD-virus (FMDV) in ruminant species adds further complications to disease control....... There have been significant discrepancies in reports regarding the pathogenesis of FMDV infection in cattle with specific emphasis on the anatomical sites involved in early and persistent virus replication. In this study, collection of small biopsy samples from the dorsal soft palate (DSP) of live animals...... was used to investigate the level of FMDV RNA present at this site at sequential time points during the infection. Results were compared to measurements of virus excretion in samples of oropharyngeal fluid collected at corresponding time points. Possible sites of virus persistence were investigated through...

  11. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

    Directory of Open Access Journals (Sweden)

    Kiwamu Hyodo

    2015-05-01

    Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

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

    DEFF Research Database (Denmark)

    Pedersen, Lene; Behnisch, Werner; Schmidt, Jörg

    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...

  13. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees

    Science.gov (United States)

    Bukh, Jens; Pietschmann, Thomas; Lohmann, Volker; Krieger, Nicole; Faulk, Kristina; Engle, Ronald E.; Govindarajan, Sugantha; Shapiro, Max; St. Claire, Marisa; Bartenschlager, Ralf

    2002-01-01

    The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced in vivo infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating in vivo. PMID:12391335

  14. Inhibitory effect of essential oils obtained from plants grown in Colombia on yellow fever virus replication in vitro

    Science.gov (United States)

    Meneses, Rocío; Ocazionez, Raquel E; Martínez, Jairo R; Stashenko, Elena E

    2009-01-01

    Background An antiviral drug is needed for the treatment of patients suffering from yellow fever. Several compounds present in plants can inactive in vitro a wide spectrum of animal viruses. Aim In the present study the inhibitory effect of essential oils of Lippia alba, Lippia origanoides, Oreganum vulgare and Artemisia vulgaris on yellow fever virus (YFV) replication was investigated. Methods The cytotoxicity (CC50) on Vero cells was evaluated by the MTT reduction method. The minimum concentration of the essential oil that inhibited virus titer by more than 50% (MIC) was determined by virus yield reduction assay. YFV was incubated 24 h at 4°C with essential oil before adsorption on Vero cell, and viral replication was carried out in the absence or presence of essential oil. Vero cells were exposed to essential oil 24 h at 37°C before the adsorption of untreated-virus. Results The CC50 values were less than 100 μg/mL and the MIC values were 3.7 and 11.1 μg/mL. The CC50/MIC ratio was of 22.9, 26.4, 26.5 and 8.8 for L. alba, L origanoides, O. vulgare and A. vulgaris, respectively. The presence of essential oil in the culture medium enhances the antiviral effect: L. origanoides oil at 11.1 μg/mLproduced a 100% reduction of virus yield, and the same result was observed with L. alba, O. vulgare and A. vulgaris oils at100 μg/mL. No reduction of virus yield was observed when Vero cells were treated with essential oil before the adsorption of untreated-virus. Conclusion The essential oils evaluated in the study showed antiviral activities against YFV. The mode of action seems to be direct virus inactivation. PMID:19267922

  15. Inhibitory effect of essential oils obtained from plants grown in Colombia on yellow fever virus replication in vitro.

    Science.gov (United States)

    Meneses, Rocío; Ocazionez, Raquel E; Martínez, Jairo R; Stashenko, Elena E

    2009-03-06

    An antiviral drug is needed for the treatment of patients suffering from yellow fever. Several compounds present in plants can inactive in vitro a wide spectrum of animal viruses. In the present study the inhibitory effect of essential oils of Lippia alba, Lippia origanoides, Oreganum vulgare and Artemisia vulgaris on yellow fever virus (YFV) replication was investigated. The cytotoxicity (CC(50)) on Vero cells was evaluated by the MTT reduction method. The minimum concentration of the essential oil that inhibited virus titer by more than 50% (MIC) was determined by virus yield reduction assay. YFV was incubated 24 h at 4 degrees C with essential oil before adsorption on Vero cell, and viral replication was carried out in the absence or presence of essential oil. Vero cells were exposed to essential oil 24 h at 37 degrees C before the adsorption of untreated-virus. The CC(50) values were less than 100 microg/mL and the MIC values were 3.7 and 11.1 microg/mL. The CC(50)/MIC ratio was of 22.9, 26.4, 26.5 and 8.8 for L. alba, L origanoides, O. vulgare and A. vulgaris, respectively. The presence of essential oil in the culture medium enhances the antiviral effect: L. origanoides oil at 11.1 microg/mL produced a 100% reduction of virus yield, and the same result was observed with L. alba, O. vulgare and A. vulgaris oils at 100 microg/mL. No reduction of virus yield was observed when Vero cells were treated with essential oil before the adsorption of untreated-virus. The essential oils evaluated in the study showed antiviral activities against YFV. The mode of action seems to be direct virus inactivation.

  16. Inhibitory effect of essential oils obtained from plants grown in Colombia on yellow fever virus replication in vitro

    Directory of Open Access Journals (Sweden)

    Martínez Jairo R

    2009-03-01

    Full Text Available Abstract Background An antiviral drug is needed for the treatment of patients suffering from yellow fever. Several compounds present in plants can inactive in vitro a wide spectrum of animal viruses. Aim In the present study the inhibitory effect of essential oils of Lippia alba, Lippia origanoides, Oreganum vulgare and Artemisia vulgaris on yellow fever virus (YFV replication was investigated. Methods The cytotoxicity (CC50 on Vero cells was evaluated by the MTT reduction method. The minimum concentration of the essential oil that inhibited virus titer by more than 50% (MIC was determined by virus yield reduction assay. YFV was incubated 24 h at 4°C with essential oil before adsorption on Vero cell, and viral replication was carried out in the absence or presence of essential oil. Vero cells were exposed to essential oil 24 h at 37°C before the adsorption of untreated-virus. Results The CC50 values were less than 100 μg/mL and the MIC values were 3.7 and 11.1 μg/mL. The CC50/MIC ratio was of 22.9, 26.4, 26.5 and 8.8 for L. alba, L origanoides, O. vulgare and A. vulgaris, respectively. The presence of essential oil in the culture medium enhances the antiviral effect: L. origanoides oil at 11.1 μg/mLproduced a 100% reduction of virus yield, and the same result was observed with L. alba, O. vulgare and A. vulgaris oils at100 μg/mL. No reduction of virus yield was observed when Vero cells were treated with essential oil before the adsorption of untreated-virus. Conclusion The essential oils evaluated in the study showed antiviral activities against YFV. The mode of action seems to be direct virus inactivation.

  17. Equine infectious anemia virus replication is upregulated during differentiation of blood monocytes from acutely infected horses.

    OpenAIRE

    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.

  18. Derepression of a novel class of vaccinia virus genes upon DNA replication

    NARCIS (Netherlands)

    Vos, J C; Stunnenberg, H.G.

    1988-01-01

    A novel class of vaccinia virus genes, called intermediate, is expressed immediately post-replication and prior to the onset of late gene transcription. Intermediate transcription is dependent on trans-acting factors which are present in an active state in virus-infected cells prior to the onset of

  19. Bicyclams, selective antagonists of the human chemokine receptor CXCR4, potently inhibit feline immunodeficiency virus replication

    NARCIS (Netherlands)

    Horzinek, M.C.; Egberink, H.F.; Clercq, E. de; Vliet, A.L.W. van; Balzarini, J.; Bridger, G.J.; Henson, G.; Schols, D.

    1999-01-01

    Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when

  20. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L

    1999-01-01

    We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation...

  1. Replication-defective virus vaccine-induced protection of mice from genital herpes simplex virus 2 requires CD4 T cells

    Science.gov (United States)

    Morrison, Lynda A.

    2008-01-01

    Replication-defective herpes simplex virus 2 (HSV-2), used as an immunization strategy, protects against HSV-2 challenge in animal models. The roles of replication-defective virus-induced T cell subsets in control of HSV-2 infection have not been established. Mice lacking B cells (μMT) were immunized, depleted of CD4 or CD8 T cells, and then challenged intravaginally with HSV-2 to elucidate T cell subset contributions in the absence of virus-specific antibody. Immunized, CD4-depleted μMT mice developed severe infection of the genital tract and nervous system. In contrast, depletion of CD8 T cells from μMT mice did not attenuate protection. Immunized wild-type mice depleted of CD4 T cells also developed more severe HSV-2 infection than mice from which CD8 T cells were depleted. Thus, immunization with replication-defective virus induces T cell responses that effectively control HSV-2 infection in the absence of HSV-immune antibody, and CD4 T cells play the predominant role in this protective effect. PMID:18410949

  2. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    International Nuclear Information System (INIS)

    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-01-01

    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.

  3. Effect of Phosphorylation of CM2 Protein on Influenza C Virus Replication.

    Science.gov (United States)

    Goto, Takanari; Shimotai, Yoshitaka; Matsuzaki, Yoko; Muraki, Yasushi; Sho, Ri; Sugawara, Kanetsu; Hongo, Seiji

    2017-11-15

    CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, a phosphorylation site(s) at residue 78 and/or 103 of CM2 was replaced with an alanine residue(s), and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine change at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid change at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild-type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site. IMPORTANCE It is well-known that many host and viral proteins are posttranslationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins NP, M1, NS1, and the nuclear export protein (NEP), which are not integrated into the

  4. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sanada, Takahiro [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Tsukiyama-Kohara, Kyoko, E-mail: kkohara@vet.kagoshima-u.ac.jp [Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima-city, Kagoshima 890-0065 (Japan); Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Kagoshima 890-0065 (Japan); Yamamoto, Naoki [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Ezzikouri, Sayeh; Benjelloun, Soumaya [Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360 (Morocco); Murakami, Shuko; Tanaka, Yasuhito [Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Tateno, Chise [PhoenixBio Co. Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan); Kohara, Michinori, E-mail: kohara-mc@igakuken.or.jp [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

    2016-01-08

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10{sup 5} copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10{sup 4}-10{sup 6} copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10{sup 3} copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  5. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    International Nuclear Information System (INIS)

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    2016-01-01

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10 5 copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10 4 -10 6 copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10 3 copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  6. Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication.

    Directory of Open Access Journals (Sweden)

    Adam J Ericsen

    2016-12-01

    Full Text Available 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.

  7. Identification of Epstein-Barr Virus Replication Proteins in Burkitt’s Lymphoma Cells

    Directory of Open Access Journals (Sweden)

    Chris Traylen

    2015-10-01

    Full Text Available The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1. An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications.

  8. Tomato yellow leaf curl virus: No evidence for replication in the insect vector Bemisia tabaci.

    Science.gov (United States)

    Sánchez-Campos, Sonia; Rodríguez-Negrete, Edgar A; Cruzado, Lucía; Grande-Pérez, Ana; Bejarano, Eduardo R; Navas-Castillo, Jesús; Moriones, Enrique

    2016-08-01

    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.

  9. Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

    Directory of Open Access Journals (Sweden)

    Ishimaru D.

    1998-01-01

    Full Text Available 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 µg/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.

  10. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.

    Science.gov (United States)

    Martins, Laura J; Bonczkowski, Pawel; Spivak, Adam M; De Spiegelaere, Ward; Novis, Camille L; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Trypsteen, 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.

  11. Enhanced replication of herpes simplex virus type 1 in human cells

    International Nuclear Information System (INIS)

    Miller, C.S.; Smith, K.O.

    1991-01-01

    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

  12. viruses associated with human and animal influenza - a review 40

    African Journals Online (AJOL)

    DR. AMINU

    ABSTRACT. In this review, the most important viruses associated with human and animal influenza are reported. These include Influenza A,B and C. Influenza viruses are members of the family. Orthomyxoviridae. Influenza A virus being the most pathogenic and wide spread with many subtypes has constantly cause ...

  13. Viruses associated with human and animal influenza - a review ...

    African Journals Online (AJOL)

    In this review, the most important viruses associated with human and animal influenza are reported. These include Influenza A,B and C. Influenza viruses are members of the family Orthomyxoviridae. Influenza A virus being the most pathogenic and wide spread with many subtypes has constantly cause epidemics in several ...

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

    Directory of Open Access Journals (Sweden)

    Alix de Brogniez

    2016-03-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  16. The Tat protein of human immunodeficiency virus-1 enhances hepatitis C virus replication through interferon gamma-inducible protein-10

    Directory of Open Access Journals (Sweden)

    Qu Jing

    2012-04-01

    Full Text Available Abstract Background Co-infection with human immunodeficiency virus-1 (HIV-1 and hepatitis C virus (HCV is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown. Results HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10 mRNA in peripheral blood mononuclear cells (PBMCs. HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production. Conclusions HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.

  17. Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

    Directory of Open Access Journals (Sweden)

    Vidya P Nair

    2016-04-01

    Full Text Available Hepatitis E virus (HEV causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4. Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp, X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1 and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient

  18. Memory B cells and CD8⁺ lymphocytes do not control seasonal influenza A virus replication after homologous re-challenge of rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Timothy D Carroll

    Full Text Available This study sought to define the role of memory lymphocytes in the protection from homologous influenza A virus re-challenge in rhesus macaques. Depleting monoclonal antibodies (mAb were administered to the animals prior to their second experimental inoculation with a human seasonal influenza A virus strain. Treatment with either anti-CD8α or anti-CD20 mAbs prior to re-challenge had minimal effect on influenza A virus replication. Thus, in non-human primates with pre-existing anti-influenza A antibodies, memory B cells and CD8α⁺ T cells do not contribute to the control of virus replication after re-challenge with a homologous strain of influenza A virus.

  19. ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment.

    Science.gov (United States)

    Ellis-Connell, Amy L; Balgeman, Alexis J; Zarbock, Katie R; Barry, Gabrielle; Weiler, Andrea; Egan, Jack O; Jeng, Emily K; Friedrich, Thomas; Miller, Jeffrey S; Haase, Ashley T; Schacker, Timothy W; Wong, Hing C; Rakasz, Eva; O'Connor, Shelby L

    2018-02-01

    Developing biological interventions to control human immunodeficiency virus (HIV) replication in the absence of antiretroviral therapy (ART) could contribute to the development of a functional cure. As a potential alternative to ART, the interleukin-15 (IL-15) superagonist ALT-803 has been shown to boost the number and function of HIV-specific CD8 + T and NK cell populations in vitro Four simian immunodeficiency virus (SIV)-positive rhesus macaques, three of whom possessed major histocompatibility complex alleles associated with control of SIV and all of whom had received SIV vaccine vectors that had the potential to elicit CD8 + T cell responses, were given ALT-803 in three treatment cycles. The first and second cycles of treatment were separated by 2 weeks, while the third cycle was administered after a 29-week break. ALT-803 transiently elevated the total CD8 + effector and central memory T cell and NK cell populations in peripheral blood, while viral loads transiently decreased by ∼2 logs in all animals. Virus suppression was not sustained as T cells became less responsive to ALT-803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT-803, concurrent with downregulation of the IL-2/15 common γC and β chain receptors on both CD8 + T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT-803 treatment. During the third treatment cycle, responsiveness to ALT-803 was restored. CD8 + T cells and NK cells increased again 3- to 5-fold, and viral loads transiently decreased again by 1 to 2 logs. IMPORTANCE Overall, our data show that ALT-803 has the potential to be used as an immunomodulatory agent to elicit effective immune control of HIV/SIV replication. We identify mechanisms to explain why virus control is transient, so that this model can be used to define a clinically appropriate treatment regimen. Copyright © 2018 American Society for Microbiology.

  20. Replication Vesicles are Load- and Choke-Points in the Hepatitis C Virus Lifecycle

    Science.gov (United States)

    Clausznitzer, Diana; Schulze, Manuel; Hüber, Christian M.; Lenz, Simon M.; Schlöder, Johannes P.; Trippler, Martin; Bartenschlager, Ralf; Lohmann, Volker; Kaderali, Lars

    2013-01-01

    Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency. PMID:23990783

  1. Replication vesicles are load- and choke-points in the hepatitis C virus lifecycle.

    Directory of Open Access Journals (Sweden)

    Marco Binder

    Full Text Available Hepatitis C virus (HCV infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.

  2. The place of viruses in biology in light of the metabolism- versus-replication-first debate.

    Science.gov (United States)

    López-García, Purificación

    2012-01-01

    The last decade has seen a revival of old virocentric ideas. These concepts are heterogeneous, extending from proposals that consider viruses functionally as living beings and/or as descendants of viral lineages that preceded cell evolution to other claims that consider viruses and/or some viral families a fourth domain of life. While the debates about whether viruses are alive or not and whether some virus-like replicators preceded the first cells fall under the long-lasting dichotomous view on the nature and origin of life (metabolism- versus replication-first), the claim that some giant viruses form a fourth domain in an organismal tree of life is not consistent with current evidence and can be falsified.

  3. Replication Capacity of Viruses from Acute Infection Drives HIV-1 Disease Progression.

    Science.gov (United States)

    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 gag IMPORTANCE 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

  4. Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

    Directory of Open Access Journals (Sweden)

    Nunya Chotiwan

    2018-02-01

    Full Text Available We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several

  5. Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

    Science.gov (United States)

    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.

  6. Stearoyl coenzyme A desaturase 1 is associated with hepatitis C virus replication complex and regulates viral replication

    DEFF Research Database (Denmark)

    Nguyen, LN; Lim, YS; Pham, Long

    2014-01-01

    The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet...... formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while...... exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1...

  7. Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line.

    Science.gov (United States)

    Yang, Darong; Zuo, Chaohui; Wang, Xiaohong; Meng, Xianghe; Xue, Binbin; Liu, Nianli; Yu, Rong; Qin, Yuwen; Gao, Yimin; Wang, Qiuping; Hu, Jun; Wang, Ling; Zhou, Zebin; Liu, Bing; Tan, Deming; Guan, Yang; Zhu, Haizhen

    2014-04-01

    The absence of a robust cell culture system for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has limited the analysis of the virus lifecycle and drug discovery. We have established a hepatoma cell line, HLCZ01, the first cell line, to the authors' knowledge, supporting the entire lifecycle of both HBV and HCV. HBV surface antigen (HBsAg)-positive particles can be observed in the supernatant and the lumen of the endoplasmic reticulum of the cells via electron microscopy. Interestingly, HBV and HCV clinical isolates propagate in HLCZ01 cells. Both viruses replicate in the cells without evidence of overt interference. HBV and HCV entry are blocked by antibodies against HBsAg and human CD81, respectively, and the replication of HBV and HCV is inhibited by antivirals. HLCZ01 cells mount an innate immune response to virus infection. The cell line provides a powerful tool for exploring the mechanisms of virus entry and replication and the interaction between host and virus, facilitating the development of novel antiviral agents and vaccines.

  8. Promotion of Hendra Virus Replication by MicroRNA 146a

    OpenAIRE

    Stewart, Cameron R.; 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.

    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 imm...

  9. Heparin octasaccharide decoy liposomes inhibit replication of multiple viruses

    Science.gov (United States)

    Hendricks, Gabriel L.; Velazquez, Lourdes; Pham, Serena; Qaisar, Natasha; Delaney, James C.; Viswanathan, Karthik; Albers, Leila; Comolli, James C.; Shriver, Zachary; Knipe, David M.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Trevejo, Jose M.

    2016-01-01

    Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as ‘molecular sinks’ and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform. PMID:25637710

  10. The cell biology of Tobacco mosaic virus replication and movement

    Directory of Open Access Journals (Sweden)

    Chengke eLiu

    2013-02-01

    Full Text Available Successful systemic infection of a plant by Tobacco mosaic virus (TMV requires three processes that repeat over time: initial establishment and accumulation in invaded cells, intercellular movement and systemic transport. Accumulation and intercellular movement of TMV necessarily involves intracellular transport by complexes containing virus and host proteins and virus RNA during a dynamic process that can be visualized. Multiple membranes appear to assist TMV accumulation, while membranes, microfilaments and microtubules appear to assist TMV movement. Here we review cell biological studies that describe TMV-membrane, -cytoskeleton and -other host protein interactions which influence virus accumulation and movement in leaves and callus tissue. The importance of understanding the developmental phase of the infection in relationship to the observed virus-membrane or -host protein interaction is emphasized. Utilizing the latest observations of TMV-membrane and -host protein interactions within our evolving understanding of the infection ontogeny, a model for TMV accumulation and intracellular spread in a cell biological context is provided.

  11. Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus.

    Science.gov (United States)

    Csabai, Zsolt; Takács, Irma F; Snyder, Michael; Boldogkői, Zsolt; Tombácz, Dóra

    2017-09-01

    Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.

  12. Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication.

    Directory of Open Access Journals (Sweden)

    Nickolay Neznanov

    2008-04-01

    Full Text Available Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.

  13. Hsp90 inhibitors reduce influenza virus replication in cell culture

    International Nuclear Information System (INIS)

    Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin; Brownlee, George

    2008-01-01

    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

  14. Monitoring the determinants of efficient viral replication using Classical Swine Fever Virus-reporter replicons

    DEFF Research Database (Denmark)

    Risager, Peter Christian; Everett, Helen; Crooke, Helen

    2012-01-01

    Classical swine fever virus (CSFV) is the etiological agent of the severe porcine disease, classical swine fever. Unraveling the molecular determinants of efficient replication is crucial for gaining improved knowledge of the pathogenic features of this virus. Monitoring the replication competence...... of the CSFV genome within cells can be achieved using autonomously replicating constructs (replicons) containing a reporter gene that expresses a readily quantifiable enzyme. Here, a newly implemented cloning technique was applied to genome modification of the fulllength CSFV cDNA previously inserted...... proteins considered non-essential for RNA replication were constructed and these deletions were replaced with an in-frame insertion of the Renilla luciferase (Rluc) sequence. RNA transcripts from these replicons should be translated as a single functional open reading frame. Full-genome cDNAs (~10-12,3 kb...

  15. Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    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.

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

    Science.gov (United States)

    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

  18. Well-tolerated Spirulina extract inhibits influenza virus replication and reduces virus-induced mortality

    Science.gov (United States)

    Chen, Yi-Hsiang; Chang, Gi-Kung; Kuo, Shu-Ming; Huang, Sheng-Yu; Hu, I-Chen; Lo, Yu-Lun; Shih, Shin-Ru

    2016-01-01

    Influenza is one of the most common human respiratory diseases, and represents a serious public health concern. However, the high mutability of influenza viruses has hampered vaccine development, and resistant strains to existing anti-viral drugs have also emerged. Novel anti-influenza therapies are urgently needed, and in this study, we describe the anti-viral properties of a Spirulina (Arthrospira platensis) cold water extract. Anti-viral effects have previously been reported for extracts and specific substances derived from Spirulina, and here we show that this Spirulina cold water extract has low cellular toxicity, and is well-tolerated in animal models at one dose as high as 5,000 mg/kg, or 3,000 mg/kg/day for 14 successive days. Anti-flu efficacy studies revealed that the Spirulina extract inhibited viral plaque formation in a broad range of influenza viruses, including oseltamivir-resistant strains. Spirulina extract was found to act at an early stage of infection to reduce virus yields in cells and improve survival in influenza-infected mice, with inhibition of influenza hemagglutination identified as one of the mechanisms involved. Together, these results suggest that the cold water extract of Spirulina might serve as a safe and effective therapeutic agent to manage influenza outbreaks, and further clinical investigation may be warranted. PMID:27067133

  19. Well-tolerated Spirulina extract inhibits influenza virus replication and reduces virus-induced mortality.

    Science.gov (United States)

    Chen, Yi-Hsiang; Chang, Gi-Kung; Kuo, Shu-Ming; Huang, Sheng-Yu; Hu, I-Chen; Lo, Yu-Lun; Shih, Shin-Ru

    2016-04-12

    Influenza is one of the most common human respiratory diseases, and represents a serious public health concern. However, the high mutability of influenza viruses has hampered vaccine development, and resistant strains to existing anti-viral drugs have also emerged. Novel anti-influenza therapies are urgently needed, and in this study, we describe the anti-viral properties of a Spirulina (Arthrospira platensis) cold water extract. Anti-viral effects have previously been reported for extracts and specific substances derived from Spirulina, and here we show that this Spirulina cold water extract has low cellular toxicity, and is well-tolerated in animal models at one dose as high as 5,000 mg/kg, or 3,000 mg/kg/day for 14 successive days. Anti-flu efficacy studies revealed that the Spirulina extract inhibited viral plaque formation in a broad range of influenza viruses, including oseltamivir-resistant strains. Spirulina extract was found to act at an early stage of infection to reduce virus yields in cells and improve survival in influenza-infected mice, with inhibition of influenza hemagglutination identified as one of the mechanisms involved. Together, these results suggest that the cold water extract of Spirulina might serve as a safe and effective therapeutic agent to manage influenza outbreaks, and further clinical investigation may be warranted.

  20. Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

    Science.gov (United States)

    Friebe, Peter; Lohmann, Volker; Krieger, Nicole; Bartenschlager, Ralf

    2001-01-01

    Sequences in the 5′ and 3′ termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5′ end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5′ NTR required for RNA replication. We show that deletions introduced into the 5′ terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5′ NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5′ NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5′ NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus. PMID:11711595

  1. Polyamine biosynthesis and the replication of turnip yellow mosaic virus

    International Nuclear Information System (INIS)

    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 6 virions per cell and continue to produce virus in culture for at least 48 hours. [ 14 C]-Spermidine (10 μM) 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 [ 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

  2. Transfection of RNA from organ samples of infected animals represents a highly sensitive method for virus detection and recovery of classical swine fever virus.

    Science.gov (United States)

    Meyer, Denise; Schmeiser, Stefanie; Postel, Alexander; Becher, Paul

    2015-01-01

    Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.

  3. Calcium spirulan, an inhibitor of enveloped virus replication, from a blue-green alga Spirulina platensis.

    Science.gov (United States)

    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.

  4. Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes

    Science.gov (United States)

    Dembowski, Jill A.

    2017-01-01

    Herpes simplex virus type 1 (HSV-1) infects over half the human population. Much of the infectious cycle occurs in the nucleus of cells where the virus has evolved mechanisms to manipulate host processes for the production of virus. The genome of HSV-1 is coordinately expressed, maintained, and replicated such that progeny virions are produced within 4–6 hours post infection. In this study, we selectively purify HSV-1 replication forks and associated proteins from virus-infected cells and identify select viral and cellular replication, repair, and transcription factors that associate with viral replication forks. Pulse chase analyses and imaging studies reveal temporal and spatial dynamics between viral replication forks and associated proteins and demonstrate that several DNA repair complexes and key transcription factors are recruited to or near replication forks. Consistent with these observations we show that the initiation of viral DNA replication is sufficient to license late gene transcription. These data provide insight into mechanisms that couple HSV-1 DNA replication with transcription and repair for the coordinated expression and maintenance of the viral genome. PMID:28095497

  5. Equine infectious anemia virus replication is upregulated during differentiation of blood monocytes from acutely infected horses.

    Science.gov (United States)

    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

  6. Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication

    OpenAIRE

    Bateman, Allen C.; Karasin, Alexander I.; Olsen, Christopher W.

    2012-01-01

    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 fa...

  7. Accessory Genes Confer a High Replication Rate to Virulent Feline Immunodeficiency Virus

    OpenAIRE

    Troyer, Ryan M.; Thompson, Jesse; Elder, John H.; VandeWoude, Sue

    2013-01-01

    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 tw...

  8. Valganciclovir for the Suppression of Epstein-Barr Virus Replication.

    Science.gov (United States)

    Yager, Jessica E; Magaret, Amalia S; Kuntz, Steven R; Selke, Stacy; Huang, Meei-Li; Corey, Lawrence; Casper, Corey; Wald, Anna

    2017-07-15

    Epstein-Barr virus (EBV) causes infectious mononucleosis and can lead to lymphoproliferative diseases. We evaluated the effects of valganciclovir on oral EBV shedding in a randomized, double-blind, placebo-controlled study. Twenty-six men received oral valganciclovir or daily placebo for 8 weeks, followed by a 2-week "washout period" and then 8 weeks of the alternative treatment. Valganciclovir reduced the proportion of days with EBV detected from 61.3% to 17.8% (relative risk, 0.28; 95% confidence interval [CI], .21-.41; P virus detected by 0.77 logs (95% CI, .62-.91 logs; P < .001). Further investigations into the impact of valganciclovir on EBV-associated diseases are needed. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  9. Human cytomegalovirus renders cells non-permissive for replication of herpes simplex viruses

    International Nuclear Information System (INIS)

    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 [ 3 H]-labeled HSV-1-superinfected cells

  10. Expression of Factor X in BHK-21 Cells Promotes Low Pathogenic Influenza Viruses Replication

    Directory of Open Access Journals (Sweden)

    Shahla Shahsavandi

    2015-01-01

    Full Text Available A cDNA clone for factor 10 (FX isolated from chicken embryo inserted into the mammalian cell expression vector pCDNA3.1 was transfected into the baby hamster kidney (BHK-21 cell line. The generated BHK-21 cells with inducible expression of FX were used to investigate the efficacy of the serine transmembrane protease to proteolytic activation of influenza virus hemagglutinin (HA with monobasic cleavage site. Data showed that the BHK-21/FX stably expressed FX after ten serial passages. The cells could proteolytically cleave the HA of low pathogenic avian influenza virus at multiplicity of infection 0.01. Growth kinetics of the virus on BHK-21/FX, BHK-21, and MDCK cells were evaluated by titrations of virus particles in each culture supernatant. Efficient multicycle viral replication was markedly detected in the cell at subsequent passages. Virus titration demonstrated that BHK-21/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in BHK-21 or MDCK cells with TPCK-trypsin. The results indicate potential application for the BHK-21/FX in influenza virus replication procedure and related studies.

  11. Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro

    Directory of Open Access Journals (Sweden)

    Raquel Elvira Ocazionez

    2010-05-01

    Full Text Available 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 37ºC 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 μg/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 μg/mL and between 1.9-33.7 μg/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.

  12. Tissue Sites of Persistent Infection and Active Replication of Equine Infectious Anemia Virus during Acute Disease and Asymptomatic Infection in Experimentally Infected Equids

    Science.gov (United States)

    Harrold, Sharon M.; Cook, Sheila J.; Cook, R. Frank; Rushlow, Keith E.; Issel, Charles J.; Montelaro, Ronald C.

    2000-01-01

    Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic infections in long-term inapparent carriers. To characterize the in vivo site(s) of viral infection and replication, we developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral singly spliced mRNA. Animals were experimentally infected with one of two reference viruses: the animal-passaged field isolate designated EIAVWyo and the virulent cell-adapted strain designated EIAVPV. Tissues and blood cells were isolated during the initial acute disease or from asymptomatic animals and analyzed for viral DNA and RNA levels by the respective quantitative assays. The results of these experiments demonstrated that the appearance of clinical symptoms in experimentally infected equids coincided with rapid widespread seeding of viral infection and replication in a variety of tissues. During acute disease, the predominant cellular site of viral infection and replication was the spleen, which typically accounted for over 90% of the cellular viral burden. In asymptomatic animals, viral DNA and RNA persisted in virtually all tissues tested, but at extremely low levels, a finding indicative of tight but incomplete immune control of EIAV replication. During all disease states, peripheral blood mononuclear cells (PBMC) were found to harbor less than 1% of

  13. FKBP8 interact with classical swine fever virus NS5A protein and promote virus RNA replication.

    Science.gov (United States)

    Li, Helin; Zhang, Chengcheng; Cui, Hongjie; Guo, Kangkang; Wang, Fang; Zhao, Tianyue; Liang, Wulong; Lv, Qizhuang; Zhang, Yanming

    2016-02-01

    The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling and host cellular responses via to its ability to interact with various cellular proteins. FKBP8 is also reported to promote virus replication. Here, we show that NS5A specifically interacts with FKBP8 through coimmunoprecipitation and GST-pulldown studies. Additionally, confocal microscopy study showed that NS5A and FKBP8 colocalized in the cytoplasm. Overexpression of FKBP8 via the eukaryotic expression plasmid pDsRED N1 significantly promoted viral RNA synthesis. The cells knockdown of FKBP8 by lentivirus-mediated shRNA markedly decreased the virus replication when infected with CSFV. These data suggest that FKBP8 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of FKBP8 protein functions may be beneficial for developing new strategies to treat CSFV infection.

  14. West Nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion.

    Science.gov (United States)

    Ambrose, Rebecca L; Mackenzie, Jason M

    2011-03-01

    For intracellular survival it is imperative that viruses have the capacity to manipulate various cellular responses, including metabolic and biosynthetic pathways. The unfolded protein response (UPR) is induced by various external and internal stimuli, including the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Our previous studies have indicated that the replication and assembly of the flavivirus West Nile virus strain Kunjin virus (WNV(KUN)) is intimately associated with the ER. Thus, we sought to determine whether the UPR was induced during WNV(KUN) infection. WNV(KUN) induces UPR signaling during replication, which is coordinated with peak replication. Interestingly, signaling is biased toward the ATF6/IRE-1 arm of the response, with high levels of Xbp-1 activation but negligible eukaryotic translation initiation factor 2α phosphorylation and downstream transcription. We show that the PERK-mediated response may partially regulate replication, since external UPR stimulation had a limiting effect on early replication events and cells deficient for PERK demonstrated increased replication and virus release. Significantly, we show that the WNV(KUN) hydrophobic nonstructural proteins NS4A and NS4B are potent inducers of the UPR, which displayed a high correlation in inhibiting Jak-STAT signaling in response to alpha interferon (IFN-α). Sequential removal of the transmembrane domains of NS4A showed that reducing hydrophobicity decreased UPR signaling and restored IFN-α-mediated activation. Overall, these results suggest that WNV(KUN) can stimulate the UPR to facilitate replication and that the induction of a general ER stress response, regulated by hydrophobic WNV(KUN) proteins, can potentiate the inhibition of the antiviral signaling pathway.

  15. Avian H11 influenza virus isolated from domestic poultry in a Colombian live animal market.

    Science.gov (United States)

    Jiménez-Bluhm, Pedro; Karlsson, Erik A; Ciuoderis, Karl A; Cortez, Valerie; Marvin, Shauna A; Hamilton-West, Christopher; Schultz-Cherry, Stacey; Osorio, Jorge E

    2016-12-07

    Live animal markets (LAMs) are an essential source of food and trade in Latin American countries; however, they can also serve as 'hotbeds' for the emergence and potential spillover of avian influenza viruses (AIV). Despite extensive knowledge of AIV in Asian LAMs, little is known about the prevalence South American LAMs. To fill this gap in knowledge, active surveillance was carried out at the major LAM in Medellin, Colombia between February and September 2015. During this period, overall prevalence in the market was 2.67% and a North American origin H11N2 AIV most similar to a virus isolated from Chilean shorebirds asymptomatically spread through multiple bird species in the market resulting in 17.0% positivity at peak of infection. Phenotypically, the H11 viruses displayed no known molecular markers associated with increased virulence in birds or mammals, had α2,3-sialic acid binding preference, and caused minimal replication in vitro and little morbidity in vivo. However, the Colombian H11N2 virus replicated and transmitted effectively in chickens explaining the spread throughout the market. Genetic similarity to H11 viruses isolated from North and South American shorebirds suggest that the LAM occurrence may have resulted from a wild bird to domestic poultry spillover event. The ability to spread in domestic poultry as well as potential for human infection by H11 viruses highlight the need for enhanced AIV surveillance in South America in both avian species and humans.

  16. A radioimmunoassay for anti-virus antibodies in farm animals

    International Nuclear Information System (INIS)

    Rodak, L.; Smid, B.; Sedlacek, M.

    1978-01-01

    A radioimmunoassay for determination of antibodies to Aujeszky's disease virus in piq serum is described. The results show a number of advantaqes of this method over the routinely employed virus-neutralization test. The possibility of using the RIA method in diagnosing other viral diseases of farm animals is suggested. (authors)

  17. Essential Role of Rta in Lytic DNA Replication of Epstein-Barr Virus

    Science.gov (United States)

    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. PMID:23077295

  18. Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture

    Science.gov (United States)

    Lohmann, Volker; Hoffmann, Sandra; Herian, Ulrike; Penin, Francois; Bartenschlager, Ralf

    2003-01-01

    Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself. PMID:12584326

  19. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    Directory of Open Access Journals (Sweden)

    Po-Yuan Ke

    Full Text Available 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 (coreceptor. 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.

  20. Structure, function and evolution of the animal mitochondrial replicative DNA helicase.

    Science.gov (United States)

    Kaguni, Laurie S; Oliveira, Marcos T

    2016-01-01

    The mitochondrial replicative DNA helicase is essential for animal mitochondrial DNA (mtDNA) maintenance. Deleterious mutations in the gene that encodes it cause mitochondrial dysfunction manifested in developmental delays, defects and arrest, limited life span, and a number of human pathogenic phenotypes that are recapitulated in animals across taxa. In fact, the replicative mtDNA helicase was discovered with the identification of human disease mutations in its nuclear gene, and based upon its deduced amino acid sequence homology with bacteriophage T7 gene 4 protein (T7 gp4), a bi-functional primase-helicase. Since that time, numerous investigations of its structure, mechanism, and physiological relevance have been reported, and human disease alleles have been modeled in the human, mouse, and Drosophila systems. Here, we review this literature and draw evolutionary comparisons that serve to shed light on its divergent features.

  1. Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication.

    Science.gov (United States)

    Bateman, Allen C; Karasin, Alexander I; Olsen, Christopher W

    2013-03-01

    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. To optimize growth conditions for differentiated swine airway epithelial cultures and to use these cultures to examine influenza virus infection and replication. 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. 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 100nm 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 of influenza viruses in the swine respiratory epithelium, and studying other swine respiratory diseases. © 2012 Blackwell Publishing Ltd.

  2. Aspirin-like molecules that inhibit human immunodeficiency virus 1 replication

    NARCIS (Netherlands)

    Pereira, Cândida F.; Paridaen, Judith T. M. L.; Rutten, Karla; Huigen, Marleen C. D. G.; van de Bovenkamp, Marja; Middel, Jeena; Beerens, Nancy; Berkhout, Ben; Schuurman, Rob; Marnett, Lawrence J.; Verhoef, Jan; Nottet, Hans S. L. M.

    2003-01-01

    Some anti-inflammatory molecules are also known to possess anti-human immunodeficiency virus (HIV) activity. We found that o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS), a recently synthesized non-steroidal anti-inflammatory molecule can inhibit HIV-1 replication. The aim of this study was to clarify

  3. Role of human GRP75 in miRNA mediated regulation of dengue virus replication.

    Science.gov (United States)

    Kakumani, Pavan Kumar; Medigeshi, Guruprasad R; Kaur, Inderjeet; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2016-07-15

    In recent times, RNAi has emerged as an important defence system that regulates replication of pathogens in host cells. Many RNAi related host factors especially the host miRNAs play important roles in all intrinsic cellular functions, including viral infection. We have been working on identification of mammalian host factors involved in Dengue virus infection. In the present study, we identified Glucose Regulated Protein 75kDa (GRP75), as a host factor that is associated with dicer complex, in particular with HADHA (trifunctional enzyme subunit alpha, mitochondrial), an auxiliary component of dicer complex. Knockdown of GRP75 by respective siRNAs in Huh-7 cells resulted in the accumulation of dengue viral genomic RNA suggesting a role of GRP75 in regulating dengue virus replication in human cell lines. To elucidate the mode of action of GRP75, we over expressed the protein in Huh-7 cells and analysed the host miRNAs processing. The results revealed that, GRP75 is involved in processing of host miRNA, hsa-mir-126, that down regulates dengue virus replication. These findings suggest a regulatory role of human miRNA pathway especially GRP75 protein and hsa-mir-126 in dengue virus replication. These results thus provide insights into the role of miRNAs and RNAi machinery in dengue life cycle. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes

    NARCIS (Netherlands)

    Taschner, P. E.; van der Kuyl, A. C.; Neeleman, L.; Bol, J. F.

    1991-01-01

    RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins P1 and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (P1-plants), RNA2 (P2-plants) or a combination of these two cDNAs (P12-plants). All transgenic

  5. Effects of morphine on replication of herpes simplex virus type 1 and 2

    African Journals Online (AJOL)

    USER

    2009-05-17

    May 17, 2009 ... virus genome has a double strand DNA which codes over. 70 gene products. HSV infection is the most ... essential for viral replication, unlike viral DNA poly- merase. It seems that an alternative method of ... tral red was used and plaques were counted after 12 h. Determination of morphine cytotoxicity.

  6. Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication.

    Science.gov (United States)

    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

  7. New models of hepatitis E virus replication in human and porcine hepatocyte cell lines

    Science.gov (United States)

    Hepatitis E virus (HEV) causes acute, enterically-transmitted hepatitis. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studie...

  8. Replication-Deficient Particles: New Insights into the Next Generation of Bleutongue Virus Vaccines

    NARCIS (Netherlands)

    Celma, Cristina C.; Stewart, Meredith; Wernike, Kerstine; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; Clercq, De Kris; Rijn, van P.A.

    2017-01-01

    Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe haemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus

  9. Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Fangke; Chen, Yun; Shi, Jintong; Ming, Ke; Liu, Jiaguo, E-mail: liujiaguo@njau.edu.cn; Xiong, Wen; Song, Meiyun; Du, Hongxu; Wang, Yixuan; Zhang, Shuaibin; Wu, Yi; Wang, Deyun; Hu, Yuanliang

    2016-04-15

    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 5 h, 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. - Highlights: • This is the first description of the replication cycle of DHAV-1. • Firstly find that DHAV-1 adsorption saturated at 90 min post-infection. • The replication lasted around 13 h after early protein synthesis for about 5 h. • The release of DHAV-1 was in steady state after 32 h.

  10. Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes

    International Nuclear Information System (INIS)

    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

    2016-01-01

    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 5 h, 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. - Highlights: • This is the first description of the replication cycle of DHAV-1. • Firstly find that DHAV-1 adsorption saturated at 90 min post-infection. • The replication lasted around 13 h after early protein synthesis for about 5 h. • The release of DHAV-1 was in steady state after 32 h.

  11. Flavone Enhances Dengue Virus Type-2 (NGC Strain Infectivity and Replication in Vero Cells

    Directory of Open Access Journals (Sweden)

    Keivan Zandi

    2012-02-01

    Full Text Available This study investigates the effects of 2-phenyl-1-benzopyran-4-one (flavone on DENV-2 infectivity in Vero cells. Virus adsorption and attachment and intracellular virus replication were investigated using a foci forming unit assay (FFUA and quantitative RT-PCR, respectively. Addition of flavone (100 μg/mL significantly increased the number of DENV-2 foci by 35.66% ± 1.52 and 49.66% ± 2.51 when added during and after virus adsorption to the Vero cells, respectively. The average foci size after 4 days of infection increased by 33% ± 2.11 and 89% ± 2.13. The DENV-2 specific RNA copy number in the flavone-treated infected cells increased by 6.41- and 23.1-fold when compared to the mock-treated infected cells. Flavone (100 μg/mL did not promote or inhibit Vero cell proliferation. The CC50 value of flavone against Vero cells was 446 µg/mL. These results suggest that flavone might enhance dengue virus replication by acting antagonistically towards flavonoids known to inhibit dengue virus replication.

  12. [Determination of genomic and replicative RNA of hepatitis C virus in patients treated with interferon].

    Science.gov (United States)

    Zeilicoff, R; Ameigeiras, B; Ojeda, E; Isla Rodríguez, R; Grünbaum, S; Genero, M; Cappelletti, C; Tielli, G; Roatta, R; Koch, O

    1995-01-01

    We have investigated the presence of genomic and replicative RNA strands of hepatitis C virus in liver and serum. Eleven patients with proven chronic hepatitis C, received Interferon a2a 4,5 MU, three times a week during six months. RT-PCR was used with sense primer to detect the replicative strand and an antisense primer to identify genomic strand. Before treatment, genomic strands were present in liver and serum of all patients. Replicative strands were present in liver and serum in five and six cases, respectively. Seven out of eleven responded to treatment. In responders, genomic strands were absent in liver of 3 cases (43%) and replicative strands in liver of 4 (57%). In plasma genomic and replicative strands were absent in 5 (71%) and 7 (100%), respectively. In all non responders, genomic strands in liver and plasma remained present. Replicative strands in liver and plasma were present in 100% and 25%, respectively. Knodell score improved in 5 out of 7 responders and remained unchanged in 3 out of 4 non responders. In 2 out of 4 responders with genomic and replicative strands in liver, Knodell score remained unchanged or worse. In all non responders, genomic and replicative strands in liver were present and Knodell score remained unchanged or worse. Genomic and replicative strands in plasma tended to be negative after treatment in responders. Genomic strands in plasma remained present in non responders. Conversely, genomic and replicative strands in liver were present in all non responders. It seems to exist a relationship between genomic and replicative strands in liver and the same or worse Knodell score. After a follow up, it will be possible to determined whether responders who still present viral RNA in liver would be prone to a relapse.

  13. NF90 Binds the Dengue Virus RNA 3′ Terminus and Is a Positive Regulator of Dengue Virus Replication

    Science.gov (United States)

    Gehrke, Lee

    2011-01-01

    Background Viral RNA translation and replication are regulated by sequence and structural elements in the 5′ and 3′ untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5′ m7GpppG cap, and a conserved 3′-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3′ terminus. Methodology/Principal Findings Proteins eluted from a dengue 3′ SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny. Conclusions/Significance The results indicate that NF90 interacts with the 3′ SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide. PMID:21386893

  14. Inhibition of hepatitis B virus and human immunodeficiency virus (HIV-1) replication by Warscewiczia coccinea (Vahl) Kl. (Rubiaceae) ethanol extract.

    Science.gov (United States)

    Quintero, A; Fabbro, R; Maillo, M; Barrios, M; Milano, M B; Fernández, A; Williams, B; Michelangeli, F; Rangel, H R; Pujol, F H

    2011-09-01

    The primary objective of this study was to search for natural products capable of inhibiting hepatitis B virus (HBV) replication. The research design, methods and procedures included testing hydro-alcoholic extracts (n = 66) of 31 species from the Venezuelan Amazonian rain forest on the cell line HepG2 2.2.15, which constitutively produces HBV. The main outcomes and results were as follows: the species Euterpe precatoria, Jacaranda copaia, Jacaranda obtusifolia, Senna silvestris, Warscewiczia coccinea and Vochysia glaberrima exerted some degree of inhibition on HBV replication. The leaves of W. coccinea showed a significant antiviral activity: 80% inhibition with 100 µg mL⁻¹ of extract. This extract also exerted inhibition on covalently closed circular deoxyribonucleic acid (cccDNA) production and on HIV-1 replication in MT4 cells (more than 90% inhibition with 50 µg mL⁻¹ of extract). Initial fractionation using organic solvents of increasing polarity and water showed that the ethanol fraction was responsible for most of the antiviral inhibitory activities of both the viruses. It was concluded that Warscewiczia coccinea extract showed inhibition of HBV and HIV-1 replication. Bioassay-guided purification of this fraction may allow the isolation of an antiviral compound with inhibitory activity against both viruses.

  15. Treatment with PTEN-Long protein inhibits hepatitis C virus replication.

    Science.gov (United States)

    Wu, Qi; Li, Zhubing; Liu, Qiang

    2017-11-01

    Hepatitis C virus (HCV) infection is a confirmed risk factor for hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) possesses tumor suppression function that is frequently defective in HCC tumors. PTEN-Long, a translation isoform of PTEN, functions in a cell non-autonomous manner. In this study, we demonstrated that intracellular overexpression of PTEN-Long inhibits HCV replication. More importantly, we showed that treatment with extracellular PTEN-Long protein inhibits HCV replication in a dose-dependent manner. Furthermore, we showed that PTEN-Long interacts with HCV core protein and this interaction is required for HCV replication inhibition by PTEN-Long. In summary, we demonstrated, for the first time, that PTEN-Long protein, an isoform of the canonical PTEN and in the form of extracellular protein treatment, inhibits HCV replication. Our study offers an opportunity for developing additional anti-HCV agents. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Recombinant subgroup B human respiratory syncytial virus expressing enhanced green fluorescent protein efficiently replicates in primary human cells and is virulent in cotton rats.

    Science.gov (United States)

    Lemon, Ken; Nguyen, D Tien; Ludlow, Martin; Rennick, Linda J; Yüksel, Selma; van Amerongen, Geert; McQuaid, Stephen; Rima, Bert K; de Swart, Rik L; Duprex, W Paul

    2015-03-01

    Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies. Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with

  17. ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes.

    Science.gov (United States)

    Yang, Wengang; Sun, Yongnian; Hou, Xiaohong; Zhao, Yongsen; Fabrycki, Joanne; Chen, Dawei; Wang, Xiangzhu; Agarwal, Atul; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2013-07-01

    Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

  18. Up-regulation of hepatitis C virus replication by human T cell leukemia virus type I-encoded Tax protein.

    Science.gov (United States)

    Zhang, Jing; Yamada, Osamu; Kawagishi, Kenji; Yoshida, Hiroshi; Araki, Hiromasa; Yamaoka, Shoji; Hattori, Toshio; Shimotohno, Kunitada

    2007-12-05

    Co-infection of hepatitis C virus (HCV) with other blood-borne pathogens such as human T cell leukemia virus (HTLV) is common in highly endemic areas. Clinical evidence showing a correlation between HTLV-I co-infection and rapid progression of HCV-associated liver disease promoted us to investigate the effect of HTLV-I-encoded Tax protein on HCV replication. Reporter assay showed that HCV replicon-encoded luciferase expression was significantly augmented by co-transfection of the Tax-expressing plasmid. Further, HCV RNA replication in replicon cells was increased either by co-culture with cells stably expressing Tax protein (Huhtax) or by culture in the presence of Huhtax-conditioned medium, indicating that Tax could also modulate HCV replication of adjacent cells in a paracrine manner. Additionally, HCV replication in Huhtax exhibited a reduced responsiveness to interferon-alpha-induced antiviral activity. This study demonstrates the facilitation of HCV replication by Tax protein, which may partially account for severer clinical consequences of HCV-related disease in HCV/HTLV co-infected individuals.

  19. Role of ATM in the Formation of the Replication Compartment during Lytic Replication of Epstein-Barr Virus in Nasopharyngeal Epithelial Cells

    Science.gov (United States)

    Hau, Pok Man; Deng, Wen; Jia, Lin; Yang, Jie; Tsurumi, Tatsuya; Chiang, Alan Kwok Shing; Huen, Michael Shing-Yan

    2014-01-01

    ABSTRACT Epstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that lytic reactivation of EBV in latently infected cells induces an ATM-dependent DNA damage response (DDR). The involvement of ATM activation has been implicated in inducing viral lytic gene transcription to promote lytic reactivation. Its contribution to the formation of a replication compartment during lytic reactivation of EBV remains poorly defined. In this study, the role of ATM in viral DNA replication was investigated in EBV-infected nasopharyngeal epithelial cells. We observed that induction of lytic infection of EBV triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using a small interfering RNA (siRNA) approach or a specific chemical inhibitor profoundly suppressed replication of EBV DNA and production of infectious virions in EBV-infected cells induced to undergo lytic reactivation. We further showed that phosphorylation of Sp1 at the serine-101 residue is essential in promoting the accretion of EBV replication proteins at the replication compartment, which is crucial for replication of viral DNA. Knockdown of Sp1 expression by siRNA effectively suppressed the replication of viral DNA and localization of EBV replication proteins to the replication compartments. Our study supports an important role of ATM activation in lytic reactivation of EBV in epithelial cells, and phosphorylation of Sp1 is an essential process downstream of ATM activation involved in the formation of viral replication compartments. Our study revealed an essential role of the ATM-dependent DDR pathway in lytic reactivation of EBV, suggesting a potential antiviral replication strategy using specific DDR inhibitors. IMPORTANCE Epstein-Barr virus (EBV) is closely associated with human malignancies, including undifferentiated nasopharyngeal carcinoma (NPC), which has a high

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

    Science.gov (United States)

    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.

  1. Inhibition of RNA recruitment and replication of an RNA virus by acridine derivatives with known anti-prion activities.

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    Zsuzsanna Sasvari

    Full Text Available BACKGROUND: Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV, a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ and quinacrine (QC, which are active against prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii reduction of minus-strand synthesis by the tombusvirus replicase; and (iii inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. CONCLUSION/SIGNIFICANCE: Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.

  2. Inhibition of RNA Recruitment and Replication of an RNA Virus by Acridine Derivatives with Known Anti-Prion Activities

    Science.gov (United States)

    Sasvari, Zsuzsanna; Bach, Stéphane; Blondel, Marc; Nagy, Peter D.

    2009-01-01

    Background Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases. Methodology/Principal Findings Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. Conclusion/Significance Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses. PMID:19823675

  3. Replication, tissue tropisms and transmission of yellow fever virus in Aedes albopictus.

    Science.gov (United States)

    Miller, B R; Mitchell, C J; Ballinger, M E

    1989-01-01

    Experimental studies undertaken to ascertain the dynamics of yellow fever virus replication in an introduced strain (Houston) of the Asian mosquito, Aedes albopictus (Skuse), indicate that this species is an efficient vector of yellow fever virus. Replication of virus in Ae. albopictus could be detected 3 d after feeding on a suspension containing 7.2 log10 Vero cell plaque forming units (PFU) per ml of virus; peak titres (3.5 log10 PFU/mosquito) occurred 7 d after exposure. Viral antigen, visualized by immunofluorescence, was first detected in midgut cells 4 d after exposure and appeared in fat cells 7 d after exposure. The only other mosquito tissues revealing viral antigen were the salivary glands, brain, and occasionally cells of the suboesophageal ganglion. Viral antigen was not detected in any of the tissues of the reproductive tract, nor could viral genomic ribonucleic acid (RNA) be detected in these tissues by RNA-RNA molecular hybridization in situ. We detected no vertical transmission of yellow fever virus in 6180 F1 adult progeny produced from infected females. The extrinsic incubation period at 26.7 degrees C was 9 d. We conclude that the Houston strain of Ae. albopictus is a competent vector of yellow fever virus and can serve as bridging vector between the jungle yellow fever cycle and the urban cycle in New World ecosystems.

  4. INHIBITION OF MAYARO VIRUS REPLICATION BY PROSTAGLANDIN A1 IN Aedes albopictus CELLS

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    Barbosa Joel Antonio

    1998-01-01

    Full Text Available Prostaglandin A1 (PGA1 inhibits Mayaro virus replication in Aedes albopictus cells at nontoxic doses to uninfected cells. At 10 µg/ml, PGA1 decreases virus production by 90%. The presence of PGA1 during virus adsorption, with no treatment after infection, reduces virus yield by 41%. Antiviral activity is observed even when treatment starts at one or two hours post-infection. However, in cells pre-treated with PGA1 during 24 hours, virus replication is not impaired. Thus, events ocurring during initial stages of infection and after virus adsorption and penetration must be the target of PGA1 action. SDS-PAGE analysis of 35S-methionine labelled proteins shows that PGA1 inhibits the synthesis of viral proteins and induces the synthesis of polypeptides with molecular weight of 70 kDa, 57 kDa and 23 kDa. In cells pre-treated with actinomycin D the induction of those proteins is suppressed. In addition, actinomycin D treatment prevents PGA1antiviral activity, indicating that PGA1-induced stress proteins are probably involved in this mechanism.

  5. Identification of a cluster of HIV-1 controllers infected with low replicating viruses.

    Science.gov (United States)

    Casado, Concepción; Pernas, Maria; Sandonis, Virginia; Alvaro-Cifuentes, Tamara; Olivares, Isabel; Fuentes, Rosa; Martínez-Prats, Lorena; Grau, Eulalia; Ruiz, Lidia; Delgado, Rafael; Rodríguez, Carmen; del Romero, Jorge; López-Galíndez, Cecilio

    2013-01-01

    Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70's-early 80's). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.

  6. The host factor RAD51 is involved in mungbean yellow mosaic India virus (MYMIV) DNA replication.

    Science.gov (United States)

    Suyal, Geetika; Mukherjee, Sunil K; Choudhury, Nirupam R

    2013-09-01

    Geminiviruses replicate their single-stranded genomes with the help of only a few viral factors and various host cellular proteins primarily by rolling-circle replication (RCR) and/or recombination-dependent replication. AtRAD51 has been identified, using the phage display technique, as a host factor that potentially interacts with the Rep protein of mungbean yellow mosaic India virus (MYMIV), a member of the genus Begomovirus. In this study, we demonstrate the interaction between MYMIV Rep and a host factor, AtRAD51, using yeast two-hybrid and β-galactosidase assays, and this interaction was confirmed using a co-immunoprecipitation assay. The AtRAD51 protein complemented the rad51∆ mutation of Saccharomyces cerevisiae in an ex vivo yeast-based geminivirus DNA replication restoration assay. The semiquantitative RT-PCR and northern hybridization data revealed a higher level of expression of the Rad51 transcript in MYMIV-infected mungbean than in uninfected, healthy plants. Our findings provide evidence for a possible cross-talk between RAD51 and MYMIV Rep, which essentially controls viral DNA replication in plants, presumably in conjunction with other host factors. The present study demonstrates for the first time the involvement of a eukaryotic RAD51 protein in MYMIV replication, and this is expected to shed light on the machinery involved in begomovirus DNA replication.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Early low-titer neutralizing antibodies impede HIV-1 replication and select for virus escape.

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    Katharine J Bar

    Full Text Available Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC(50 selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1-V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical

  9. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    Energy Technology Data Exchange (ETDEWEB)

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

    2015-01-15

    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. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  10. Intrinsic Innate Immunity Fails To Control Herpes Simplex Virus and Vesicular Stomatitis Virus Replication in Sensory Neurons and Fibroblasts

    Science.gov (United States)

    Rosato, Pamela C.

    2014-01-01

    ABSTRACT Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG), wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly, a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings, TG neurons responded to IFN-β pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in γ34.5, while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFN-β. Taken together, these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFN-β, and HSV-1 combats this response through γ34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFN-β signaling in establishing an antiviral state. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that establishes a lifelong latent infection in neurons. Reactivation from latency can cause cold sores, blindness, and death from encephalitis. Humans with deficiencies in innate immunity have significant problems controlling HSV infections. In this study, we therefore sought to elucidate the role of neuronal innate immunity in the control of viral infection. Using neurons isolated from mice, we found that the intrinsic capacity of neurons to restrict virus replication was unaffected by the presence

  11. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

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    Jessica C Casciano

    Full Text Available Many viruses modulate calcium (Ca2+ signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC, and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus

  12. Peretinoin, an Acyclic Retinoid, Inhibits Hepatitis B Virus Replication by Suppressing Sphingosine Metabolic Pathway In Vitro

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    Kazuhisa Murai

    2018-01-01

    Full Text Available Hepatocellular carcinoma (HCC frequently develops from hepatitis C virus (HCV and hepatitis B virus (HBV infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA, and all-trans-retinoic acid (ATRA, had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1 to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC.

  13. Microscopic Visualisation of Zoonotic Arbovirus Replication in Tick Cell and Organ Cultures Using Semliki Forest Virus Reporter Systems

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    Lesley Bell-Sakyi

    2016-09-01

    Full Text Available Ticks are vectors and reservoirs of many arboviruses pathogenic for humans or domestic animals; in addition, during bloodfeeding they can acquire and harbour pathogenic arboviruses normally transmitted by other arthropods such as mosquitoes. Tick cell and organ cultures provide convenient tools for propagation and study of arboviruses, both tick-borne and insect-borne, enabling elucidation of virus-tick cell interaction and yielding insight into the mechanisms behind vector competence and reservoir potential for different arbovirus species. The mosquito-borne zoonotic alphavirus Semliki Forest virus (SFV, which replicates well in tick cells, has been isolated from Rhipicephalus, Hyalomma, and Amblyomma spp. ticks removed from mammalian hosts in East Africa; however nothing is known about any possible role of ticks in SFV epidemiology. Here we present a light and electron microscopic study of SFV infecting cell lines and organ cultures derived from African Rhipicephalus spp. ticks. As well as demonstrating the applicability of these culture systems for studying virus-vector interactions, we provide preliminary evidence to support the hypothesis that SFV is not normally transmitted by ticks because the virus does not infect midgut cells.

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

    Science.gov (United States)

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

    2014-11-01

    Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. H2AX phosphorylation and DNA damage kinase activity are dispensable for herpes simplex virus replication.

    Science.gov (United States)

    Botting, Carolyn; Lu, Xu; Triezenberg, Steven J

    2016-01-27

    Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM(-/-) cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection.

  16. Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

    Science.gov (United States)

    Wargo, Andrew R.; Scott, Robert J.; Kerr, Benjamin; Kurath, Gael

    2017-01-01

    Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV

  17. Analysis of classical swine fever virus RNA replication determinants using replicons

    DEFF Research Database (Denmark)

    Risager, Peter Christian; Fahnøe, Ulrik; Gullberg, Maria

    2013-01-01

    Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome (BAC), was modified by targeted......), as well as by detection of the CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for the replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain...

  18. A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication

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    Mary E. Piper

    2010-03-01

    Full Text Available Rift Valley fever virus (RVFV is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication.

  19. A novel system for identification of inhibitors of rift valley Fever virus replication.

    Science.gov (United States)

    Piper, Mary E; Gerrard, Sonja R

    2010-03-01

    Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication.

  20. Lipid Tales of Viral Replication and Transmission.

    Science.gov (United States)

    Altan-Bonnet, Nihal

    2017-03-01

    Positive-strand RNA viruses are the largest group of RNA viruses on Earth and cellular membranes are critical for all aspects of their life cycle, from entry and replication to exit. In particular, membranes serve as platforms for replication and as carriers to transmit these viruses to other cells, the latter either as an envelope surrounding a single virus or as the vesicle containing a population of viruses. Notably, many animal and human viruses appear to induce and exploit phosphatidylinositol 4-phosphate/cholesterol-enriched membranes for replication, whereas many plant and insect-vectored animal viruses utilize phosphatidylethanolamine/cholesterol-enriched membranes for the same purpose; and phosphatidylserine-enriched membrane carriers are widely used by both single and populations of viruses for transmission. Here I discuss the implications for viral pathogenesis and therapeutic development of this remarkable convergence on specific membrane lipid blueprints for replication and transmission. Published by Elsevier Ltd.

  1. Conditional virus replication as an approach to a safe live attenuated human immunodeficiency virus vaccine

    NARCIS (Netherlands)

    Berkhout, Ben; Verhoef, Koen; Marzio, Giuseppe; Klaver, Bep; Vink, Monique; Zhou, Xue; Das, Atze T.

    2002-01-01

    Despite intensive efforts, no safe and effective vaccine has been developed for the prophylaxis of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Studies with the simian immunodeficiency virus (SIV)/macaque model demonstrated that live attenuated viruses are the most

  2. Bluetongue virus nonstructural protein NS3/NS3a is not essential for virus replication

    NARCIS (Netherlands)

    Gennip, van H.G.P.; Water, van de S.G.P.; Rijn, van P.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

  3. Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Barajas, Daniel; Xu, Kai; Sharma, Monika; Wu, Cheng-Yu; Nagy, Peter D., E-mail: pdnagy2@uky.edu

    2014-12-15

    Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells. - Highlights: • Tombusvirus p33 replication protein interacts with FFAT-domain host protein. • Tombusvirus replication leads to upregulation of phospholipids. • Tombusvirus replication depends on de novo lipid synthesis. • Deletion of FFAT-domain host protein enhances TBSV replication. • TBSV rewires host phospholipid synthesis.

  4. Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

    Science.gov (United States)

    Saito, Akatsuki; Henning, Matthew S.; Serrao, Erik; Dubose, Brittany N.; Teng, Samantha; Huang, Jing; Li, Xiangming; Saito, Namiko; Roy, Saumendra Prasad; Siddiqui, Mohammad Adnan; Ahn, Jinwoo; Tsuji, Moriya; Hatziioannou, Theodora; Engelman, Alan N.

    2016-01-01

    ABSTRACT Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4+ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. IMPORTANCE CPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for

  5. Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo.

    Science.gov (United States)

    Saito, Akatsuki; Henning, Matthew S; Serrao, Erik; Dubose, Brittany N; Teng, Samantha; Huang, Jing; Li, Xiangming; Saito, Namiko; Roy, Saumendra Prasad; Siddiqui, Mohammad Adnan; Ahn, Jinwoo; Tsuji, Moriya; Hatziioannou, Theodora; Engelman, Alan N; Yamashita, Masahiro

    2016-08-01

    Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4(+) T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. CPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and

  6. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  7. Uracil DNA glycosylase BKRF3 contributes to Epstein-Barr virus DNA replication through physical interactions with proteins in viral DNA replication complex.

    Science.gov (United States)

    Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei; Chen, Mei-Ru

    2014-08-01

    Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at

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

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Jokinen, Jenny; Lukashevich, Igor S. [Department of Pharmacology and Toxicology, School of Medicine, Center for Predictive Medicine and Emerging Infectious Diseases, University of Louisville, Louisville, KY (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2014-11-15

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

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

    Science.gov (United States)

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-02

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

  11. Intermolecular RNA Recombination Occurs at Different Frequencies in Alternate Forms of Brome Mosaic Virus RNA Replication Compartments

    Directory of Open Access Journals (Sweden)

    Hernan Garcia-Ruiz

    2018-03-01

    Full Text Available Positive-strand RNA viruses replicate their genomes in membrane-bound replication compartments. Brome mosaic virus (BMV replicates in vesicular invaginations of the endoplasmic reticulum membrane. BMV has served as a productive model system to study processes like virus-host interactions, RNA replication and recombination. Here we present multiple lines of evidence showing that the structure of the viral RNA replication compartments plays a fundamental role and that recruitment of parental RNAs to a common replication compartment is a limiting step in intermolecular RNA recombination. We show that a previously defined requirement for an RNA recruitment element on both parental RNAs is not to function as a preferred crossover site, but in order for individual RNAs to be recruited into the replication compartments. Moreover, modulating the form of the replication compartments from spherular vesicles (spherules to more expansive membrane layers increased intermolecular RNA recombination frequency by 200- to 1000-fold. We propose that intermolecular RNA recombination requires parental RNAs to be recruited into replication compartments as monomers, and that recruitment of multiple RNAs into a contiguous space is much more common for layers than for spherules. These results could explain differences in recombination frequencies between viruses that replicate in association with smaller spherules versus larger double-membrane vesicles and convoluted membranes.

  12. Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

    Science.gov (United States)

    Soto-Acosta, Rubén; Bautista-Carbajal, Patricia; Syed, Gulam H; Siddiqui, Aleem; Del Angel, Rosa M

    2014-09-01

    Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Yang, Hanchun, E-mail: yanghanchun1@cau.edu.cn [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Hu, Hongbo, E-mail: hongbo@cau.edu.cn [College of Food Science and Nutritional Engineering, China Agricultural University, Beijing (China)

    2012-08-01

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/{beta}-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome-lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  14. Luteolin restricts dengue virus replication through inhibition of the proprotein convertase furin.

    Science.gov (United States)

    Peng, Minhua; Watanabe, Satoru; Chan, Kitti Wing Ki; He, Qiuyan; Zhao, Ya; Zhang, Zhongde; Lai, Xiaoping; Luo, Dahai; Vasudevan, Subhash G; Li, Geng

    2017-07-01

    In many countries afflicted with dengue fever, traditional medicines are widely used as panaceas for illness, and here we describe the systematic evaluation of a widely known natural product, luteolin, originating from the "heat clearing" class of herbs. We show that luteolin inhibits the replication of all four serotypes of dengue virus, but the selectivity of the inhibition was weak. In addition, ADE-mediated dengue virus infection of human cell lines and primary PBMCs was inhibited. In a time-of-drug-addition study, luteolin was found to reduce infectious virus particle formation, but not viral RNA synthesis, in Huh-7 cells. During the virus life cycle, the host protease furin cleaves the pr moiety from prM protein of immature virus particles in the trans-Golgi network to produce mature virions. Analysis of virus particles from luteolin-treated cells revealed that prM was not cleaved efficiently. Biochemical interrogation of human furin showed that luteolin inhibited the enzyme activity in an uncompetitive manner, with Ki value of 58.6 μM, suggesting that treatment may restrict the virion maturation process. Luteolin also exhibited in vivo antiviral activity in mice infected with DENV, causing reduced viremia. Given the mode of action of luteolin and its widespread source, it is possible that it can be tested in combination with other dengue virus inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1 in the Suppression of Influenza A Virus Replication.

    Directory of Open Access Journals (Sweden)

    Xin Liu

    Full Text Available Influenza A virus causes annual epidemics and occasional pandemics in humans. Here, we investigated four members of the fibroblast growth factor receptor (FGFR family; FGFR1 to 4, and examined their expression patterns in human lung epithelial cells A549 with influenza A virus infection. We identified a functional role of FGFR1 in influenza A/Puerto Rico/8/1934 (PR8 and A/Anhui/01/2005 (H5N1 virus replication. Our results showed that FGFR1 silencing by siRNA interference promoted influenza A/PR8 and H5N1 virus replication in A549 cells, while lentivirus-mediated exogenous FGFR1 expression significantly suppressed influenza A virus replication; however, FGFR4 did not have the same effects. Moreover, FGFR1 phosphorylation levels were downregulated in A549 cells by influenza A virus infection, while the repression of FGFR1 kinase using PD173074, a potent and selective FGFR1 inhibitor, could enhance virus replication. Furthermore, we found that FGFR1 inhibits influenza virus internalization, but not binding, during viral entry. These results suggested that FGFR1 specifically antagonizes influenza A virus replication, probably by blocking viral entry.

  16. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

    Directory of Open Access Journals (Sweden)

    Philippa M Beard

    Full Text Available Vaccinia virus (VACV is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

  17. Rapid Titration of Measles and Other Viruses: Optimization with Determination of Replication Cycle Length

    Science.gov (United States)

    Grigorov, Boyan; Rabilloud, Jessica; Lawrence, Philip; Gerlier, Denis

    2011-01-01

    Background Measles virus (MV) is a member of the Paramyxoviridae family and an important human pathogen causing strong immunosuppression in affected individuals and a considerable number of deaths worldwide. Currently, measles is a re-emerging disease in developed countries. MV is usually quantified in infectious units as determined by limiting dilution and counting of plaque forming unit either directly (PFU method) or indirectly from random distribution in microwells (TCID50 method). Both methods are time-consuming (up to several days), cumbersome and, in the case of the PFU assay, possibly operator dependent. Methods/Findings A rapid, optimized, accurate, and reliable technique for titration of measles virus was developed based on the detection of virus infected cells by flow cytometry, single round of infection and titer calculation according to the Poisson's law. The kinetics follow up of the number of infected cells after infection with serial dilutions of a virus allowed estimation of the duration of the replication cycle, and consequently, the optimal infection time. The assay was set up to quantify measles virus, vesicular stomatitis virus (VSV), and human immunodeficiency virus type 1 (HIV-1) using antibody labeling of viral glycoprotein, virus encoded fluorescent reporter protein and an inducible fluorescent-reporter cell line, respectively. Conclusion Overall, performing the assay takes only 24–30 hours for MV strains, 12 hours for VSV, and 52 hours for HIV-1. The step-by-step procedure we have set up can be, in principle, applicable to accurately quantify any virus including lentiviral vectors, provided that a virus encoded gene product can be detected by flow cytometry. PMID:21915289

  18. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.

    Science.gov (United States)

    Romero-Brey, Inés; Merz, Andreas; Chiramel, Abhilash; Lee, Ji-Young; Chlanda, Petr; Haselman, Uta; Santarella-Mellwig, Rachel; Habermann, Anja; Hoppe, Simone; Kallis, Stephanie; Walther, Paul; Antony, Claude; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

    2012-01-01

    All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

  19. The role of PTEN - HCV core interaction in hepatitis C virus replication

    OpenAIRE

    Wu, Qi; Li, Zhubing; Mellor, Paul; Zhou, Yan; Anderson, Deborah H.; Liu, Qiang

    2017-01-01

    Hepatitis C virus (HCV) infection leads to severe liver diseases including hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumour suppressor, is frequently mutated or deleted in HCC tumors. PTEN has previously been demonstrated to inhibit HCV secretion. In this study, we determined the effects of PTEN on the other steps in HCV life cycle, including entry, translation, and replication. We showed that PTEN inhibits HCV entry through its lipid ph...

  20. Genome of the Acidianus bottle-shaped virus and insights into the replication and packaging mechanisms

    DEFF Research Database (Denmark)

    Peng, Xu; Basta, Tamara; Häring, Monika

    2007-01-01

    of the bacteriophage varphi29 and the human adenovirus. The region contains the genes for a putative protein-primed DNA polymerase, and a small putative RNA with a predicted secondary structure closely similar to that of the prohead RNA of bacteriophage varphi29. The apparent similarities in the putative mechanisms...... of DNA replication and packaging of ABV to those of bacterial and eukaryal viruses are most consistent with the concept of a primordial gene pool as a source of viral genes....

  1. Differential virulence mechanisms of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) include host entry and virus replication kinetics

    Science.gov (United States)

    Penaranda, M.M.D.; Purcell, M.K.; Kurath, G.

    2009-01-01

    Host specificity is a phenomenon exhibited by all viruses. For the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV), differential specificity of virus strains from the U and M genogroups has been established both in the field and in experimental challenges. In rainbow trout (Oncorhynchus mykiss), M IHNV strains are consistently more prevalent and more virulent than U IHNV. The basis of the differential ability of these two IHNV genogroups to cause disease in rainbow trout was investigated in live infection challenges with representative U and M IHNV strains. When IHNV was delivered by intraperitoneal injection, the mortality caused by U IHNV increased, indicating that the low virulence of U IHNV is partly due to inefficiency in entering the trout host. Analyses of in vivo replication showed that U IHNV consistently had lower prevalence and lower viral load than M IHNV during the course of infection. In analyses of the host immune response, M IHNV-infected fish consistently had higher and longer expression of innate immune-related genes such as Mx-1. This suggests that the higher virulence of M IHNV is not due to suppression of the immune response in rainbow trout. Taken together, the results support a kinetics hypothesis wherein faster replication enables M IHNV to rapidly achieve a threshold level of virus necessary to override the strong host innate immune response. ?? 2009 SGM.

  2. Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus.

    Science.gov (United States)

    Puglia, Ana L P; Rezende, Alexandre G; Jorge, Soraia A C; Wagner, Renaud; Pereira, Carlos A; Astray, Renato M

    2013-11-01

    Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

    Science.gov (United States)

    Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

    2017-09-15

    Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35

  4. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    International Nuclear Information System (INIS)

    Bernard, Eric; Hamel, Rodolphe; Neyret, Aymeric; Ekchariyawat, Peeraya; Molès, Jean-Pierre; Simmons, Graham; Chazal, Nathalie; Desprès, Philippe

    2015-01-01

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV

  5. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, Eric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Hamel, Rodolphe [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Neyret, Aymeric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Ekchariyawat, Peeraya [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Molès, Jean-Pierre [INSERM U1058, UM1, CHU Montpellier (France); Simmons, Graham [Blood Systems Research Institute, San Francisco, CA 94118 (United States); Chazal, Nathalie [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Desprès, Philippe [Unité Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris (France); and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  6. Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication

    Science.gov (United States)

    Jiang, Wei; Sheng, Chunjie; Gu, Xiuling; Liu, Dong; Yao, Chen; Gao, Shijuan; Chen, Shuai; Huang, Yinghui; Huang, Wenlin; Fang, Min

    2016-01-01

    Influenza A virus (IAV) is a major human pathogen with the potential to become pandemic. IAV contains only eight RNA segments; thus, the virus must fully exploit the host cellular machinery to facilitate its own replication. In an effort to comprehensively characterize the host machinery taken over by IAV in mammalian cells, we generated stable A549 cell lines with over-expression of the viral non-structural protein (NS1) to investigate the potential host factors that might be modulated by the NS1 protein. We found that the viral NS1 protein directly interacted with cellular Rac1 and facilitated viral replication. Further research revealed that NS1 down-regulated Rac1 activity via post-translational modifications. Therefore, our results demonstrated that IAV blocked Rac1-mediated host cell signal transduction through the NS1 protein to facilitate its own replication. Our findings provide a novel insight into the mechanism of IAV replication and indicate new avenues for the development of potential therapeutic targets. PMID:27869202

  7. Replication and interaction of herpes simplex virus and human papillomavirus in differentiating host epithelial tissue

    International Nuclear Information System (INIS)

    Meyers, Craig; Andreansky, Samita S.; Courtney, Richard J.

    2003-01-01

    We have investigated the interactions and consequences of superinfecting and coreplication of human papillomavirus (HPV) and herpes simplex virus (HSV) in human epithelial organotypic (raft) culture tissues. In HPV-positive tissues, HSV infection and replication induced significant cytopathic effects (CPE), but the tissues were able to recover and maintain a certain degree of tissue integrity and architecture. HPV31b not only maintained the episomal state of its genomic DNA but also maintained its genomic copy number even during times of extensive HSV-induced CPE. E2 transcripts encoded by HPV31b were undetectable even though HPV31b replication was maintained in HSV- infected raft tissues. Expression of HPV31b oncogenes (E6 and E7) was also repressed but to a lesser degree than was E2 expression. The extent of CPE induced by HSV is dependent on the magnitude of HPV replication and gene expression at the time of HSV infection. During active HSV infection, HPV maintains its genomic copy number even though genes required for its replication were repressed. These studies provide new insight into the complex interaction between two common human sexually transmitted viruses in an in vitro system, modeling their natural host tissue in vivo

  8. MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

    Science.gov (United States)

    Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

    2017-01-01

    An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

  9. Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Anuj Sharma

    2018-01-01

    Full Text Available Background: Chikungunya virus (CHIKV is a re-emerging pathogen that has caused widespread outbreaks affecting millions of people around the globe. Currently, there is no specific therapeutic drug against CHIKV, with symptomatic treatment only to manage the disease. Pi3-akt signaling has been implicated in infection of several viruses including that of CHIKV. Effect of Pi3-akt signaling inhibitors on CHIKV replication was evaluated in this study. Methods: Human primary dermal fibroblast cells were treated with inhibitors of the Pi3-akt signaling pathway. Suppression of CHIKV replication was evaluated as reduction in virus titer in cell supernatants. Effect of miltefosine (MF on CHIKV replication was evaluated in pre and post treatment regimen. Inhibition of virus replication was determined by cell growth, virus titer and western blot. Results: Inhibition of Akt-phosphorylation significantly inhibited CHIKV replication. No effect on CHIKV replication was observed after treatment with Pi3-kinase and mTOR activation inhibitors. Further, MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens. Conclusion: Data suggests that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection. This is the first study to show inhibition of CHIKV replication by MF, and presents a case for further development of MF as an anti-CHIKV drug.

  10. Mutations that abrogate transactivational activity of the feline leukemia virus long terminal repeat do not affect virus replication

    International Nuclear Information System (INIS)

    Abujamra, Ana L.; Faller, Douglas V.; Ghosh, Sajal K.

    2003-01-01

    The U3 region of the LTR of oncogenic Moloney murine leukemia virus (Mo-MuLV) and feline leukemia viruses (FeLV) have been previously reported to activate expression of specific cellular genes in trans, such as MHC class I, collagenase IV, and MCP-1, in an integration-independent manner. It has been suggested that transactivation of these specific cellular genes by leukemia virus U3-LTR may contribute to the multistage process of leukemogenesis. The U3-LTR region, necessary for gene transactivational activity, also contains multiple transcription factor-binding sites that are essential for normal virus replication. To dissect the promoter activity and the gene transactivational activity of the U3-LTR, we conducted mutational analysis of the U3-LTR region of FeLV-A molecular clone 61E. We identified minimal nucleotide substitution mutants on the U3 LTR that did not disturb transcription factor-binding sites but abrogated its ability to transactivate the collagenase gene promoter. To determine if these mutations actually have altered any uncharacterized important transcription factor-binding site, we introduced these U3-LTR mutations into the full-length infectious molecular clone 61E. We demonstrate that the mutant virus was replication competent but could not transactivate cellular gene expression. These results thus suggest that the gene transactivational activity is a distinct property of the LTR and possibly not related to its promoter activity. The cellular gene transactivational activity-deficient mutant FeLV generated in this study may also serve as a valuable reagent for testing the biological significance of LTR-mediated cellular gene activation in the tumorigenesis caused by leukemia viruses

  11. Replication of influenza A virus in swine umbilical cord epithelial stem-like cells.

    Science.gov (United States)

    Khatri, Mahesh; Chattha, Kuldeep S

    2015-01-01

    In this study, we describe the isolation and characterization of epithelial stem-like cells from the swine umbilical cord and their susceptibility to influenza virus infection. Swine umbilical cord epithelial stem cells (SUCECs) expressed stem cell and pluripotency associated markers such as SSEA-1, SSEA-4, TRA 1-60 and TRA 1-81 and Oct4. Morphologically, cells displayed polygonal morphology and were found to express epithelial markers; pancytokeratin, cytokeratin-18 and occludin; mesenchymal cell markers CD44, CD90 and haematopoietic cell marker CD45 were not detected on these cells. The cells had extensive proliferation and self- renewal properties. The cells also possessed immunomodulatory activity and inhibited the proliferation of T cells. Also, higher levels of anti-inflammatory cytokine IL-10 were detected in SUCEC-T cell co-cultures. The cells were multipotent and differentiated into lung epithelial cells when cultured in epithelial differentiation media. We also examined if SUCECs are susceptible to infection with influenza virus. SUCECs expressed sialic acid receptors, used by influenza virus for binding to cells. The 2009 pandemic influenza virus and swine influenza virus replicated in these cells. SUCECs due to their differentiation and immunoregulatory properties will be useful as cellular therapy in a pig model for human diseases. Additionally, our data indicate that influenza virus can infect SUCECs and may transmit influenza virus from mother to fetus through umbilical cord and transplantation of influenza virus-infected stem cells may transmit infection to recipients. Therefore, we propose that umbilical cord cells, in addition to other agents, should also be tested for influenza virus before cryopreservation for future use as a cell therapy for disease conditions.

  12. Minigenomes, transcription and replication competent virus-like particles and beyond: reverse genetics systems for filoviruses and other negative stranded hemorrhagic fever viruses.

    Science.gov (United States)

    Hoenen, Thomas; Groseth, Allison; de Kok-Mercado, Fabian; Kuhn, Jens H; Wahl-Jensen, Victoria

    2011-08-01

    Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Borna disease virus phosphoprotein modulates epigenetic signaling in neurons to control viral replication.

    Science.gov (United States)

    Bonnaud, Emilie M; Szelechowski, Marion; Bétourné, Alexandre; Foret, Charlotte; Thouard, Anne; Gonzalez-Dunia, Daniel; Malnou, Cécile E

    2015-06-01

    Understanding the modalities of interaction of neurotropic viruses with their target cells represents a major challenge that may improve our knowledge of many human neurological disorders for which viral origin is suspected. Borna disease virus (BDV) represents an ideal model to analyze the molecular mechanisms of viral persistence in neurons and its consequences for neuronal homeostasis. It is now established that BDV ensures its long-term maintenance in infected cells through a stable interaction of viral components with the host cell chromatin, in particular, with core histones. This has led to our hypothesis that such an interaction may trigger epigenetic changes in the host cell. Here, we focused on histone acetylation, which plays key roles in epigenetic regulation of gene expression, notably for neurons. We performed a comparative analysis of histone acetylation patterns of neurons infected or not infected by BDV, which revealed that infection decreases histone acetylation on selected lysine residues. We showed that the BDV phosphoprotein (P) is responsible for these perturbations, even when it is expressed alone independently of the viral context, and that this action depends on its phosphorylation by protein kinase C. We also demonstrated that BDV P inhibits cellular histone acetyltransferase activities. Finally, by pharmacologically manipulating cellular acetylation levels, we observed that inhibiting cellular acetyl transferases reduces viral replication in cell culture. Our findings reveal that manipulation of cellular epigenetics by BDV could be a means to modulate viral replication and thus illustrate a fascinating example of virus-host cell interaction. Persistent DNA viruses often subvert the mechanisms that regulate cellular chromatin dynamics, thereby benefitting from the resulting epigenetic changes to create a favorable milieu for their latent and persistent states. Here, we reasoned that Borna disease virus (BDV), the only RNA virus known to

  14. The virus–receptor interaction in the replication of feline immunodeficiency virus (FIV)☆

    Science.gov (United States)

    Willett, Brian J; Hosie, Margaret J

    2013-01-01

    The feline and human immunodeficiency viruses (FIV and HIV) target helper T cells selectively, and in doing so they induce a profound immune dysfunction. The primary determinant of HIV cell tropism is the expression pattern of the primary viral receptor CD4 and co-receptor(s), such as CXCR4 and CCR5. FIV employs a distinct strategy to target helper T cells; a high affinity interaction with CD134 (OX40) is followed by binding of the virus to its sole co-receptor, CXCR4. Recent studies have demonstrated that the way in which FIV interacts with its primary receptor, CD134, alters as infection progresses, changing the cell tropism of the virus. This review examines the contribution of the virus–receptor interaction to replication in vivo as well as the significance of these findings to the development of vaccines and therapeutics. PMID:23992667

  15. Pesti Des Petits ruminants virus infection in animals

    Directory of Open Access Journals (Sweden)

    Chauhan H.C.

    2009-08-01

    Full Text Available For centuries morbillivirus infections have had a huge impact on both human beings and animals. Morbilliviruses are highly contagious pathogens that cause some of the most devastating viral diseases of humans and animals world wide. They include measles virus (MV, canine distemper virus (CDV, rinderpest virus (RPV and peste des petits ruminants (PPRV virus. Furthermore, new emerging infectious diseases of morbilliviruses with significant ecological consequences of marine mammals have been discovered in the past decades. Phocid distemper virus (PDV in seals and the cetacean morbillivirus (CMV have been found in dolphins, whales and porpoises. Peste des petits ruminants (PPR is a highly contagious ,infectious , an acute or sub acute viral disease of domestic and wild small ruminants characterized by fever, oculonasal discharges, stomatitis, conjunctivitis, gastroenteritis and pneumonia. Goats are more severely affected than sheep. It is also known as pseudorinderpest of small ruminants, pest of small ruminants, pest of sheep and goats, kata, stomatitis- pneumoentritis syndrome, contagious pustular stomatitis and pneumoentritis complex. It is one of the major notifiable diseases of the World Organization for Animal Health (OIE. [Vet. World 2009; 2(4.000: 150-155

  16. RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication

    NARCIS (Netherlands)

    Feenstra, F.; Gennip, van H.G.P.; Water, van de S.G.P.; Rijn, van P.A.

    2014-01-01

    Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges.

  17. Emerging animal viruses: real threats or simple bystanders?

    Directory of Open Access Journals (Sweden)

    Eduardo Furtado Flores

    2013-10-01

    Full Text Available The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV, is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae, tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV, was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. The origin of SBV is still a matter of debate but it may be a reassortant from previous known bunyaviruses Shamonda and Satuperi. Hepatitis E virus (HEV, family Hepeviridae is a long known agent of human acute hepatitis and in 1997 was first identified in pigs. Current data indicates that swine HEV is spread worldwide, mainly associated with subclinical infection. Two of the four HEV genotypes are zoonotic and may be transmitted between swine and human by contaminated water and undercooked pork meat. The

  18. Ebola Virus Replication and Disease Without Immunopathology in Mice Expressing Transgenes to Support Human Myeloid and Lymphoid Cell Engraftment.

    Science.gov (United States)

    Spengler, Jessica R; Lavender, Kerry J; Martellaro, Cynthia; Carmody, Aaron; Kurth, Andreas; Keck, James G; Saturday, Greg; Scott, Dana P; Nichol, Stuart T; Hasenkrug, Kim J; Spiropoulou, Christina F; Feldmann, Heinz; Prescott, Joseph

    2016-10-15

    The study of Ebola virus (EBOV) pathogenesis in vivo has been limited to nonhuman primate models or use of an adapted virus to cause disease in rodent models. Herein we describe wild-type EBOV (Makona variant) infection of mice engrafted with human hematopoietic CD34 + stem cells (Hu-NSG™-SGM3 mice; hereafter referred to as SGM3 HuMice). SGM3 HuMice support increased development of myeloid immune cells, which are primary EBOV targets. In SGM3 HuMice, EBOV replicated to high levels, and disease was observed following either intraperitoneal or intramuscular inoculation. Despite the high levels of viral antigen and inflammatory cell infiltration in the liver, the characteristic histopathology of Ebola virus disease was not observed, and this absence of severe immunopathology may have contributed to the recovery and survival of some of the animals. Future investigations into the underlying mechanisms of the atypical disease presentation in SGM3 HuMice will provide additional insights into the immunopathogenesis of severe EBOV disease. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  19. Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

    Science.gov (United States)

    Kirui, James; Bucci, Michael D.; Poole, Daniel S.

    2014-01-01

    ABSTRACT Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved “P[F/P]AAAPP” sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These

  20. Histone Deacetylase Inhibitors Improve the Replication of Oncolytic Herpes Simplex Virus in Breast Cancer Cells

    Science.gov (United States)

    Cody, James J.; Markert, James M.; Hurst, Douglas R.

    2014-01-01

    New therapies are needed for metastatic breast cancer patients. Oncolytic herpes simplex virus (oHSV) is an exciting therapy being developed for use against aggressive tumors and established metastases. Although oHSV have been demonstrated safe in clinical trials, a lack of sufficient potency has slowed the clinical application of this approach. We utilized histone deacetylase (HDAC) inhibitors, which have been noted to impair the innate antiviral response and improve gene transcription from viral vectors, to enhance the replication of oHSV in breast cancer cells. A panel of chemically diverse HDAC inhibitors were tested at three different doses (LD50) for their ability to modulate the replication of oHSV in breast cancer cells. Several of the tested HDAC inhibitors enhanced oHSV replication at low multiplicity of infection (MOI) following pre-treatment of the metastatic breast cancer cell line MDA-MB-231 and the oHSV-resistant cell line 4T1, but not in the normal breast epithelial cell line MCF10A. Inhibitors of class I HDACs, including pan-selective compounds, were more effective for increasing oHSV replication compared to inhibitors that selectively target class II HDACs. These studies demonstrate that select HDAC inhibitors increase oHSV replication in breast cancer cells and provides support for pre-clinical evaluation of this combination strategy. PMID:24651853

  1. Plasticity of DNA replication initiation in Epstein-Barr virus episomes.

    Directory of Open Access Journals (Sweden)

    Paolo Norio

    2004-06-01

    Full Text Available In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD to study the latent duplication of Epstein-Barr virus (EBV episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size. These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.

  2. Histone deacetylase inhibitors improve the replication of oncolytic herpes simplex virus in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    James J Cody

    Full Text Available New therapies are needed for metastatic breast cancer patients. Oncolytic herpes simplex virus (oHSV is an exciting therapy being developed for use against aggressive tumors and established metastases. Although oHSV have been demonstrated safe in clinical trials, a lack of sufficient potency has slowed the clinical application of this approach. We utilized histone deacetylase (HDAC inhibitors, which have been noted to impair the innate antiviral response and improve gene transcription from viral vectors, to enhance the replication of oHSV in breast cancer cells. A panel of chemically diverse HDAC inhibitors were tested at three different doses (LD50 for their ability to modulate the replication of oHSV in breast cancer cells. Several of the tested HDAC inhibitors enhanced oHSV replication at low multiplicity of infection (MOI following pre-treatment of the metastatic breast cancer cell line MDA-MB-231 and the oHSV-resistant cell line 4T1, but not in the normal breast epithelial cell line MCF10A. Inhibitors of class I HDACs, including pan-selective compounds, were more effective for increasing oHSV replication compared to inhibitors that selectively target class II HDACs. These studies demonstrate that select HDAC inhibitors increase oHSV replication in breast cancer cells and provides support for pre-clinical evaluation of this combination strategy.

  3. Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

    Science.gov (United States)

    Brettmann, Erin A; Shaik, Jahangheer S; Zangger, Haroun; Lye, Lon-Fye; Kuhlmann, F Matthew; Akopyants, Natalia S; Oschwald, Dayna M; Owens, Katherine L; Hickerson, Suzanne M; Ronet, Catherine; Fasel, Nicolas; Beverley, Stephen M

    2016-10-25

    Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

  4. Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

    Science.gov (United States)

    Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H; Veits, Jutta; Gischke, Marcel; Mettenleiter, Thomas C; Abdelwhab, Elsayed M

    2018-02-14

    Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

  5. Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells

    Directory of Open Access Journals (Sweden)

    Anne Dittrich

    2018-02-01

    Full Text Available Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L to enhance replication in mammals and retained replication efficiency in the original avian host.

  6. Viral gene products and replication of the human immunodeficiency type 1 virus.

    Science.gov (United States)

    Morrow, C D; Park, J; Wakefield, J K

    1994-05-01

    The acquired immunodeficiency syndrome (AIDS) epidemic represents a modern-day plague that has not only resulted in a tragic loss of people from a wide spectrum of society but has reshaped our viewpoints regarding health care, the treatment of infectious diseases, and social issues regarding sexual behavior. There is little doubt now that the cause of the disease AIDS is a virus known as the human immunodeficiency virus (HIV). The HIV virus is a member of a large family of viruses termed retroviruses, which have as a hallmark the capacity to convert their RNA genome into a DNA form that then undergoes a process of integration into the host cell chromosome, followed by the expression of the viral genome and translation of viral proteins in the infected cell. This review describes the organization of the HIV-1 viral genome, the expression of viral proteins, as well as the functions of the accessory viral proteins in HIV replication. The replication of the viral genome is divided into two phases, the early phase and the late phase. The early phase consists of the interaction of the virus with the cell surface receptor (CD4 molecule in most cases), the uncoating and conversion of the viral RNA genome into a DNA form, and the integration into the host cell chromosome. The late phase consists of the expression of the viral proteins from the integrated viral genome, the translation of viral proteins, and the assembly and release of the virus. Points in the HIV-1 life cycle that are targets for therapeutic intervention are also discussed.

  7. Reverse genetics with animal viruses. NSV reverse genetics

    International Nuclear Information System (INIS)

    Mebatsion, T.

    2005-01-01

    New strategies to genetically manipulate the genomes of several important animal pathogens have been established in recent years. This article focuses on the reverse genetics techniques, which enables genetic manipulation of the genomes of non-segmented negative-sense RNA viruses. Recovery of a negative-sense RNA virus entirely from cDNA was first achieved for rabies virus in 1994. Since then, reverse genetic systems have been established for several pathogens of medical and veterinary importance. Based on the reverse genetics technique, it is now possible to design safe and more effective live attenuated vaccines against important viral agents. In addition, genetically tagged recombinant viruses can be designed to facilitate serological differentiation of vaccinated animals from infected animals. The approach of delivering protective immunogens of different pathogens using a single vector was made possible with the introduction of the reverse genetics system, and these novel broad-spectrum vaccine vectors have potential applications in improving animal health in developing countries. (author)

  8. Animal models of human respiratory syncytial virus disease

    NARCIS (Netherlands)

    Bem, Reinout A.; Domachowske, Joseph B.; Rosenberg, Helene F.

    2011-01-01

    Infection with the human pneumovirus pathogen, respiratory syncytial virus (hRSV), causes a wide spectrum of respiratory disease, notably among infants and the elderly. Laboratory animal studies permit detailed experimental modeling of hRSV disease and are therefore indispensable in the search for

  9. Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication Is Revealed by Complete Genome Sequencing

    Science.gov (United States)

    Eckerle, Lance D.; Becker, Michelle M.; Halpin, Rebecca A.; Li, Kelvin; Venter, Eli; Lu, Xiaotao; Scherbakova, Sana; Graham, Rachel L.; Baric, Ralph S.; Stockwell, Timothy B.; Spiro, David J.; Denison, Mark R.

    2010-01-01

    Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and

  10. Construction of self-replicating subgenomic West Nile virus replicons for screening antiviral compounds.

    Science.gov (United States)

    Alcaraz-Estrada, Sofia L; Reichert, Erin Donohue; Padmanabhan, Radhakrishnan

    2013-01-01

    Mosquito-borne flavivirus RNA genomes encode one long open reading frame flanking 5'- and 3'-untranslated regions (5'- and 3'-UTRs) which contain cis-acting RNA elements playing important roles for viral RNA translation and replication. The viral RNA encodes a single polyprotein, which is processed into three structural proteins and seven nonstructural (NS) proteins. The regions coding for the seven NS proteins are sufficient for replication of the RNA. The sequences encoding the structural genes can be deleted except for two short regions. The first one encompasses 32 amino acid (aa) residues from the N-terminal coding sequence of capsid (C) and the second, 27 aa region from the C-terminus of envelope (E) protein. The deleted region can be substituted with a gene coding for a readily quantifiable reporter to give rise to a subgenomic reporter replicon. Replicons containing a variety of reporter genes and marker genes for construction of stable mammalian cell lines are valuable reagents for studying the effects of mutations in translation and/or replication in isolation from processes like the entry and assembly of the virus particles. Here we describe the construction of two West Nile virus (WNV) replicons by overlap extension PCR and standard recombinant DNA techniques. One has a Renilla luciferase (Rluc) reporter gene followed by an internal ribosome entry site (element) for cap-independent translation of the open reading frame encompassing the carboxy-terminal sequence of E to NS5. The second replicon has in tandem the Rluc gene, foot and mouth disease virus 2A, and neomycin phosphotransferase gene that allows establishment of a stable mammalian cell line expressing the Rluc reporter in the presence of the neomycin analog, G418. The stable replicon-expressing Vero cell line has been used for cell-based screening and determination of EC50 values for antiviral compounds that inhibited WNV replication.

  11. Alpha/beta interferon receptor deficiency in mice significantly enhances susceptibility of the animals to pseudorabies virus infection.

    Science.gov (United States)

    Wei, Jingyun; Ma, Yanmei; Wang, Long; Chi, Xiaojuan; Yan, Ruoxiang; Wang, Song; Li, Xinxin; Chen, Xiaoyong; Shao, Wenhan; Chen, Ji-Long

    2017-05-01

    Pseudorabies virus, one of the neurotropic viruses, can infect numerous mammals. In particular, pseudorabies virus infection of swine occurs worldwide, and is a major threat to swine industry. However, the mechanism underlying the interaction between pseudorabies virus and host innate immune system is not fully understood. Here, we investigated the involvement of interferon α/β (IFN-α/β) receptor (IFNAR) in the pathogenesis of pseudorabies virus in a mouse model. The results showed that IFNAR-deficient (IFNAR -/- ) mice were highly susceptible to the virus infection, as evidenced by markedly reduced survival rate of infected animals and increased viral replication. The expression of IFN-α/β and relevant interferon-stimulated genes in IFNAR -/- mice was significantly lower than that in wild-type (WT) littermates after the viral infection. Moreover, in response to the virus challenge, IFNAR -/- mice displayed elevated levels of inflammatory cytokines including interleukin 6 (IL-6) and IL-1β, and IFNAR -/- cells showed increased phosphorylation of STAT3. Collectively, these data reveal that the IFNAR -/- mice are more sensitive to pseudorabies virus infection than WT animals, and excessive IL-6/STAT3 response in IFNAR -/- mice may contribute to the pathogenesis. Our findings suggest that type I IFNs/IFNAR-dependent homeostatic control of the innate immunity is required for host defense against pseudorabies virus infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The role of microRNAs in hepatitis C virus RNA replication.

    Science.gov (United States)

    Conrad, K Dominik; Niepmann, Michael

    2014-05-01

    Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5' end of the 5' untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA's 5' end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5'-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.

  13. Deletion of the M2-2 gene from avian metapneumovirus subgroup C impairs virus replication and immunogenicity in Turkeys.

    Science.gov (United States)

    Yu, Qingzhong; Estevez, Carlos N; Roth, Jason P; Hu, Haixia; Zsak, Laszlo

    2011-06-01

    The second matrix (M2) gene of avian metapneumovirus subgroup C (aMPV-C) contains two overlapping open reading frames (ORFs), encoding two putative proteins, M2-1 and M2-2. Both proteins are believed to be involved in viral RNA transcription or replication. To further characterize the function of the M2-2 protein in virus replication, the non-overlapping region of the M2-2 ORF was deleted from an infectious cDNA clone of the aMPV-C strain, and a viable virus was rescued by using reverse genetics technology. The recombinant virus, raMPV-C ΔM2-2, was characterized in vitro and in vivo. In Vero cells, raMPV-C ΔM2-2 replicated slightly less efficiently than the parental virus, 10-fold reduction at 48-h post-infection. The raMPV-C ΔM2-2 virus induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental virus infection. In specific-pathogen-free (SPF) turkeys, raMPV-C ΔM2-2 was attenuated and caused no clinical signs of disease. Less than 20% of the inoculated birds shed detectable virus in tracheal tissue during the first 5 days post-infection, and no virus shedding was detected afterward. Forty percent of infected birds produced a weak antibody response at 14 days post-infection. Upon challenge with a virulent aMPV-C strain, more than 80% of the raMPV-C ΔM2-2-inoculated birds showed typical disease signs and virus shedding in tracheal tissue. These results suggest that the M2-2 protein of aMPV-C virus is not essential for virus replication in vitro, but is required for sufficient virus replication to maintain pathogenicity and immunogenicity in the natural host.

  14. Development of viable TAP-tagged dengue virus for investigation of host-virus interactions in viral replication.

    Science.gov (United States)

    Poyomtip, Teera; Hodge, Kenneth; Matangkasombut, Ponpan; Sakuntabhai, Anavaj; Pisitkun, Trairak; Jirawatnotai, Siwanon; Chimnaronk, Sarin

    2016-03-01

    Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.

  15. Macrophages as target cells for Mayaro virus infection: involvement of reactive oxygen species in the inflammatory response during virus replication

    Directory of Open Access Journals (Sweden)

    MARIANA G. CAVALHEIRO

    Full Text Available ABSTRACT Alphaviruses among the viruses that cause arthritis, consisting in a public health problem worldwide by causing localized outbreaks, as well as large epidemics in humans. Interestingly, while the Old World alphaviruses are arthritogenic, the New World alphaviruses cause encephalitis. One exception is Mayaro virus (MAYV, which circulates exclusively in South America but causes arthralgia and is phylogenetically related to the Old World alphaviruses. Although MAYV-induced arthritis in humans is well documented, the molecular and cellular factors that contribute to its pathogenesis are completely unknown. In this study, we demonstrated for the first time that macrophages, key players in arthritis development, are target cells for MAYV infection, which leads to cell death through apoptosis. We showed that MAYV replication in macrophage induced the expression of TNF, a cytokine that would contribute to pathogenesis of MAYV fever, since TNF promotes an inflammatory profile characteristic of arthritis. We also found a significant increase in the production of reactive oxygen species (ROS at early times of infection, which coincides with the peak of virus replication and precedes TNF secretion. Treatment of the cells with antioxidant agents just after infection completely abolished TNF secretion, indicating an involvement of ROS in inflammation induced during MAYV infection.

  16. Pterodontic Acid Isolated from Laggera pterodonta Inhibits Viral Replication and Inflammation Induced by Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Wenda Guan

    2017-10-01

    Full Text Available Laggera pterodonta (DC. Benth. is a traditional Chinese medicine. The previous study revealed that the crude extracts of this herb could inhibit influenza virus infection, but its anti-influenza components and underlying mechanism of action remain unknown. Column chromatography was performed to isolate components from the plant. Activity against influenza virus of the compound was determined by CPE inhibition assay. Neuraminidase (NA inhibition was measured by chemiluminescence assay. The anti-virus and anti-inflammation effects were determined using dual-luciferase reporter assay, immunofluorescence, quantitative real-time PCR and luminex assay. Pterodontic acid was isolated from L. pterodonta, which showed selective anti-viral activities to H1 subtype of human influenza A virus. Meanwhile, the NA activity was not obviously inhibited by the compound. Further experiments exhibited that the compound can suppress the activation of NF-κB signal pathway and export of viral RNP complexes from the nucleus. In addition, it can significantly attenuate expression of the pro-inflammatory molecules IL-6, MIP-1β, MCP-1, and IP-10 induced by human influenza A virus (H1N1 and similarly downregulate expression of cytokines and chemokines induced by avian influenza A virus (H9N2. This study showed that in vitro antiviral activity of pterodontic acid is most probably associated with inhibiting the replication of influenza A virus by blocking nuclear export of viral RNP complexes, and attenuating the inflammatory response by inhibiting activation of the NF-κB pathway. Pterodontic acid might be a potential antiviral agent against influenza A virus.

  17. Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses.

    Science.gov (United States)

    Meng, Fandan; Punyadarsaniya, Darsaniya; Uhlenbruck, Sabine; Hennig-Pauka, Isabel; Schwegmann-Wessels, Christel; Ren, Xiaofeng; Dürrwald, Ralf; Herrler, Georg

    2013-11-13

    Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.

  18. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Science.gov (United States)

    de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent

    2013-01-01

    Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  19. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    Directory of Open Access Journals (Sweden)

    Benoît de Chassey

    Full Text Available Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1 appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  20. A new model to produce infectious hepatitis C virus without the replication requirement.

    Directory of Open Access Journals (Sweden)

    Miriam Triyatni

    2011-04-01

    Full Text Available Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV. Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b. Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i HCV bicistronic particles (HCVbp containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii HCV reporter particles (HCVrp containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii HCV wild-type particles (HCVwt containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1. Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV

  1. A new model to produce infectious hepatitis C virus without the replication requirement.

    Science.gov (United States)

    Triyatni, Miriam; Berger, Edward A; Saunier, Bertrand

    2011-04-01

    Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity

  2. Control of African swine fever virus replication by small interfering RNA targeting the A151R and VP72 genes.

    Science.gov (United States)

    Keita, Djénéba; Heath, Livio; Albina, Emmanuel

    2010-01-01

    African swine fever virus (ASFV) is the unique member of the Asfarviridae family and Asfivirus genus. It is an enveloped double-stranded DNA arbovirus that replicates in the cell cytoplasm, similar to poxviruses. There is no vaccine and no treatment available to control this virus. We describe the use of small interfering RNA (siRNA) targeting the A151R and VP72 (B646L) genes to control the ASFV replication in vitro. Results suggest that siRNA targeting the A151R and VP72 genes can reduce both the virus replication and its levels of messenger RNA transcripts. The reduction was up to 4 log(10) copies on the virus titre and up to 3 log(10) copies on virus RNA transcripts levels. The combination of multiple siRNA did not improve the antiviral effect significantly, compared with use of individual siRNAs. The function of the A151R gene product in the virus replication cycle is yet unclear, but is essential. We also demonstrate that it is possible to inhibit, using small interfering RNA, a virus that replicates exclusively in the cell cytoplasm in specific viral factories.

  3. Comparison of complete genome sequences of dog rabies viruses isolated from China and Mexico reveals key amino acid changes that may be associated with virus replication and virulence.

    Science.gov (United States)

    Yu, Fulai; Zhang, Guoqing; Zhong, Xiangfu; Han, Na; Song, Yunfeng; Zhao, Ling; Cui, Min; Rayner, Simon; Fu, Zhen F

    2014-07-01

    Rabies is a global problem, but its impact and prevalence vary across different regions. In some areas, such as parts of Africa and Asia, the virus is prevalent in the domestic dog population, leading to epidemic waves and large numbers of human fatalities. In other regions, such as the Americas, the virus predominates in wildlife and bat populations, with sporadic spillover into domestic animals. In this work, we attempted to investigate whether these distinct environments led to selective pressures that result in measurable changes within the genome at the amino acid level. To this end, we collected and sequenced the full genome of two isolates from divergent environments. The first isolate (DRV-AH08) was from China, where the virus is present in the dog population and the country is experiencing a serious epidemic. The second isolate (DRV-Mexico) was taken from Mexico, where the virus is present in both wildlife and domestic dog populations, but at low levels as a consequence of an effective vaccination program. We then combined and compared these with other full genome sequences to identify distinct amino acid changes that might be associated with environment. Phylogenetic analysis identified strain DRV-AH08 as belonging to the China-I lineage, which has emerged to become the dominant lineage in the current epidemic. The Mexico strain was placed in the D11 Mexico lineage, associated with the West USA-Mexico border clade. Amino acid sequence analysis identified only 17 amino acid differences in the N, G and L proteins. These differences may be associated with virus replication and virulence-for example, the short incubation period observed in the current epidemic in China.

  4. Leflunomide/teriflunomide inhibit Epstein-Barr virus (EBV)- induced lymphoproliferative disease and lytic viral replication.

    Science.gov (United States)

    Bilger, Andrea; Plowshay, Julie; Ma, Shidong; Nawandar, Dhananjay; Barlow, Elizabeth A; Romero-Masters, James C; Bristol, Jillian A; Li, Zhe; Tsai, Ming-Han; Delecluse, Henri-Jacques; Kenney, Shannon C

    2017-07-04

    EBV infection causes mononucleosis and is associated with specific subsets of B cell lymphomas. Immunosuppressed patients such as organ transplant recipients are particularly susceptible to EBV-induced lymphoproliferative disease (LPD), which can be fatal. Leflunomide (a drug used to treat rheumatoid arthritis) and its active metabolite teriflunomide (used to treat multiple sclerosis) inhibit de novo pyrimidine synthesis by targeting the cellular dihydroorotate dehydrogenase, thereby decreasing T cell proliferation. Leflunomide also inhibits the replication of cytomegalovirus and BK virus via both "on target" and "off target" mechanisms and is increasingly used to treat these viruses in organ transplant recipients. However, whether leflunomide/teriflunomide block EBV replication or inhibit EBV-mediated B cell transformation is currently unknown. We show that teriflunomide inhibits cellular proliferation, and promotes apoptosis, in EBV-transformed B cells in vitro at a clinically relevant dose. In addition, teriflunomide prevents the development of EBV-induced lymphomas in both a humanized mouse model and a xenograft model. Furthermore, teriflunomide inhibits lytic EBV infection in vitro both by preventing the initial steps of lytic viral reactivation, and by blocking lytic viral DNA replication. Leflunomide/teriflunomide might therefore be clinically useful for preventing EBV-induced LPD in patients who have high EBV loads yet require continued immunosuppression.

  5. A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells.

    Science.gov (United States)

    Ersing, Ina; Nobre, Luis; Wang, Liang Wei; Soday, Lior; Ma, Yijie; Paulo, Joao A; Narita, Yohei; Ashbaugh, Camille W; Jiang, Chang; Grayson, Nicholas E; Kieff, Elliott; Gygi, Steven P; Weekes, Michael P; Gewurz, Benjamin E

    2017-05-16

    Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. MicroRNA 130a Regulates both Hepatitis C Virus and Hepatitis B Virus Replication through a Central Metabolic Pathway.

    Science.gov (United States)

    Duan, Xiaoqiong; Li, Shilin; Holmes, Jacinta A; Tu, Zeng; Li, Yujia; Cai, Dachuan; Liu, Xiao; Li, Wenting; Yang, Chunhui; Jiao, Baihai; Schaefer, Esperance A; Fusco, Dahlene N; Salloum, Shadi; Chen, Limin; Lin, Wenyu; Chung, Raymond T

    2018-04-01

    Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE We identified

  7. Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmidt, J; Luz, A

    1999-01-01

    ) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary...... delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta......, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant....

  8. Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

    Directory of Open Access Journals (Sweden)

    Weihong Pan

    Full Text Available Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway. This mutant HSV-1 was named as Signal-Smart 1 (SS1. A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05, while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a "prototype" for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

  9. Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

    Science.gov (United States)

    Pan, Weihong; Bodempudi, Vidya; Esfandyari, Tuba; Farassati, Faris

    2009-08-04

    Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication) is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway). This mutant HSV-1 was named as Signal-Smart 1 (SS1). A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05), while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a "prototype" for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

  10. PKR Activation Favors Infectious Pancreatic Necrosis Virus Replication in Infected Cells

    Directory of Open Access Journals (Sweden)

    Amr A.A. Gamil

    2016-06-01

    Full Text Available The double-stranded RNA-activated protein kinase R (PKR is a Type I interferon (IFN stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells.

  11. The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals.

    Science.gov (United States)

    Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

    2016-04-20

    H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.

  12. Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication.

    Directory of Open Access Journals (Sweden)

    Saki Kondo

    Full Text Available Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs, are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

  13. The N terminus of the influenza B virus nucleoprotein is essential for virus viability, nuclear localization, and optimal transcription and replication of the viral genome.

    Science.gov (United States)

    Sherry, Lee; Smith, Matt; Davidson, Sophie; Jackson, David

    2014-11-01

    The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However, NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50-amino-acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension; however, this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. The truncation of more than three amino acids prevented virus recovery, suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in the nuclear localization of BNP, with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first 10 residues of BNP demonstrated few differences in nuclear localization; however, the viruses exhibited significant reductions in viral mRNA transcription and genome replication, resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall, our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP but also for regulating viral mRNA transcription and genome replication. The multifunctional NP of influenza viruses has roles throughout the viral replication cycle; therefore, it is essential for virus viability. Despite high levels of homology between the NP of influenza A and B viruses, the NP of influenza B virus

  14. A Respiratory Syncytial Virus Vaccine Vectored by a Stable Chimeric and Replication-Deficient Sendai Virus Protects Mice without Inducing Enhanced Disease.

    Science.gov (United States)

    Wiegand, Marian Alexander; Gori-Savellini, Gianni; Gandolfo, Claudia; Papa, Guido; Kaufmann, Christine; Felder, Eva; Ginori, Alessandro; Disanto, Maria Giulia; Spina, Donatella; Cusi, Maria Grazia

    2017-05-15

    Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated. IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity

  15. Well begun is half done: Rubella virus perturbs autophagy signaling, thereby facilitating the construction of viral replication compartments.

    Science.gov (United States)

    Orosz, László; Megyeri, Klára

    2016-04-01

    The rubella virus is the causative agent of postnatal German measles and the congenital rubella syndrome. The majority of the rubella virus replication complexes originate from the endomembrane system. The rubella virus perturbs the signaling pathways regulating the formation of autophagic membranes in the infected cells, including the Ras/Raf/MEK/ERK and PI3K/Akt pathways. It is widely accepted that these pathways inhibit autophagy. In contrast, the class III PI3K enzymes are essential for autophagy initiation. By manipulating the Ras/Raf/MEK/ERK, class I PI3K/Akt and class III PI3K axes of signal transduction, the rubella virus may differentially regulate the autophagic cascade, with consequent stimulation of the initiation and strong suppression of the later phases. Dysregulation of autophagy by this virus can have a significant impact on the construction of replication compartments by regulating membrane trafficking. We hypothesize that the rubella virus perturbs the autophagic process in order to prevent the degradation of the virus progeny, and to ensure its replication by hijacking omegasomes for the construction of the replication complexes. The virus is therefore able to utilize an antiviral mechanism to its own advantage. Therapeutic modalities targeting the autophagic process may help to ameliorate the serious consequences of the congenital rubella syndrome. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

    Directory of Open Access Journals (Sweden)

    Ana Paula Carneiro Salgado

    2013-08-01

    Full Text Available Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV and Cowpox (CPXV and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

  17. Baculovirus resistance in codling moth (Cydia pomonella L.) caused by early block of virus replication.

    Science.gov (United States)

    Asser-Kaiser, Sabine; Radtke, Pit; El-Salamouny, Said; Winstanley, Doreen; Jehle, Johannes A

    2011-02-20

    An up to 10,000-fold resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV) was observed in field populations of codling moth, C. pomonella, in Europe. Following different experimental approaches, a modified peritrophic membrane, a modified midgut receptor, or a change of the innate immune response could be excluded as possible resistance mechanisms. When CpGV replication was traced by quantitative PCR in different tissues of susceptible and resistant insects after oral and intra-hemocoelic infection, no virus replication could be detected in any of the tissues of resistant insects, suggesting a systemic block prior to viral DNA replication. This conclusion was corroborated by fluorescence microscopy using a modified CpGV (bacCpGV(hsp-eGFP)) carrying enhanced green fluorescent gene (eGFP), which showed that infection in resistant insects did not spread. In conclusion, the different lines of evidence indicate that CpGV can enter but not replicate in the cells of resistant codling moth larvae. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Hepatitis B Virus-Encoded MicroRNA Controls Viral Replication.

    Science.gov (United States)

    Yang, Xi; Li, Hongfeng; Sun, Huahui; Fan, Hongxia; Hu, Yaqi; Liu, Min; Li, Xin; Tang, Hua

    2017-05-15

    MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding, functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and Northern blotting. HBV-miR-3 is located at nucleotides (nt) 373 to 393 of the HBV genome and was generated from 3.5-kb, 2.4-kb, and 2.1-kb HBV in a classic miRNA biogenesis (Drosha-Dicer-dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV + hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released into the circulation by exosomes and HBV virions, and HBV-miR-3 expression had a positive correlation with HBV titers in the sera of patients in the acute phase of HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg, and replication of HBV. HBV-miR-3 targets the unique site of the HBV 3.5-kb transcript to specifically reduce HBc protein expression, levels of pregenomic RNA (pgRNA), and HBV replication intermediate (HBV-RI) generation but does not affect the HBV DNA polymerase level, thus suppressing HBV virion production (replication). This may explain the low levels of HBV virion generation with abundant subviral particles lacking core during HBV replication, which may contribute to the development of persistent infection in patients. Taken together, our findings shed light on novel mechanisms by which HBV-encoded miRNA controls the process of self-replication by regulating HBV transcript during infection. IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV) that can become a long-term, chronic infection and lead to cirrhosis or liver cancer. HBV is a small DNA virus that belongs to the hepadnavirus family, with virions and subviral forms of particles that lack a core. MicroRNA (miRNA), a small (∼22-nt) noncoding RNA

  19. Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera

    Directory of Open Access Journals (Sweden)

    Pettis Jeffery S

    2009-12-01

    Full Text Available Abstract Background For years, the understanding of the pathogenetic mechanisms that underlie honey bee viral diseases has been severely hindered because of the lack of a cell culture system for virus propagation. As a result, it is very imperative to develop new methods that would permit the in vitro pathogenesis study of honey bee viruses. The identification of virus replication is an important step towards the understanding of the pathogenesis process of viruses in their respective hosts. In the present study, we developed a strand-specific RT-PCR-based method for analysis of Deformed Wing Virus (DWV replication in honey bees and in honey bee parasitic mites, Varroa Destructor. Results The results shows that the method developed in our study allows reliable identification of the virus replication and solves the problem of falsely-primed cDNA amplifications that commonly exists in the current system. Using TaqMan real-time quantitative RT-PCR incorporated with biotinylated primers and magnetic beads purification step, we characterized the replication and tissue tropism of DWV infection in honey bees. We provide evidence for DWV replication in the tissues of wings, head, thorax, legs, hemolymph, and gut of honey bees and also in Varroa mites. Conclusion The strategy reported in the present study forms a model system for studying bee virus replication, pathogenesis and immunity. This study should be a significant contribution to the goal of achieving a better understanding of virus pathogenesis in honey bees and to the design of appropriate control measures for bee populations at risk to virus infections.

  20. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    International Nuclear Information System (INIS)

    Li Guiqiu; Gu Hongxi; Li Di; Xu Weizhen

    2007-01-01

    The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies

  1. Lycorine reduces mortality of human enterovirus 71-infected mice by inhibiting virus replication

    Directory of Open Access Journals (Sweden)

    Qin Chuan

    2011-10-01

    Full Text Available Abstract Human enterovirus 71 (EV71 infection causes hand, foot and mouth disease in children under 6 years old and this infection occasionally induces severe neurological complications. No vaccines or drugs are clinical available to control EV71 epidemics. In present study, we show that treatment with lycorine reduced the viral cytopathic effect (CPE on rhabdomyosarcoma (RD cells by inhibiting virus replication. Analysis of this inhibitory effect of lycorine on viral proteins synthesis suggests that lycorine blocks the elongation of the viral polyprotein during translation. Lycorine treatment of mice challenged with a lethal dose of EV71 resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. When mice were infected with a moderate dose of EV71, lycorine treatment was able to protect them from paralysis. Lycorine may be a potential drug candidate for the clinical treatment of EV71-infected patients.

  2. Different effects of phorbol ester derivates on human immunodeficiency virus 1 replication in lymphocytic and monocytic human cells

    International Nuclear Information System (INIS)

    Kalvatchev, Z.; Walder, R.; Garzaro, D.

    1997-01-01

    The mode of action of the phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA) on the human immunodeficiency virus 1 (HIV-l) replication in human lymphocytes and monocytes was studied. PDBu and PMA appear to have similar effects on the regulation of HIV-l replication in acutely infected cells. Here we show a significantly increased replication of HIV-l induced by PDBu and PMA in Molt-4 and Jurkat cells, but a reduced replication in THP-l and U-937 cells. Moreover, quantitatively different activity of the two derivatives in relation to HIV-l replication was observed. PDBu proved to be a stronger stimulator or suppressor of HIV-l replication as compared to PMA. Although the precise mechanism of the activation of HIV-l replication by phorbol ester derivatives is not clear, it can be assumed that the hydrophilicity of PDBu may cause its stronger effect. (authors)

  3. Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements.

    Science.gov (United States)

    Marschalek, Adriane; Finke, Stefan; Schwemmle, Martin; Mayer, Daniel; Heimrich, Bernd; Stitz, Lothar; Conzelmann, Karl-Klaus

    2009-02-01

    Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and relies on the canonical 5'-end-dependent translation of capped viral mRNAs. Here, we have used internal ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2 IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-beta) promoter, and increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2 IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-alpha receptor (IFNAR(-/-)). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demonstrated only in newborn IFNAR(-/-) mice. Translational control of individual genes is a promising strategy to attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study the function of IRES elements in detail.

  4. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line.

    Science.gov (United States)

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G; Wenzel, Jürgen J; Johne, Reimar

    2016-09-29

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules ( CEACAM ) 5 and 6 , and an upregulation of the syndecan 2 ( SDC2 ) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown.

  5. Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus.

    Science.gov (United States)

    Sehgal, O P; Sinha, R C; Gellatly, D L; Ivanov, I; AbouHaidar, M G

    1993-04-01

    Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.

  6. A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication

    OpenAIRE

    Piper, Mary E.; Gerrard, Sonja R.

    2010-01-01

    Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly ...

  7. Olive baboons: a non-human primate model for testing dengue virus type 2 replication.

    Science.gov (United States)

    Valdés, Iris; Gil, Lázaro; Castro, Jorge; Odoyo, Damián; Hitler, Rikoi; Munene, Elephas; Romero, Yaremis; Ochola, Lucy; Cosme, Karelia; Kariuki, Thomas; Guillén, Gerardo; Hermida, Lisset

    2013-12-01

    This study evaluated the use of a non-human primate, the olive baboon (Papio anubis), as a model of dengue infection. Olive baboons closely resemble humans genetically and physiologically and have been used extensively for assessing novel vaccine formulations. Two doses of dengue virus type 2 (DENV-2) were tested in baboons: 10(3) and 10(4) pfu. Similarly, African green monkeys received the same quantity of virus and acted as positive controls. Following exposure, high levels of viremia were detected in both animal species. There was a trend to detect more days of viremia and more homogeneous viral titers in animals receiving the low viral dose. In addition, baboons infected with the virus generally exhibited positive virus isolation 1 day later than African green monkeys. Humoral responses consisting of antiviral and neutralizing antibodies were detected in all animals after infection. We conclude that baboons provide an alternative non-human primate species for experimental DENV-2 infection and we recommend their use for further tests of vaccines, administering the lowest dose assayed: 10(3) pfu. Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  8. Regulation of herpes simplex virus type 1 replication in Vero cells by Psychotria serpens: relationship to gene expression, DNA replication, and protein synthesis.

    Science.gov (United States)

    Kuo, Y C; Chen, C C; Tsai, W J; Ho, Y H

    2001-08-01

    Inhibitory effects of ethanolic extracts from seven Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. From a bioassay-guided fractionation procedure, PS-A-6 was isolated from Psychotria serpens (P. serpens), which suppressed HSV-1 multiplication in Vero cells without apparent cytotoxicity. Time-of-addition experiments suggested that the inhibitory action of PS-A-6 on HSV-1 replication was not through blocking of virus adsorption. In an attempt to further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to viral multiplication was examined, including viral gene expression, DNA replication, and structural protein synthesis. The results indicated that gB mRNA and protein expression in Vero cells were impeded by PS-A-6. Southern blot analysis showed that HSV-1 DNA replication in Vero cells was arrested by PS-A-6. In addition, PS-A-6 decreased thymidine kinase (tk) and ICP27 mRNA expression in the cells. The mechanisms of antiviral action of PS-A-6 seem to be mediated, at least in part, through inhibition of early transcripts of HSV-1, such as tk and ICP27 mRNAs, arresting HSV-1 DNA synthesis and gB gene expression in Vero cells. Plans are underway for the isolation of pure compounds from PS-A-6 and elucidation of their mechanism of action.

  9. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Lovmand, J

    1993-01-01

    Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching t......RNA(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus...... can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site....

  10. Neuronal Interferon Signaling Is Required for Protection against Herpes Simplex Virus Replication and Pathogenesis

    Science.gov (United States)

    Rosato, Pamela C.; Leib, David A.

    2015-01-01

    Interferon (IFN) responses are critical for controlling herpes simplex virus 1 (HSV-1). The importance of neuronal IFN signaling in controlling acute and latent HSV-1 infection remains unclear. Compartmentalized neuron cultures revealed that mature sensory neurons respond to IFNβ at both the axon and cell body through distinct mechanisms, resulting in control of HSV-1. Mice specifically lacking neural IFN signaling succumbed rapidly to HSV-1 corneal infection, demonstrating that IFN responses of the immune system and non-neuronal tissues are insufficient to confer survival following virus challenge. Furthermore, neurovirulence was restored to an HSV strain lacking the IFN-modulating gene, γ34.5, despite its expected attenuation in peripheral tissues. These studies define a crucial role for neuronal IFN signaling for protection against HSV-1 pathogenesis and replication, and they provide a novel framework to enhance our understanding of the interface between host innate immunity and neurotropic pathogens. PMID:26153886

  11. Chaetocin reactivates the lytic replication of Epstein-Barr virus from latency via reactive oxygen species.

    Science.gov (United States)

    Zhang, Shilun; Yin, Juan; Zhong, Jiang

    2017-01-01

    Oxidative stress, regarded as a negative effect of free radicals in vivo, takes place when organisms suffer from harmful stimuli. Some viruses can induce the release of reactive oxygen species (ROS) in infected cells, which may be closely related with their pathogenicity. In this report, chaetocin, a fungal metabolite reported to have antimicrobial and cytostatic activity, was studied for its effect on the activation of latent Epstein-Barr virus (EBV) in B95-8 cells. We found that chaetocin remarkably up-regulated EBV lytic transcription and DNA replication at a low concentration (50 nmol L -1 ). The activation of latent EBV was accompanied by an increased cellular ROS level. N-acetyl-L-cysteine (NAC), an ROS inhibitor, suppressed chaetocin-induced EBV activation. Chaetocin had little effect on histone H3K9 methylation, while NAC also significantly reduced H3K9 methylation. These results suggested that chaetocin reactivates latent EBV primarily via ROS pathways.

  12. Evaluation of seasonal influenza vaccines for H1N1pdm09 and type B viruses based on a replication-incompetent PB2-KO virus.

    Science.gov (United States)

    Ui, Hiroki; Yamayoshi, Seiya; Uraki, Ryuta; Kiso, Maki; Oishi, Kohei; Murakami, Shin; Mimori, Shigetaka; Kawaoka, Yoshihiro

    2017-04-04

    Vaccination is the first line of protection against influenza virus infection in humans. Although inactivated and live-attenuated vaccines are available, each vaccine has drawbacks in terms of immunogenicity and safety. To overcome these issues, our group has developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2-expressing cells. Here we generated PB2-KO viruses possessing the hemagglutinin (HA) and neuraminidase (NA) segments from H1N1pdm09 or type B viruses and tested their vaccine potential. The two PB2-KO viruses propagated efficiently in PB2-expressing cells, and expressed chimeric HA as expected. Virus-specific IgG and IgA antibodies were detected in mice immunized with the viruses, and the immunized mice showed milder clinical signs and/or lower virus replication levels in the respiratory tract upon virus challenge. Our results indicate that these PB2-KO viruses have potential as vaccine candidates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models.

    Science.gov (United States)

    Gong, Xiao-Qian; Sun, Ying-Feng; Ruan, Bao-Yang; Liu, Xiao-Min; Wang, Qi; Yang, Hai-Ming; Wang, Shuai-Yong; Zhang, Peng; Wang, Xiu-Hui; Shan, Tong-Ling; Tong, Wu; Zhou, Yan-Jun; Li, Guo-Xin; Zheng, Hao; Tong, Guang-Zhi; Yu, Hai

    2017-06-01

    Swine influenza viruses have been circulating in pigs throughout world and might be potential threats to human health. PA-X protein is a newly discovered protein produced from the PA gene by ribosomal frameshifting and the effects of PA-X on the 1918 H1N1, the pandemic 2009 H1N1, the highly pathogenic avian H5N1 and the avian H9N2 influenza viruses have been reported. However, the role of PA-X in the pathogenesis of swine influenza virus is still unknown. In this study, we rescued the H1N1 wild-type (WT) classical swine influenza virus (A/Swine/Guangdong/1/2011 (H1N1)) and H1N1 PA-X deficient virus containing mutations at the frameshift motif, and compared their replication properties and pathogenicity of swine influenza virus in vitro and in vivo. Our results show that the expression of PA-X inhibits virus replication and polymerase activity in cultured cells and decreases virulence in mouse models. Therefore, our study demonstrates that PA-X protein acts as a negative virulence regulator for classical H1N1 swine influenza virus and decreases virulence by inhibiting viral replication and polymerase activity, deepening our understanding of the pathogenesis of swine influenza virus. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Molecular basis of pathogenesis of emerging viruses infecting aquatic animals

    Directory of Open Access Journals (Sweden)

    Lang Gui

    2018-01-01

    Full Text Available Aquatic vertebrates are very abundant in the world, and they are of tremendous importance in providing global food security and nutrition. However, emergent and resurgent viruses, such as ranavirus (e.g., Rana grylio virus, RGV and Andriasd avidianus ranavirus, ADRV, herpesvirus (e.g., Carassius carassius herpesvirus, CaHV, reovirus (e.g., grass carp reovirus 109, GCRV-109, Scophthal musmaximus reovirus, SMReV and Micropterus salmoides reovirus, MsReV, and rhabdovirus (e.g., Siniper cachuatsi rhabdovirus, SCRV and Scophthal musmaximus rhabdovirus, SMRV can cause severe diseases in aquaculture animals and wild lower vertebrates, such as frogs, giant salamanders, fish, and so on. Here, we will briefly describe the symptoms produced by the aforementioned viruses and the molecular basis of the virus–host interactions. This manuscript aims to provide an overview of viral diseases in lower vertebrates with an emphasis on visible symptomatic manifestations and pathogenesis.

  15. Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

    Science.gov (United States)

    Byrd, Chelsea M; Grosenbach, Douglas W; Berhanu, Aklile; Dai, Dongcheng; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Yang, Guang; Tyavanagimatt, Shanthakumar; Harver, Chris; Wineinger, Kristin A; Page, Jessica; Stavale, Eric; Stone, Melialani A; Fuller, Kathleen P; Lovejoy, Candace; Leeds, Janet M; Hruby, Dennis E; Jordan, Robert

    2013-04-01

    Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

  16. Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein.

    Science.gov (United States)

    Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long

    2017-01-01

    Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID 50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.

  17. Replication-competent recombinant porcine reproductive and respiratory syndrome (PRRS) viruses expressing indicator proteins and antiviral cytokines.

    Science.gov (United States)

    Sang, Yongming; Shi, Jishu; Sang, Wenjing; Rowland, Raymond R R; Blecha, Frank

    2012-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more

  18. Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

    Energy Technology Data Exchange (ETDEWEB)

    Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States); Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo (Egypt); Che, Xibing; Sung, Phillip; Sommer, Marvin H. [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States); Hay, John [Department of Microbiology and Immunology, School of Medicine and Biomedical Science, University at Buffalo, Buffalo, NY (United States); Arvin, Ann M. [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States)

    2016-05-15

    VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.

  19. Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

    Science.gov (United States)

    Milrot, Elad; Mutsafi, Yael; Fridmann-Sirkis, Yael; Shimoni, Eyal; Rechav, Katya; Gurnon, James R; Van Etten, James L; Minsky, Abraham

    2016-01-01

    The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway. © 2015 John Wiley & Sons Ltd.

  20. Antiviral efficacy against hepatitis B virus replication of oleuropein isolated from Jasminum officinale L. var. grandiflorum.

    Science.gov (United States)

    Zhao, Guiqin; Yin, Zhifeng; Dong, Junxing

    2009-09-07

    Jasminum officinale L. var. grandiflorum (JOG) is a folk medicine used for the treatment of hepatitis in south of China. Phytochemical studies showed that secoiridoid glycosides are the typical constituents of this plant. The present study was undertaken to evaluate the effect of oleuropein (Ole) derived from the flowers of JOG on hepatitis B virus (HBV) replication in HepG2 2.2.15 cell line in vitro and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by ELISA. DHBV in duck serum was analyzed by dot blot. Ole blocks effectively HBsAg secretion in HepG2 2.2.15 cells in a dose-dependent manner (IC(50)=23.2 microg/ml). Ole (80 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. Ole therefore warrants further investigation as a potential therapeutic agent for HBV infection.

  1. Thiazolides as novel antiviral agents. 1. Inhibition of hepatitis B virus replication.

    Science.gov (United States)

    Stachulski, Andrew V; Pidathala, Chandrakala; Row, Eleanor C; Sharma, Raman; Berry, Neil G; Iqbal, Mazhar; Bentley, Joanne; Allman, Sarah A; Edwards, Geoffrey; Helm, Alison; Hellier, Jennifer; Korba, Brent E; Semple, J Edward; Rossignol, Jean-Francois

    2011-06-23

    We report the syntheses and activities of a wide range of thiazolides [viz., 2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis B virus replication, with QSAR analysis of our results. The prototypical thiazolide, nitazoxanide [2-hydroxybenzoyl-N-(5-nitrothiazol-2-yl)amide, NTZ] 1 is a broad spectrum antiinfective agent effective against anaerobic bacteria, viruses, and parasites. By contrast, 2-hydroxybenzoyl-N-(5-chlorothiazol-2-yl)amide 3 is a novel, potent, and selective inhibitor of hepatitis B replication (EC(50) = 0.33 μm) but is inactive against anaerobes. Several 4'- and 5'-substituted thiazolides show good activity against HBV; by contrast, some related salicyloylanilides show a narrower spectrum of activity. The ADME properties of 3 are similar to 1; viz., the O-acetate is an effective prodrug, and the O-aryl glucuronide is a major metabolite. The QSAR study shows a good correlation of observed EC(90) for intracellular virions with thiazolide structural parameters. Finally we discuss the mechanism of action of thiazolides in relation to the present results.

  2. Characterization of Aedes aegypti innate-immune pathways that limit Chikungunya virus replication.

    Directory of Open Access Journals (Sweden)

    Melanie McFarlane

    2014-07-01

    Full Text Available Replication of arboviruses in their arthropod vectors is controlled by innate immune responses. The RNA sequence-specific break down mechanism, RNA interference (RNAi, has been shown to be an important innate antiviral response in mosquitoes. In addition, immune signaling pathways have been reported to mediate arbovirus infections in mosquitoes; namely the JAK/STAT, immune deficiency (IMD and Toll pathways. Very little is known about these pathways in response to chikungunya virus (CHIKV infection, a mosquito-borne alphavirus (Togaviridae transmitted by aedine species to humans resulting in a febrile and arthralgic disease. In this study, the contribution of several innate immune responses to control CHIKV replication was investigated. In vitro experiments identified the RNAi pathway as a key antiviral pathway. CHIKV was shown to repress the activity of the Toll signaling pathway in vitro but neither JAK/STAT, IMD nor Toll pathways were found to mediate antiviral activities. In vivo data further confirmed our in vitro identification of the vital role of RNAi in antiviral defence. Taken together these results indicate a complex interaction between CHIKV replication and mosquito innate immune responses and demonstrate similarities as well as differences in the control of alphaviruses and other arboviruses by mosquito immune pathways.

  3. Heat Shock Protein 90 Ensures Efficient Mumps Virus Replication by Assisting with Viral Polymerase Complex Formation.

    Science.gov (United States)

    Katoh, Hiroshi; Kubota, Toru; Nakatsu, Yuichiro; Tahara, Maino; Kidokoro, Minoru; Takeda, Makoto

    2017-03-15

    Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication. IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a

  4. The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes.

    Directory of Open Access Journals (Sweden)

    Kun Zhang

    2017-04-01

    Full Text Available RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+ RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes.

  5. The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes

    Science.gov (United States)

    Yang, Meng; Liu, Songyu; Li, Zhenggang; Wang, Xianbing; Han, Chenggui; Yu, Jialin

    2017-01-01

    RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes. PMID:28388677

  6. Post-transcriptional inhibition of hepatitis C virus replication through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-03-01

    Full Text Available Abstract Background Hepatitis C Virus (HCV infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN α and ribavirin. This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV. Results This study was design to examine the ability of exogenous small interfering RNAs (siRNAs to block the replication of HCV in human liver cells. In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase. siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064. We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858 showed 58% and 88% reduction in viral titer respectively. Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 μM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.

  7. Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection.

    Science.gov (United States)

    Cui, Hongguang; Wang, Aiming

    2016-05-15

    The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature

  8. Diverse circular replication-associated protein encoding viruses circulating in invertebrates within a lake ecosystem.

    Science.gov (United States)

    Dayaram, Anisha; Galatowitsch, Mark L; Argüello-Astorga, Gerardo R; van Bysterveldt, Katherine; Kraberger, Simona; Stainton, Daisy; Harding, Jon S; Roumagnac, Philippe; Martin, Darren P; Lefeuvre, Pierre; Varsani, Arvind

    2016-04-01

    Over the last five years next-generation sequencing has become a cost effective and efficient method for identifying known and unknown microorganisms. Access to this technique has dramatically changed the field of virology, enabling a wide range of environmental viral metagenome studies to be undertaken of organisms and environmental samples from polar to tropical regions. These studies have led to the discovery of hundreds of highly divergent single stranded DNA (ssDNA) virus-like sequences encoding replication-associated proteins. Yet, few studies have explored how viruses might be shared in an ecosystem through feeding relationships. Here we identify 169 circular molecules (160 CRESS DNA molecules, nine circular molecules) recovered from a New Zealand freshwater lake, that we have tentatively classified into 51 putatively novel species and five previously described species (DflaCV-3, -5, -6, -8, -10). The CRESS DNA viruses identified in this study were recovered from molluscs (Echyridella menzeisii, Musculium novaezelandiae, Potamopyrgus antipodarum and Physella acuta) and insect larvae (Procordulia grayi, Xanthocnemis zealandica, and Chironomus zealandicus) collected from Lake Sarah, as well as from the lake water and benthic sediments. Extensive diversity was observed across most CRESS DNA molecules recovered. The putative capsid protein of one viral species was found to be most similar to those of members of the Tombusviridae family, thus expanding the number of known RNA-DNA hybrid viruses in nature. We noted a strong association between the CRESS DNA viruses and circular molecules identified in the water and browser organisms (C. zealandicus, P. antipodarum and P. acuta), and between water sediments and undefended prey species (C. zealandicus). However, we were unable to find any significant correlation of viral assemblages to the potential feeding relationships of the host aquatic invertebrates. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. JC virus agnoprotein enhances large T antigen binding to the origin of viral DNA replication: evidence for its involvement in viral DNA replication.

    Science.gov (United States)

    Saribas, A Sami; White, Martyn K; Safak, Mahmut

    2012-11-10

    Agnoprotein is required for the successful completion of the JC virus (JCV) life cycle and was previously shown to interact with JCV large T-antigen (LT-Ag). Here, we further characterized agnoprotein's involvement in viral DNA replication. Agnoprotein enhances the DNA binding activity of LT-Ag to the viral origin (Ori) without directly interacting with DNA. The predicted amphipathic α-helix of agnoprotein plays a major role in this enhancement. All three phenylalanine (Phe) residues of agnoprotein localize to this α-helix and Phe residues in general are known to play critical roles in protein-protein interaction, protein folding and stability. The functional relevance of all Phe residues was investigated by mutagenesis. When all were mutated to alanine (Ala), the mutant virus (F31AF35AF39A) replicated significantly less efficiently than each individual Phe mutant virus alone, indicating the importance of Phe residues for agnoprotein function. Collectively, these studies indicate a close involvement of agnoprotein in viral DNA replication. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Host cell proteins interacting with the 3' end of TGEV coronavirus genome influence virus replication.

    Science.gov (United States)

    Galán, Carmen; Sola, Isabel; Nogales, Aitor; Thomas, Benjamin; Akoulitchev, Alexandre; Enjuanes, Luis; Almazán, Fernando

    2009-09-01

    Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specific recognition of RNA cis-acting signals located at the ends of the viral genome. To identify cellular proteins involved in coronavirus RNA synthesis, transmissible gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for RNA affinity protein purification. Ten proteins were preferentially pulled down with either the 5' or 3' ends of the genome and identified by proteomic analysis. Nine of them, including members of the heterogeneous ribonucleoprotein family of proteins (hnRNPs), the poly(A)-binding protein (PABP), the p100 transcriptional co-activator protein and two aminoacyl-tRNA synthetases, showed a preferential binding to the 3' end of the genome, whereas only the polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5' end of the genome. The potential function of the 3' end-interacting proteins in virus replication was studied by analyzing the effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP, hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) caused a significant 2 to 3-fold reduction of viral RNA synthesis. Interestingly, the silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in viral RNA synthesis in both systems. These data suggest that PABP, hnRNP Q, and EPRS play a positive role in virus infection that could be mediated through their interaction with the viral 3' end, and that GAPDH has a negative effect on viral infection.

  11. Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity.

    Science.gov (United States)

    Martinez, Zachary S; Castro, Edison; Seong, Chang-Soo; Cerón, Maira R; Echegoyen, Luis; Llano, Manuel

    2016-10-01

    Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4(+) T cells. However, these compounds did not inhibit infection by single-round infection vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In contrast, analysis of single-round infection VSV-G-pseudotyped HIV-1 produced in the presence of compound 1 or 2 showed a complete lack of infectivity in human CD4(+) T cells, suggesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of virion-associated reverse transcriptase activity, ratifying the effect of the fullerene derivatives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infectivity, suggesting a protease-independent mechanism of action. Highlighting the potential therapeutic relevance of fullerene derivatives, these compounds block infection by HIV-1 resistant to protease and maturation inhibitors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Antiviral Activity of Resveratrol against Human and Animal Viruses.

    Science.gov (United States)

    Abba, Yusuf; Hassim, Hasliza; Hamzah, Hazilawati; Noordin, Mohamed Mustapha

    2015-01-01

    Resveratrol is a potent polyphenolic compound that is being extensively studied in the amelioration of viral infections both in vitro and in vivo. Its antioxidant effect is mainly elicited through inhibition of important gene pathways like the NF-κβ pathway, while its antiviral effects are associated with inhibitions of viral replication, protein synthesis, gene expression, and nucleic acid synthesis. Although the beneficial roles of resveratrol in several viral diseases have been well documented, a few adverse effects have been reported as well. This review highlights the antiviral mechanisms of resveratrol in human and animal viral infections and how some of these effects are associated with the antioxidant properties of the compound.

  13. Glycoprotein from street rabies virus BD06 induces early and robust immune responses when expressed from a non-replicative adenovirus recombinant.

    Science.gov (United States)

    Wang, Shuchao; Sun, Chenglong; Zhang, Shoufeng; Zhang, Xiaozhuo; Liu, Ye; Wang, Ying; Zhang, Fei; Wu, Xianfu; Hu, Rongliang

    2015-09-01

    The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines.

  14. Animal models for Ebola and Marburg virus infections

    Science.gov (United States)

    Nakayama, Eri; Saijo, Masayuki

    2013-01-01

    Ebola and Marburg hemorrhagic fevers (EHF and MHF) are caused by the Filoviridae family, Ebolavirus and Marburgvirus (ebolavirus and marburgvirus), respectively. These severe diseases have high mortality rates in humans. Although EHF and MHF are endemic to sub-Saharan Africa. A novel filovirus, Lloviu virus, which is genetically distinct from ebolavirus and marburgvirus, was recently discovered in Spain where filoviral hemorrhagic fever had never been reported. The virulence of this virus has not been determined. Ebolavirus and marburgvirus are classified as biosafety level-4 (BSL-4) pathogens and Category A agents, for which the US government requires preparedness in case of bioterrorism. Therefore, preventive measures against these viral hemorrhagic fevers should be prepared, not only in disease-endemic regions, but also in disease-free countries. Diagnostics, vaccines, and therapeutics need to be developed, and therefore the establishment of animal models for EHF and MHF is invaluable. Several animal models have been developed for EHF and MHF using non-human primates (NHPs) and rodents, which are crucial to understand pathophysiology and to develop diagnostics, vaccines, and therapeutics. Rhesus and cynomolgus macaques are representative models of filovirus infection as they exhibit remarkably similar symptoms to those observed in humans. However, the NHP models have practical and ethical problems that limit their experimental use. Furthermore, there are no inbred and genetically manipulated strains of NHP. Rodent models such as mouse, guinea pig, and hamster, have also been developed. However, these rodent models require adaptation of the virus to produce lethal disease and do not mirror all symptoms of human filovirus infection. This review article provides an outline of the clinical features of EHF and MHF in animals, including humans, and discusses how the animal models have been developed to study pathophysiology, vaccines, and therapeutics. PMID:24046765

  15. Animal models for Ebola and Marburg virus infections

    Directory of Open Access Journals (Sweden)

    Eri eNakayama

    2013-09-01

    Full Text Available Ebola and Marburg hemorrhagic fevers (EHF and MHF are caused by the Filoviridae family, Ebolavirus and Marburgvirus (ebolavirus and marburgvirus, respectively. These severe diseases have high mortality rates in humans. Although EHF and MHF are endemic to sub-Saharan Africa. A novel filovirus, Lloviu virus, which is genetically distinct from ebolavirus and marburgvirus, was recently discovered in Spain where filoviral hemorrhagic fever had never been reported. The virulence of this virus has not been determined. Ebolavirus and marburgvirus are classified as biosafety level-4 (BSL-4 pathogens and Category A agents, for which the US government requires preparedness in case of bioterrorism. Therefore, preventive measures against these viral hemorrhagic fevers should be prepared, not only in disease-endemic regions, but also in disease-free countries. Diagnostics, vaccines, and therapeutics need to be developed, and therefore the establishment of animal models for EHF and MHF is invaluable. Several animal models have been developed for EHF and MHF using nonhuman primates (NHPs and rodents, which are crucial to understand pathophysiology and to develop diagnostics, vaccines, and therapeutics. Rhesus and cynomolgus macaques are representative models of filovirus infection as they exhibit remarkably similar symptoms to those observed in humans. However, the NHP models have practical and ethical problems that limit their experimental use. Furthermore, there are no inbred and genetically manipulated strains of NHP. Rodent models such as mouse, guinea pig, and hamster, have also been developed. However, these rodent models require adaptation of the virus to produce lethal disease and do not mirror all symptoms of human filovirus infection. This review article provides an outline of the clinical features of EHF and MHF in animals, including humans, and discusses how the animal models have been developed to study pathophysiology, vaccines, and therapeutics.

  16. Replication of the Shrimp Virus WSSV Depends on Glutamate-Driven Anaplerosis.

    Directory of Open Access Journals (Sweden)

    Chun-Yuan Li

    Full Text Available Infection with the white spot syndrome virus (WSSV induces a metabolic shift in shrimp that resembles the "Warburg effect" in mammalian cells. This effect is triggered via activation of the PI3K-Akt-mTOR pathway, and it is usually accompanied by the activation of other metabolic pathways that provide energy and direct the flow of carbon and nitrogen. Here we show that unlike the glutamine metabolism (glutaminolysis seen in most cancer cells to double deaminate glutamine to produce glutamate and the TCA cycle intermediate α-ketoglutarate (α-KG, at the WSSV genome replication stage (12 hpi, although glutaminase (GLS expression was upregulated, only glutamate was taken up by the hemocytes of WSSV-infected shrimp. At the same time, we observed an increase in the activity of the two enzymes that convert glutamate to α-KG, glutamate dehydrogenase (GDH and aspartate aminotransferase (ASAT. α-ketoglutarate concentration was also increased. A series of inhibition experiments suggested that the up-regulation of GDH is regulated by mTORC2, and that the PI3K-mTORC1 pathway is not involved. Suppression of GDH and ASAT by dsRNA silencing showed that both of these enzymes are important for WSSV replication. In GDH-silenced shrimp, direct replenishment of α-KG rescued both ATP production and WSSV replication. From these results, we propose a model of glutamate-driven anaplerosis that fuels the TCA cycle via α-KG and ultimately supports WSSV replication.

  17. Effect of fatty acids on arenavirus replication: inhibition of virus production by lauric acid.

    Science.gov (United States)

    Bartolotta, S; García, C C; Candurra, N A; Damonte, E B

    2001-01-01

    To study the functional involvement of cellular membrane properties on arenavirus infection, saturated fatty acids of variable chain length (C10-C18) were evaluated for their inhibitory activity against the multiplication of Junin virus (JUNV). The most active inhibitor was lauric acid (C12), which reduced virus yields of several attenuated and pathogenic strains of JUNV in a dose dependent manner, without affecting cell viability. Fatty acids with shorter or longer chain length had a reduced or negligible anti-JUNV activity. Lauric acid did not inactivate virion infectivity neither interacted with the cell to induce a state refractory to virus infection. From mechanistic studies, it can be concluded that lauric acid inhibited a late maturation stage in the replicative cycle of JUNV. Viral protein synthesis was not affected by the compound, but the expression of glycoproteins in the plasma membrane was diminished. A direct correlation between the inhibition of JUNV production and the stimulation of triacylglycerol cell content was demonstrated, and both lauric-acid induced effects were dependent on the continued presence of the fatty acid. Thus, the decreased insertion of viral glycoproteins into the plasma membrane, apparently due to the increased incorporation of triacylglycerols, seems to cause an inhibition of JUNV maturation and release.

  18. Oct4+ stem/progenitor swine lung epithelial cells are targets for influenza virus replication.

    Science.gov (United States)

    Khatri, Mahesh; Goyal, Sagar M; Saif, Yehia M

    2012-06-01

    We isolated stem/progenitor epithelial cells from the lungs of 4- to 6-week-old pigs. The epithelial progenitor colony cells were surrounded by mesenchymal stromal cells. The progenitor epithelial colony cells expressed stem cell markers such as octamer binding transcription factor 4 (Oct4) and stage-specific embryonic antigen 1 (SSEA-1), as well as the epithelial markers pancytokeratin, cytokeratin-18, and occludin, but not mesenchymal (CD44, CD29, and CD90) and hematopoietic (CD45) markers. The colony cells had extensive self-renewal potential and had the capacity to undergo differentiation to alveolar type I- and type II-like pneumocytes. Additionally, these cells expressed sialic acid receptors and supported the active replication of influenza virus, which was accompanied by cell lysis. The lysis of progenitor epithelial cells by influenza virus may cause a marked reduction in the potential of progenitor cells for self renewal and for their ability to differentiate into specialized cells of the lung. These observations suggest the possible involvement of lung stem/progenitor cells in influenza virus infection.

  19. Landscape Utilisation, Animal Behaviour and Hendra Virus Risk.

    Science.gov (United States)

    Field, H E; Smith, C S; de Jong, C E; Melville, D; Broos, A; Kung, N; Thompson, J; Dechmann, D K N

    2016-03-01

    Hendra virus causes sporadic fatal disease in horses and humans in eastern Australia. Pteropid bats (flying-foxes) are the natural host of the virus. The mode of flying-fox to horse transmission remains unclear, but oro-nasal contact with flying-fox urine, faeces or saliva is the most plausible. We used GPS data logger technology to explore the landscape utilisation of black flying-foxes and horses to gain new insight into equine exposure risk. Flying-fox foraging was repetitious, with individuals returning night after night to the same location. There was a preference for fragmented arboreal landscape and non-native plant species, resulting in increased flying-fox activity around rural infrastructure. Our preliminary equine data logger study identified significant variation between diurnal and nocturnal grazing behaviour that, combined with the observed flying-fox foraging behaviour, could contribute to Hendra virus exposure risk. While we found no significant risk-exposing difference in individual horse movement behaviour in this study, the prospect warrants further investigation, as does the broader role of animal behaviour and landscape utilisation on the transmission dynamics of Hendra virus.

  20. A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

    Science.gov (United States)

    Hoffmann, Julia; Schneider, Carola; Heinbockel, Lena; Brandenburg, Klaus; Reimer, Rudolph; Gabriel, Gülsah

    2014-04-01

    Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

    Science.gov (United States)

    Wang, Zihua; Wu, Li; Cheng, Xin; Liu, Shizhu; Li, Baosheng; Li, Haijun; Kang, Fubiao; Wang, Junping; Xia, Huan; Ping, Caiyan; Nassal, Michael; Sun, Dianxing

    2013-01-01

    Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better

  2. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

    Directory of Open Access Journals (Sweden)

    Zihua Wang

    Full Text Available Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV, a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg RNA which is also required as bicistronic mRNA for the capsid (core protein and the reverse transcriptase (Pol; their open reading frames (ORFs overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES. We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR and humanized Renilla green fluorescent protein (hrGFP produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to

  3. Conserved elements within the genome of foot-and mouth disease virus; their influence on virus replication

    DEFF Research Database (Denmark)

    Kjær, Jonas; Poulsen, Line D.; Vinther, Jeppe

    Objectives: Several conserved elements within the genome of foot-and-mouth disease virus (FMDV) have been identified, e.g. the IRES. Such elements can be crucial for the efficient replication of the genomic RNA. Previously, SHAPE analysis of the entire FMDV genome (Poulsen et al., 2016 submitted......). It is believed that this “cleavage” is achieved by ribosomal skipping, in which the 2A peptide prevents the ribosome from linking the next amino acid (aa) to the growing polypeptide. The nature of this “cleavage” has so far not been investigated in the context of the full-length FMDV RNA within cells. Through...... mutations, to disrupt the 3Dpol RNA secondary structure, were generated in a FMDV replicon containing Gaussia luciferase. 2) Sequence changes encoding selected modifications of the 2A peptide (as described by Donnelly et al., 2001) were introduced into a full-length FMDV cDNA and in a FMDV replicon c...

  4. Replication of West Nile virus, Rabensburg lineage in mammalian cells is restricted by temperature

    Directory of Open Access Journals (Sweden)

    Aliota Matthew T

    2012-12-01

    Full Text Available Abstract Background The genus Flavivirus currently consists of approximately 80 single-strand positive-sense RNA viruses. These replicate in a range of hosts including myriad vertebrate, insect, and tick species. As a consequence of this broad host range, the majority of flaviviruses can be propagated in most vertebrate and insect cell cultures. This ability to infect arthropods and vertebrates usually is essential for maintenance of these viruses in nature. But recently, there has been the discovery of a number of flaviviruses that infect mosquitoes but not vertebrates. It remains largely unknown why certain flaviviruses infect vertebrates and mosquitoes while others infect mosquitoes or vertebrates exclusively. Methods Here, we initiated in vitro host range studies of Rabensburg virus (RABV, an intermediate between the mosquito-specific and horizontally transmitted flaviviruses, to provide information on the factor(s that underlie the varying host range of flaviviruses. RABV is an intermediate between the mosquito-specific and horizontally transmitted flaviviruses because it does not infect mammalian or avian cell cultures, house sparrows, or chickens, but it does share genetic characteristics with the Japanese Encephalitis serogroup of flaviviruses. Results In vitro growth kinetic assays revealed the complete abrogation of RABV growth on Vero and E6 cells incubated at temperatures 35°C and higher, but surprisingly RABV infected, replicated efficiently, and displayed overt cytopathic effects (CPE on Vero and E6 cell cultures incubated below 35°C. In contrast, RABV was fully viable, replicated efficiently, and displayed overt CPE on C6/36 cells incubated at 28°C or 37°C, thus implicating temperature as an important factor limiting the host range of RABV. Conclusions These data are critical for further study to more fully identify the determinants that mediate the evolution of biological transmission among flaviviruses. It also will be

  5. In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication.

    Science.gov (United States)

    Freitas, Ferdinando B; Frouco, Gonçalo; Martins, Carlos; Leitão, Alexandre; Ferreira, Fernando

    2016-10-01

    African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro. Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (-66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF

  6. Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-Driven Replication Capacity.

    Science.gov (United States)

    Naidoo, Vanessa L; Mann, Jaclyn K; Noble, Christie; Adland, Emily; Carlson, Jonathan M; Thomas, Jake; Brumme, Chanson J; Thobakgale-Tshabalala, Christina F; Brumme, Zabrina L; Brockman, Mark A; Goulder, Philip J R; Ndung'u, Thumbi

    2017-09-01

    In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter ( n = 53) and transmitter ( n = 44) mothers ( P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses ( n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers ( P HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities. IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT bottleneck. This study advances our understanding of the genetic bottleneck in MTCT by revealing that viruses transmitted to infants have a lower replicative ability as well as a higher similarity to the population consensus (in this case HIV subtype C) than those of their mothers. Furthermore, the observation that "consensus-like" virus sequences

  7. The eukaryotic elongation factor 1A is critical for genome replication of the paramyxovirus respiratory syncytial virus.

    Directory of Open Access Journals (Sweden)

    Ting Wei

    Full Text Available The eukaryotic translation factor eEF1A assists replication of many RNA viruses by various mechanisms. Here we show that down-regulation of eEF1A restricts the expression of viral genomic RNA and the release of infectious virus, demonstrating a biological requirement for eEF1A in the respiratory syncytial virus (RSV life cycle. The key proteins in the replicase/transcriptase complex of RSV; the nucleocapsid (N protein, phosphoprotein (P and matrix (M protein, all associate with eEF1A in RSV infected cells, although N is the strongest binding partner. Using individually expressed proteins, N, but not P or M bound to eEF1A. This study demonstrates a novel interaction between eEF1A and the RSV replication complex, through binding to N protein, to facilitate genomic RNA synthesis and virus production.

  8. The eukaryotic elongation factor 1A is critical for genome replication of the paramyxovirus respiratory syncytial virus.

    Science.gov (United States)

    Wei, Ting; Li, Dongsheng; Marcial, Daneth; Khan, Moshin; Lin, Min-Hsuan; Snape, Natale; Ghildyal, Reena; Harrich, David; Spann, Kirsten

    2014-01-01

    The eukaryotic translation factor eEF1A assists replication of many RNA viruses by various mechanisms. Here we show that down-regulation of eEF1A restricts the expression of viral genomic RNA and the release of infectious virus, demonstrating a biological requirement for eEF1A in the respiratory syncytial virus (RSV) life cycle. The key proteins in the replicase/transcriptase complex of RSV; the nucleocapsid (N) protein, phosphoprotein (P) and matrix (M) protein, all associate with eEF1A in RSV infected cells, although N is the strongest binding partner. Using individually expressed proteins, N, but not P or M bound to eEF1A. This study demonstrates a novel interaction between eEF1A and the RSV replication complex, through binding to N protein, to facilitate genomic RNA synthesis and virus production.

  9. Detergent-resistant membrane association of NS2 and E2 during hepatitis C virus replication.

    Science.gov (United States)

    Shanmugam, Saravanabalaji; Saravanabalaji, Dhanaranjani; Yi, MinKyung

    2015-04-01

    Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these

  10. Novel bisegmented virus (picobirnavirus of animals, birds and humans

    Directory of Open Access Journals (Sweden)

    Anjan Mondal

    2014-04-01

    Full Text Available Picobirnaviruses (PBVs are novel group of small, nonenveloped, bisegmented and double stranded RNA viruses. PBVs have been identified in the faeces of a broad range of hosts by several international research groups. Since attempts to culture PBV in vitro have not been made to date and no animal model of infection and disease exists. Laboratory diagnosis relies upon electron microscopy, the detection of the double stranded RNA bisegmented genome by polyacrylamide gel electrophoresis and reverse transcription polymerase chain reaction. PBVs have been identified in both normal and diarrheic faeces. Although their pathogenicity is still unclear, their potential needs further investigation.

  11. 42 CFR 71.56 - African rodents and other animals that may carry the monkeypox virus.

    Science.gov (United States)

    2010-10-01

    ... the monkeypox virus. 71.56 Section 71.56 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND... and other animals that may carry the monkeypox virus. (a) What actions are prohibited? What animals... transmitting or carrying the monkeypox virus. Such products include, but are not limited to, fully taxidermied...

  12. Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection.

    Science.gov (United States)

    Oaks, J L; Long, M T; Baszler, T V

    2004-09-01

    Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred selectively within the lesion and not in other tissues. These findings suggest that EIAV-associated neurologic disease is the direct result of viral replication.

  13. The bovine papilloma virus E1 protein has ATPase activity essential to viral DNA replication and efficient transformation in cells.

    Science.gov (United States)

    MacPherson, P; Thorner, L; Parker, L M; Botchan, M

    1994-10-01

    The bovine papilloma virus (BPV) E1 protein essential to viral DNA replication has recently been shown to associate via direct protein-DNA interactions with the viral origin of replication and to be an ATP-dependent helicase. We show here that in accordance with the latter function, the E1 gene product has intrinsic ATPase activity. Mutations placed throughout the nucleotide binding consensus element abolish the ATPase activity of E1 and render BPV genomes harboring such mutations defective for episomal replication and impaired for oncogenic transformation.

  14. Generation of a Replication-Competent, Propagation-Deficient Virus Vector Based on the Transmissible Gastroenteritis Coronavirus Genome

    OpenAIRE

    Ortego, Javier; Escors, David; Laude, Hubert; Enjuanes, Luis

    2002-01-01

    Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E+ packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic...

  15. Inhibition of dengue virus replication by diisopropyl chrysin-7-yl phosphate.

    Science.gov (United States)

    Du, Jiang; Chen, Zhe; Zhang, Ting; Wang, Jianmin; Jin, Qi

    2016-08-01

    Dengue fever is a tropical disease and caused by dengue virus (DENV), which is transmitted by mosquitoes and infects about 400 million people annually. With the development of international trade and travel, China is facing a growing threat. Over 40 thousands of people were infected during the 2014 DENV outbreak in Guangdong. Neither licensed vaccine nor therapeutic drug has been available. In this report, we isolated two clinical DENV strains. The full-length genome was sequenced and characterized. We also applied a flavonoid, CPI, into an anti-DENV assay. Replication of viral RNA and expression of viral protein was all strongly inhibited. These results indicated that CPI may serve as potential protective agents in the treatment of patients with chronic DENV infection.

  16. Research advances in microRNAs in regulating hepatitis C virus replication and antiviral therapy

    Directory of Open Access Journals (Sweden)

    CUI Xianghua

    2015-05-01

    Full Text Available Hepatitis C virus (HCV infection is one of the most common causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. MicroRNAs (miRNAs, a class of small, non-coding RNA, are involved in a variety of physiological and pathological processes in human bodies. The mechanism by which miRNAs regulate HCV replication is described, and the effects of liver-specific microRNA-122 antagonists on hepatitis C antiviral therapy are discussed. Our study indicates that miRNAs play an important regulatory role in HCV expression. Targeting miRNAs may be a potential therapeutic approach for treating HCV infection, but further studies are still in need.

  17. Replication modes of Maize streak virus mutants lacking RepA or the RepA-pRBR interaction motif.

    Science.gov (United States)

    Ruschhaupt, Moritz; Martin, Darren P; Lakay, Francisco; Bezuidenhout, Marion; Rybicki, Edward P; Jeske, Holger; Shepherd, Dionne N

    2013-08-01

    The plant-infecting mastreviruses (family Geminiviridae) express two distinct replication-initiator proteins, Rep and RepA. Although RepA is essential for systemic infectivity, little is known about its precise function. We therefore investigated its role in replication using 2D-gel electrophoresis to discriminate the replicative forms of Maize streak virus (MSV) mutants that either fail to express RepA (RepA(-)), or express RepA that is unable to bind the plant retinoblastoma related protein, pRBR. Whereas amounts of viral DNA were reduced in two pRBR-binding deficient RepA mutants, their repertoires of replicative forms changed only slightly. While a complete lack of RepA expression was also associated with reduced viral DNA titres, the only traces of replicative intermediates of RepA(-) viruses were those indicative of recombination-dependent replication. We conclude that in MSV, RepA, but not RepA-pRBR binding, is necessary for single-stranded DNA production and efficient rolling circle replication. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Honeysuckle aqueous extract and induced let-7a suppress dengue virus type 2 replication and pathogenesis.

    Science.gov (United States)

    Lee, Ying-Ray; Yeh, Siao-Fen; Ruan, Xiao-Ming; Zhang, Hao; Hsu, Sheng-Da; Huang, Hsien-Da; Hsieh, Chang-Chi; Lin, Yee-Shin; Yeh, Trai-Ming; Liu, Hsiao-Sheng; Gan, Dai-Di

    2017-02-23

    Honeysuckle (Lonicera japonica Thunb.), a traditional Chinese herb, has widely been used to treat pathogen infection. However, the underlying-mechanism remains elusive. To reveal the host microRNA (miRNA) profile with the anti-viral activity after honeysuckle treatment. Here we reveal the differentially expressed miRNAs by Solexa ® deep sequencing from the blood of human and mice after the aqueous extract treatment. Among these overexpressed innate miRNAs both in human and mice, let-7a is able to target the NS1 region (nt 3313-3330) of dengue virus (DENV) serotypes 1, 2 and 4 predicated by the target predication software. We confirmed that let-7a could target DENV2 at the predicated NS1 sequence and suppress DENV2 replication demonstrated by luciferase-reporter activity, RT-PCR, real-time PCR, Western blotting and plaque assay. ICR-suckling mice consumed honeysuckle aqueous extract either before or after intracranial injection with DENV2 showed decreased levels of NS1 RNA and protein expression accompanied with alleviated disease symptoms, decreased virus load, and prolonged survival time. Similar results were observed when DENV2-infected mice were intracranially injected with let-7a. We reveal that honeysuckle attenuates DENV replication and related pathogenesis in vivo through induction of let-7a expression. This study opens a new direction for prevention and treatment of DENV infection through induction of the innate miRNA let-7a by honeysuckle. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  19. Hepatitis B virus infection and replication in human bone marrow mesenchymal stem cells

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    Ma Ruiping

    2011-10-01

    Full Text Available Abstract Background Hepatitis B virus (HBV infection is a blood borne infectious disease that affects the liver. Human bone marrow mesenchymal stem cells (BMSCs may serve as a cell source for adult stem cell transplantation in liver repair. However, the susceptibility of human BMSCs to HBV infection is poorly understood. The aim of this study was to investigate the infection and replication of HBV in cultures of human BMSCs. Results Human BMSCs were confirmed using flow cytometry. Intracellular HBV DNA was detected at d 2 after infection and maintained at relatively high levels from d 6 to d 12. The maximal level of intracellular HBV DNA was 9.37 × 105 copies/mL. The extracellular HBV DNA was observed from d 3 to d 15, and the levels ranged from 3.792 × 102 copies/mL to 4.067 × 105 copies/mL. HBsAg in the culture medium was detected from d 2 to d 16. HBeAg secretion was positive from d 5 to d 13. HBcAg constantly showed positive signals in approximately 7%-20% of BMSCs from 2 days after exposure. Intracellular HBV covalently closed circular DNA (cccDNA could be detected as early as 2 days postinfection, and strong signals were obtained with increasing time. Conclusion HBV can infect and replicate in human BMSCs. Human BMSCs may be a useful tool for investigating HBV life-cycle and the mechanism of initial virus-cell interactions.

  20. Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication

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    Christopher M. Murphy

    2016-09-01

    Full Text Available The hepatitis B virus (HBV regulatory protein X (HBx activates gene expression from the HBV covalently closed circular DNA (cccDNA genome. Interaction of HBx with the DDB1-CUL4-ROC1 (CRL4 E3 ligase is critical for this function. Using substrate-trapping proteomics, we identified the structural maintenance of chromosomes (SMC complex proteins SMC5 and SMC6 as CRL4HBx substrates. HBx expression and HBV infection degraded the SMC5/6 complex in human hepatocytes in vitro and in humanized mice in vivo. HBx targets SMC5/6 for ubiquitylation by the CRL4HBx E3 ligase and subsequent degradation by the proteasome. Using a minicircle HBV (mcHBV reporter system with HBx-dependent activity, we demonstrate that SMC5/6 knockdown, or inhibition with a dominant-negative SMC6, enhance HBx null mcHBV-Gluc gene expression. Furthermore, SMC5/6 knockdown rescued HBx-deficient HBV replication in human hepatocytes. These results indicate that a primary function of HBx is to degrade SMC5/6, which restricts HBV replication by inhibiting HBV gene expression.

  1. SELECTIVE INHIBITION OF HEPATITIS C VIRUS REPLICATION BY ALPHA-ZAM, A NIGELLA SATIVA SEED FORMULATION.

    Science.gov (United States)

    Oyero, Olufunmilayo G; Toyama, Masaaki; Mitsuhiro, Naoki; Onifade, Abdulfatah A; Hidaka, Akemi; Okamoto, Mika; Baba, Masanori

    2016-01-01

    Hepatitis C virus (HCV) infection became curable because of the development of direct acting antivirals (DAAs). However, the high cost of DAAs has greatly impeded their potential impact on the treatment of HCV infection. As a result, hepatitis C will continue to cause substantial morbidity, and mortality among chronically infected individuals in low and middle income countries. Thus, urgent need exists for developing cheaper drugs available to hepatitis C patients in these countries. Alpha-zam, an indigenous herbal formulation from Nigella sativa seed, was examined for its anti-HCV activity and cytotoxicity in genotype 1b HCV replicon cells. The antiviral activity was determined by luciferase expression and viral RNA synthesis, while the cytotoxicity was assessed by viable cell number and glyceraldehyde-3-phosphate dehydrogenase RNA synthesis in the replicon cells. Alpha-zam was found to be a selective inhibitor of HCV replication. The 50% effective dilution and 50% cytotoxic dilution of Alpha-zam were 761- and < 100-fold, respectively, in the subgenomic replicon cells LucNeo#2. Its selective inhibition of HCV was also confirmed by HCV RNA levels in LucNeo#2 and in the full-genome HCV replicon cells NNC#2 using real-time reverse transcriptase polymerase chain reaction. Furthermore, the anti-HCV activity of Alpha-zam was not due to the induction of interferon. Alpha-zam selectively inhibits HCV replication and therefore has potential for a novel antiviral agent against HCV infection.

  2. Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

    Science.gov (United States)

    Marques, Rafael E; Guabiraba, Rodrigo; Del Sarto, Juliana L; Rocha, Rebeca F; Queiroz, Ana Luiza; Cisalpino, Daniel; Marques, Pedro E; Pacca, Carolina C; Fagundes, Caio T; Menezes, Gustavo B; Nogueira, Maurício L; Souza, Danielle G; Teixeira, Mauro M

    2015-08-01

    Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5. © 2015 John Wiley & Sons Ltd.

  3. Hepatitis B Virus Stimulated Fibronectin Facilitates Viral Maintenance and Replication through Two Distinct Mechanisms.

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    Sheng Ren

    Full Text Available Fibronectin (FN is a high molecular weight extracellular matrix protein that functions in cell adhesion, growth, migration, and embryonic development. However, little is known about the role of FN during viral infection. In the present study, we found significantly higher levels of FN in sera, and liver tissues from hepatitis B virus (HBV patients relative to healthy individuals. HBV expression enhanced FN mRNA and protein levels in the hepatic cell lines Huh7 and HepG2. HBV infection of susceptible HepG2-sodium taurocholate co-transporting polypeptide cells also increased FN expression. We also found that transcriptional factor specificity protein 1 was involved in the induction of FN by HBV. Knockdown of FN expression significantly inhibited HBV DNA replication and protein synthesis through activating endogenous IFN-α production. In addition, FN interacted with the transforming growth factor β-activated protein kinase 1 (TAK1 and TAK1-binding protein complex and attenuated interferon signaling by inhibiting TAK1 phosphorylation. Furthermore, the nuclear translocation of NF-κB/p65 was found to be inhibited by FN. We also observed that FN promoted HBV enhancers to support HBV expression. These results suggest novel functions of endogenous FN involved in immune evasion and maintenance of HBV replication.

  4. Antiviral Effect of Pterocarpus indicus Willd Leaves Extract Against Replication of Dengue Virus (DENV In Vitro

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    Beti Ernawati Dewi

    2018-01-01

    Full Text Available Dengue hemorrhagic fever (DHF is major public health problem in tropical and subtropical areas of the world with lack of approved vaccines and effective antiviral therapies. With no current treatment for illness attributed to dengue virus (DENV infection other than supportive care, therapeutic strategies that use natural extract was developed. Indonesia have many plants that potential for antiviral drµgs such as Pterocarpus indicus Willd (P. indicus. The objective of this study was to determine the effect of P. indicus to inhibit DENV replication. We used a well-differentiated hepatocytes-derived cellular carcinoma cell line (Huh-7 it-1 cells to determine and select antiviral activity. The toxicity effects were determined by MTT assay. Then, the suppression of DENV replication was determined by Focus assay. Dengue infected cells with DMSO were used as control. We found that crude extract (Pi, hexane (Pi.1 and ethyl acetate (Pi.2 extract showed strong inhibition with high selectivity index (SI of 1,392; 285.36 and 168.56 respectively.  Sub fraction of Pi.1 and Pi.2 still showed strong inhibition with high SI.  Further sub-sub fraction of Pi.2 such as Pi.2.12 and Pi.2.12.1 still showed inhibition of DENV replication but there was reduction of SI value. The mechanism experiment of Pi.2.12, we found that Pi 2.12 more profound to inhibit in the post infection stage that entry or pre-infection. We conclude that the sub-fraction of Pi.2.12 has potential antiviral activity against DV infection in vitro. Further studies are still needed to investigate the pure compound of Pi.2.12 that inhibit and have advantages in the future as alternative for treatment of DENV infection.

  5. Taraxacum officinale and Urtica dioica extracts inhibit dengue virus serotype 2 replication in vitro.

    Science.gov (United States)

    Flores-Ocelotl, María R; Rosas-Murrieta, Nora H; Moreno, Diego A; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Domínguez, Fabiola; Santos-López, Gerardo

    2018-03-16

    Urtica dioica, Taraxacum officinale, Calea integrifolia and Caesalpinia pulcherrima are widely used all over the world for treatment of different illnesses. In Mexico, these plants are traditionally used to alleviate or counteract rheumatism and inflammatory muscle diseases. In the present study we evaluated the activity of aqueous and methanolic extracts of these four plants, on the replication of dengue virus serotype 2 (DENV2). Extraction process was carried out in a Soxtherm® system at 60, 85 and 120 °C; a chemical fractionation in silica gel chromatography was performed and compounds present in the active fractions were identified by HPLC-DAD-ESI/MSn. The cytotoxic concentration and the inhibitory effect of extracts or fractions on the DENV2 replication were analyzed in the BHK-21 cell line (plaque forming assay). The half maximal inhibitory concentration (IC 50 ) and the selectivity index (SI) were calculated for the extracts and fractions. The methanolic extracts at 60 °C of T. officinale and U. dioica showed the higher inhibitory effects on DENV2 replication. After the chemical fractionation, the higher activity fraction was found for U. dioica and T. officinale, presenting IC 50 values of 165.7 ± 3.85 and 126.1 ± 2.80 μg/ml, respectively; SI values were 5.59 and 6.01 for each fraction. The compounds present in T. officinale, were luteolin and caffeoylquinic acids derivatives and quercertin diclycosides. The compounds in the active fraction of U. dioica, were, chlorogenic acid, quercertin derivatives and flavonol glycosides (quercetin and kaempferol). Two fractions from U. dioica and T. officinale methanolic extracts with anti-dengue activity were found. The compounds present in both fractions were identified, several recognized molecules have demonstrated activity against other viral species. Subsequent biological analysis of the molecules, alone or in combination, contained in the extracts will be carried out to develop therapeutics

  6. Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo.

    Science.gov (United States)

    Lei, Yan-Chang; Hao, You-Hua; Zhang, Zheng-Mao; Tian, Yong-Jun; Wang, Bao-Ju; Yang, Yan; Zhao, Xi-Ping; Lu, Meng-Ji; Gong, Fei-Li; Yang, Dong-Liang

    2006-07-28

    To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

  7. The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

    Science.gov (United States)

    Rawat, Siddhartha

    2014-01-01

    ABSTRACT Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an

  8. Seroprevalence of bovine immunodeficiency virus and bovine leukemia virus in draught animals in Cambodia.

    Science.gov (United States)

    Meas, S; Ohashi, K; Tum, S; Chhin, M; Te, K; Miura, K; Sugimoto, C; Onuma, M

    2000-07-01

    Since bovine immunodeficiency virus (BIV), known as bovine lentivirus, has been detected in dairy and beef cattle in various countries around the world, a prevalence study of antibodies to BIV and bovine leukemia virus (BLV) was conducted in draught animals in five provinces in Cambodia, where protozoan parasite infections were suspected in some animals. To clarify the status of draught animals including Haryana, Brahman, mixed-breed, local breed cattle and muscle water buffaloes, a total of 544 cattle and 42 buffaloes were tested, and 26.3 and 16.7%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting. There were 5.3% positive for anti-BLV antibodies detected by immunodiffusion test among the cattle, but no reactors among buffaloes and no dual infection for both BIV and BLV was determined in this study. Peripheral blood mononuclear cells from BIV-seropositive cattle were found to have BIV-provirus DNA, as detected by polymerase chain reaction and subsequent Southern blot hybridization. This is the first evidence for the presence of BIV and BLV infections in draught animals in tropical countries such as Cambodia. This wide distribution of BIV suggests its association with problems in animal health as reported worldwide, and that a primary BIV infection can predispose death of affected animals by other aggressive pathogens or stresses.

  9. Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies.

    Science.gov (United States)

    Peng, Bo; Wang, Liqun Rejean; Gómez-Román, Victor Raúl; Davis-Warren, Alberta; Montefiori, David C; Kalyanaraman, V S; Venzon, David; Zhao, Jun; Kan, Elaine; Rowell, Thomas J; Murthy, Krishna K; Srivastava, Indresh; Barnett, Susan W; Robert-Guroff, Marjorie

    2005-08-01

    A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1(MN)env/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIV(SF162) gp140deltaV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1(MN)env/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1(SF162) gp140deltaV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful.

  10. Simian Immunodeficiency Virus (SIV-Specific Chimeric Antigen Receptor-T Cells Engineered to Target B Cell Follicles and Suppress SIV Replication

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    Kumudhini Preethi Haran

    2018-03-01

    Full Text Available There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh cells using antiviral chimeric antigen receptor (CAR T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure of HIV and SIV infections.

  11. Inhibition of porcine transmissible gastroenteritis virus (TGEV) replication in mini-pigs by shRNA.

    Science.gov (United States)

    Zhou, Junfang; Huang, Fen; Hua, Xiuguo; Cui, Li; Zhang, Wen; Shen, Yan; Yan, Yijia; Chen, Piren; Ding, Dezhong; Mou, Jing; Chen, Qi; Lan, Daoliang; Yang, Zhibiao

    2010-04-01

    Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis (TGE), characterized by high mortality and severely retarded growth in piglets that dramatically affects the porcine industry. Previously, we have identified two shRNA-expressing plasmids pEGFP-U6/P1 and pEGFP-U6/P2 that target RNA-dependent RNA polymerase (RdRP) gene of TGEV with more than 95% of virus inhibition in vitro. In this study, inhibition of the TGEV replication by pEGFP-U6/P1 and pEGFP-U6/P2 was tested in mini-pigs. SPF mini-pigs at 25 days old were injected with the shRNA-expressing plasmids and then infected with TGEV. The results from the analyses of clinical signs, histopathology, indirect immunofluorescence (IIF) and RT-PCR show that the two shRNA-expressing plasmids could significantly decrease the quantity of TGEV in different organs and protect mini-pigs from TGEV infection. These findings illustrate the prospect for TGEV-specific shRNAs to be new anti-TGEV agents. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  12. Foamy virus vectors expressing anti-HIV transgenes efficiently block HIV-1 replication.

    Science.gov (United States)

    Taylor, Jason A; Vojtech, Lucia; Bahner, Ingrid; Kohn, Donald B; Laer, Dorothee Von; Russell, David W; Richard, Robert E

    2008-01-01

    Gene therapy has the potential to control human immunodeficiency virus (HIV) in patients who do not respond to traditional antiviral therapy. In this study, we tested foamy virus (FV) vectors expressing three anti-HIV transgenes, both individually and in a combination vector. The transgenes tested in this study are RevM10, a dominant negative version of the viral rev protein, Sh1, a short hairpin RNA directed against a conserved overlapping sequence of tat and rev, and membrane-associated C46 (maC46), a membrane-attached peptide that blocks HIV cell entry. FV vectors efficiently transduce hematopoietic stem cells and, unlike lentivirus (LV) vectors, do not share viral proteins with HIV. The titers of the FV vectors described in this study were not affected by anti-HIV transgenes. On a direct comparison of FV vectors expressing the individual transgenes, entry inhibition using the maC46 transgene was found to be the most effective at blocking HIV replication. A clinically relevant FV vector expressing three anti-HIV transgenes effectively blocked HIV infection in primary macrophages derived from transduced, peripheral blood CD34-selected cells and in a cell line used for propagating HIV, CEMx174. These results suggest that there are potential benefits of using FV vectors in HIV gene therapy.

  13. RNAi-based inhibition of porcine reproductive and respiratory syndrome virus replication in transgenic pigs.

    Science.gov (United States)

    Li, Li; Li, Qiuyan; Bao, Yonghua; Li, Jinxiu; Chen, Zhisheng; Yu, Xiuling; Zhao, Yaofeng; Tian, Kegong; Li, Ning

    2014-02-10

    Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease causing heavy losses to the swine industry worldwide. Many studies have shown that transient delivery of small interfering RNA (siRNA) or adenovirus-mediated RNA interfere (RNAi) could potentially inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in vivo and in vitro. Here, we applied RNAi to produce transgenic (TG) pigs that constitutively expressed PRRSV-specific siRNA derived from small hairpin RNA (shRNA). First, we evaluated siRNA expression in the founding and F1 generation pigs and confirmed stable transmission. Then, we detected the expression of IFN-β and protein kinase R (PKR) and found no difference among TG, non-transgenic (NTG), and wild-type pigs. Lastly, the F1 generation pigs, including TG and NTG piglets, were challenged with 3×10⁴·⁵ TCID₅₀ of JXA1, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Our results showed that the in vivo siRNA expression substantially reduced the serum HP-PRRSV titers and increased survival time by 3 days when TG pigs were compared with the NTG controls. These data suggested that RNAi-based genetic modification might be used to breed viral-resistant livestock with stable siRNA expression with no complications of siRNA toxicity. Copyright © 2013. Published by Elsevier B.V.

  14. Swine influenza H1N1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human H1N1 influenza virus.

    Science.gov (United States)

    Khatri, Mahesh; Dwivedi, Varun; Krakowka, Steven; Manickam, Cordelia; Ali, Ahmed; Wang, Leyi; Qin, Zhuoming; Renukaradhya, Gourapura J; Lee, Chang-Won

    2010-11-01

    Pigs are capable of generating reassortant influenza viruses of pandemic potential, as both the avian and mammalian influenza viruses can infect pig epithelial cells in the respiratory tract. The source of the current influenza pandemic is H1N1 influenza A virus, possibly of swine origin. This study was conducted to understand better the pathogenesis of H1N1 influenza virus and associated host mucosal immune responses during acute infection in humans. Therefore, we chose a H1N1 swine influenza virus, Sw/OH/24366/07 (SwIV), which has a history of transmission to humans. Clinically, inoculated pigs had nasal discharge and fever and shed virus through nasal secretions. Like pandemic H1N1, SwIV also replicated extensively in both the upper and lower respiratory tracts, and lung lesions were typical of H1N1 infection. We detected innate, proinflammatory, Th1, Th2, and Th3 cytokines, as well as SwIV-specific IgA antibody in lungs of the virus-inoculated pigs. Production of IFN-γ by lymphocytes of the tracheobronchial lymph nodes was also detected. Higher frequencies of cytotoxic T lymphocytes, γδ T cells, dendritic cells, activated T cells, and CD4+ and CD8+ T cells were detected in SwIV-infected pig lungs. Concomitantly, higher frequencies of the immunosuppressive T regulatory cells were also detected in the virus-infected pig lungs. The findings of this study have relevance to pathogenesis of the pandemic H1N1 influenza virus in humans; thus, pigs may serve as a useful animal model to design and test effective mucosal vaccines and therapeutics against influenza virus.

  15. Newcastle disease virus induces stable formation ofbona fidestress granules to facilitate viral replication through manipulating host protein translation.

    Science.gov (United States)

    Sun, Yingjie; Dong, Luna; Yu, Shengqing; Wang, Xiaoxu; Zheng, Hang; Zhang, Pin; Meng, Chunchun; Zhan, Yuan; Tan, Lei; Song, Cuiping; Qiu, Xusheng; Wang, Guijun; Liao, Ying; Ding, Chan

    2017-04-01

    Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide. Treatment with nocodazole, a microtubule disruption drug, led to the formation of relatively small and circular granules, indicating that NDV infection induces canonical SGs. Furthermore, the role of SGs on NDV replication was investigated by knockdown of TIA-1 and TIA-1-related (TIAR) protein, the 2 critical components involved in SG formation from the HeLa cells, followed by NDV infection. Results showed that depletion of TIA-1 or TIAR inhibited viral protein synthesis, reduced extracellular virus yields, but increased global protein translation. FISH revealed that NDV-induced SGs contained predominantly cellular mRNA rather than viral mRNA. Deletion of TIA-1 or TIAR reduced NP mRNA levels in polysomes. These results demonstrate that NDV triggers stable formation of bona fide SGs, which benefit viral protein translation and virus replication by arresting cellular mRNA.-Sun, Y., Dong, L., Yu, S., Wang, X., Zheng, H., Zhang, P., Meng, C., Zhan, Y., Tan, L., Song, C., Qiu, X., Wang, G., Liao, Y., Ding, C. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation. © FASEB.

  16. GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.

    Science.gov (United States)

    Nain, Minu; Mukherjee, Sriparna; Karmakar, Sonali Porey; Paton, Adrienne W; Paton, James C; Abdin, M Z; Basu, Anirban; Kalia, Manjula; Vrati, Sudhanshu

    2017-03-15

    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target. IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric

  17. Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus.

    Science.gov (United States)

    Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

    2014-01-01

    Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a plant RNA virus, the Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on plant-virus interaction are discussed.

  18. Deletion analysis of cis- and trans-acting elements involved in replication of alfalfa mosaic virus RNA 3 in vivo

    NARCIS (Netherlands)

    van der Kuyl, A. C.; Neeleman, L.; Bol, J. F.

    1991-01-01

    DNA copies of alfalfa mosaic virus (AIMV) RNA 3 were transcribed in vitro into RNA molecules with deletions in coding and noncoding sequences. The replication of these transcripts was studied in protoplasts from transgenic tobacco plants expressing DNA copies of AIMV RNAs 1 and 2. Deletions in the

  19. Modifications of the 3 '-UTR stem-loop of infectious bursal disease virus are allowed without influencing replication or virulence

    NARCIS (Netherlands)

    Boot, H.J.; Pritz-Verschuren, S.B.E.

    2004-01-01

    Many questions regarding the initiation of replication and translation of the segmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) remain to be solved. Computer analysis shows that the non-polyadenylated extreme 3'-untranslated regions (UTRs) of the coding strand of both

  20. Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution

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    Nauwynck Hans J

    2010-02-01

    Full Text Available Abstract Background Throughout the history of human influenza pandemics, pigs have been considered the most likely "mixing vessel" for reassortment between human and avian influenza viruses (AIVs. However, the replication efficiencies of influenza viruses from various hosts, as well as the expression of sialic acid (Sia receptor variants in the entire porcine respiratory tract have never been studied in detail. Therefore, we established porcine nasal, tracheal, bronchial and lung explants, which cover the entire porcine respiratory tract with maximal similarity to the in vivo situation. Subsequently, we assessed virus yields of three porcine, two human and six AIVs in these explants. Since our results on virus replication were in disagreement with the previously reported presence of putative avian virus receptors in the trachea, we additionally studied the distribution of sialic acid receptors by means of lectin histochemistry. Human (Siaα2-6Gal and avian virus receptors (Siaα2-3Gal were identified with Sambucus Nigra and Maackia amurensis lectins respectively. Results Compared to swine and human influenza viruses, replication of the AIVs was limited in all cultures but most strikingly in nasal and tracheal explants. Results of virus titrations were confirmed by quantification of infected cells using immunohistochemistry. By lectin histochemistry we found moderate to abundant expression of the human-like virus receptors in all explant systems but minimal binding of the lectins that identify avian-like receptors, especially in the nasal, tracheal and bronchial epithelium. Conclusions The species barrier that restricts the transmission of influenza viruses from one host to another remains preserved in our porcine respiratory explants. Therefore this system offers a valuable alternative to study virus and/or host properties required for adaptation or reassortment of influenza viruses. Our results indicate that, based on the expression of Sia

  1. Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication

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    Reh Juliane

    2011-08-01

    Full Text Available Abstract Background Foamy viruses (FVs unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol protein interactions, ensures Pol particle encapsidation. Results Several Prototype FV (PFV Gag-Pol fusion protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85PR-RT and p40IN Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71Gag

  2. Antiviral Activity of Resveratrol against Human and Animal Viruses

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    Yusuf Abba

    2015-01-01

    Full Text Available Resveratrol is a potent polyphenolic compound that is being extensively studied in the amelioration of viral infections both in vitro and in vivo. Its antioxidant effect is mainly elicited through inhibition of important gene pathways like the NF-κβ pathway, while its antiviral effects are associated with inhibitions of viral replication, protein synthesis, gene expression, and nucleic acid synthesis. Although the beneficial roles of resveratrol in several viral diseases have been well documented, a few adverse effects have been reported as well. This review highlights the antiviral mechanisms of resveratrol in human and animal viral infections and how some of these effects are associated with the antioxidant properties of the compound.

  3. Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication.

    Science.gov (United States)

    Pudupakam, R S; Kenney, Scott P; Córdoba, Laura; Huang, Yao-Wei; Dryman, Barbara A; Leroith, Tanya; Pierson, F William; Meng, Xiang-Jin

    2011-10-01

    The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.

  4. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  5. Differential replication of avian influenza H9N2 viruses in human alveolar epithelial A549 cells

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    Peiris Malik

    2010-03-01

    Full Text Available Abstract Avian influenza virus H9N2 isolates cause a mild influenza-like illness in humans. However, the pathogenesis of the H9N2 subtypes in human remains to be investigated. Using a human alveolar epithelial cell line A549 as host, we found that A/Quail/Hong Kong/G1/97 (H9N2/G1, which shares 6 viral "internal genes" with the lethal A/Hong Kong/156/97 (H5N1/97 virus, replicates efficiently whereas other H9N2 viruses, A/Duck/Hong Kong/Y280/97 (H9N2/Y280 and A/Chicken/Hong Kong/G9/97 (H9N2/G9, replicate poorly. Interestingly, we found that there is a difference in the translation of viral protein but not in the infectivity or transcription of viral genes of these H9N2 viruses in the infected cells. This difference may possibly be explained by H9N2/G1 being more efficient on viral protein production in specific cell types. These findings suggest that the H9N2/G1 virus like its counterpart H5N1/97 may be better adapted to the human host and replicates efficiently in human alveolar epithelial cells.

  6. Establishment of a mouse melanoma model system for the efficient infection and replication of human adenovirus type 5-based oncolytic virus.

    Science.gov (United States)

    Kang, Sujin; Kim, Joo-Hang; Kim, So Young; Kang, Dongxu; Je, Suyeon; Song, Jae J

    2014-10-24

    Due to poor adenoviral infectivity and replication in mouse tumor cell types compared with human tumor cell types, use of human-type adenoviral vectors in mouse animal model systems was limited. Here, we demonstrate enhanced infectivity and productive replication of adenovirus in mouse melanoma cells following introduction of both the Coxsackievirus and adenovirus receptor (CAR) and E1B-55K genes. Introduction of CAR into B16BL6 or B16F10 cells increased the infectivity of GFP-expressing adenovirus; however, viral replication was unaffected. We demonstrated a dramatic increase of adenoviral replication (up to 100-fold) in mouse cells via E1B-55K expression and subsequent viral spreading in mouse tissue. These results reveal for the first time that human adenovirus type 5 (Ad5)-based oncolytic virus can be applied to immunocompetent mouse with the introduction of CAR and E1B-55K to syngenic mouse cell line. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.

    Science.gov (United States)

    Wang, M; Park, J S; Ishiai, M; Hurwitz, J; Lee, S H

    2000-12-01

    Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) alpha and delta. A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alpha-primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.

  8. Replication-Competent Recombinant Porcine Reproductive and Respiratory Syndrome (PRRS Viruses Expressing Indicator Proteins and Antiviral Cytokines

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    Frank Blecha

    2012-01-01

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129. Coding regions of exogenous genes, which included Renilla luciferase (Rluc, green and red fluorescent proteins (GFP and DsRed, respectively and several interferons (IFNs, were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 » IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101 in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV vaccines, which are capable of reversing subverted innate immune responses and

  9. Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination.

    Science.gov (United States)

    Herate, Cecile; Vigne, Clarisse; Guenzel, Carolin A; Lambele, Marie; Rouyez, Marie-Christine; Benichou, Serge

    2016-04-12

    Through incorporation into virus particles, the HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhancement of virus infectivity in established CD4-positive cell lines via a nonenzymatic mechanism. We report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV-1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte-derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2- and RPA32-depleted T lymphocytes. Reciprocally, viruses produced in UNG2-depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination. Our data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV-1.

  10. Large Animal Models for Foamy Virus Vector Gene Therapy

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    Peter A. Horn

    2012-12-01

    Full Text Available Foamy virus (FV vectors have shown great promise for hematopoietic stem cell (HSC gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit.

  11. The kinase inhibitor SFV785 dislocates dengue virus envelope protein from the replication complex and blocks virus assembly.

    Directory of Open Access Journals (Sweden)

    Azlinda Anwar

    Full Text Available Dengue virus (DENV is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785, which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-07-15

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  14. Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges

    Science.gov (United States)

    De Regge, Nick; Madder, Maxime; Deblauwe, Isra; Losson, Bertrand; Fassotte, Christiane; Demeulemeester, Julie; Smeets, François; Tomme, Marie; Cay, Ann Brigitte

    2014-01-01

    Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21–24 and 33–36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV. PMID:24466312

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

    Science.gov (United States)

    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. © The Author 2016. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

    Science.gov (United States)

    Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

    2017-12-13

    Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that

  17. Hepatitis B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal, Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis▿ †

    OpenAIRE

    Lamontagne, Jason; Pinkerton, Mark; Block, Timothy M.; Lu, Xuanyong

    2009-01-01

    Hepatitis B and C viruses (HBV and HCV, respectively) are different and distinct viruses, but there are striking similarities in their disease potential. Infection by either virus can cause chronic hepatitis, liver cirrhosis, and ultimately, liver cancer, despite the fact that no pathogenetic mechanisms are known which are shared by the two viruses. Our recent studies have suggested that replication of either of these viruses upregulates a cellular protein called serine protease inhibitor Kaz...

  18. Antiviral Activity of Hatay Propolis Against Replication of Herpes Simplex Virus Type 1 and Type 2

    Science.gov (United States)

    Yildirim, Ayse; Duran, Gulay Gulbol; Duran, Nizami; Jenedi, Kemal; Bolgul, Behiye Sezgin; Miraloglu, Meral; Muz, Mustafa

    2016-01-01

    Background Propolis is a bee product widely used in folk medicine and possessing many pharmacological properties. In this study we aimed to investigate: i) the antiviral activities of Hatay propolis samples against HSV-1 and HSV-2 in HEp-2 cell line, and ii) the presence of the synergistic effects of propolis with acyclovir against these viruses. Material/Methods All experiments were carried out in HEp-2 cell cultures. Proliferation assays were performed in 24-well flat bottom microplates. We inoculated 1×105 cells per ml and RPMI 1640 medium with 10% fetal calf serum into each well. Studies to determine cytotoxic effect were performed. To investigate the presence of antiviral activity of propolis samples, different concentrations of propolis (3200, 1600, 800, 400, 200, 100, 75, 50, and 25 μg/mL) were added into the culture medium. The amplifications of HSV-1 and HSV-2 DNA were performed by real-time PCR method. Acyclovir (Sigma, USA) was chosen as a positive control. Cell morphology was evaluated by scanning electron microscopy (SEM). Results The replication of HSV-1 and HSV-2 was significantly suppressed in the presence of 25, 50, and 100 μg/mL of Hatay propolis. We found that propolis began to inhibit HSV-1 replication after 24 h of incubation and propolis activity against HSV-2 was found to start at 48 h following incubation. The activity of propolis against both HSV-1 and HSV-2 was confirmed by a significant decrease in the number of viral copies. Conclusions We determined that Hatay propolis samples have important antiviral effects compared with acyclovir. In particular, the synergy produced by antiviral activity of propolis and acyclovir combined had a stronger effect against HSV-1 and HSV-2 than acyclovir alone. PMID:26856414

  19. Characterization of the adenoassociated virus Rep protein complex formed on the viral origin of DNA replication

    International Nuclear Information System (INIS)

    Li Zengi; Brister, J. Rodney; Im, Dong-Soo; Muzyczka, Nicholas

    2003-01-01

    Interaction between the adenoassociated virus (AAV) replication proteins, Rep68 and 78, and the viral terminal repeats (TRs) is mediated by a DNA sequence termed the Rep-binding element (RBE). This element is necessary for Rep-mediated unwinding of duplex DNA substrates, directs Rep catalyzed cleavage of the AAV origin of DNA replication, and is required for viral transcription and proviral integration. Six discrete Rep complexes with the AAV TR substrates have been observed in vitro, and cross-linking studies suggest these complexes contain one to six molecules of Rep. However, the functional relationship between Rep oligomerization and biochemical activity is unclear. Here we have characterized Rep complexes that form on the AAV TR. Both Rep68 and Rep78 appear to form the same six complexes with the AAV TR, and ATP seems to stimulate formation of specific, higher order complexes. When the sizes of these Rep complexes were estimated on native polyacrylamide gels, the four slower migrating complexes were larger than predicted by an amount equivalent to one or two TRs. To resolve this discrepancy, the molar ratio of protein and DNA was calculated for the three largest complexes. Data from these experiments indicated that the larger complexes included multiple TRs in addition to multiple Rep molecules and that the Rep-to-TR ratio was approximately 2. The two largest complexes were also associated with increased Rep-mediated, origin cleavage activity. Finally, we characterized a second, Rep-mediated cleavage event that occurs adjacent to the normal nicking site, but on the opposite strand. This second site nicking event effectively results in double-stranded DNA cleavage at the normal nicking site

  20. Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

    Science.gov (United States)

    Liu, Yu; Zhao, Miaoxian; Gong, Mingxing; Xu, Ying; Xie, Cantao; Deng, Haohui; Li, Xueying; Wu, Hongkai; Wang, Zhanhui

    2018-04-01

    Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Determination of whether tomato spotted wilt virus replicates in Toxorhynchites amboinensis mosquitoes and the relatedness of this virus to phleboviruses (family Bunyaviridae).

    Science.gov (United States)

    Wang, M; Mitchell, C J; Hu, J S; Gonsalves, D; Calisher, C H

    1992-01-01

    Tomato spotted wilt virus (TSWV) has been reported to be morphologically, molecularly and structurally similar to viruses in the family Bunyaviridae. By various types of enzyme-linked immunosorbent assays (ELISA) and Western blot hybridizations, we tested TSWV with antibodies to 12 viruses in the Phlebovirus genus of this family. Serological relatedness was not found between TSWV and phleboviruses. However, one preparation of antibody to Arumowot virus reacted with a 53-kD protein from healthy plant extracts. Six-day-old adult Toxorhynchites amboinensis mosquitoes were inoculated with purified TSWV. Infectious virus was not detected in any of the injected insects during the 5-week test period. However, TSWV antigens were detected in these mosquitoes by ELISA at the original injected level for at least a week after injection. TSWV antigen concentration began to decrease thereafter, but remained at detectable levels for as long as 5 weeks after injection. However, there was no evidence that TSWV replicated in mosquitoes.

  2. Avian influenza viruses that cause highly virulent infections in humans exhibit distinct replicative properties in contrast to human H1N1 viruses

    Science.gov (United States)

    Simon, Philippe F.; de La Vega, Marc-Antoine; Paradis, Éric; Mendoza, Emelissa; Coombs, Kevin M.; Kobasa, Darwyn; Beauchemin, Catherine A. A.

    2016-04-01

    Avian influenza viruses present an emerging epidemiological concern as some strains of H5N1 avian influenza can cause severe infections in humans with lethality rates of up to 60%. These have been in circulation since 1997 and recently a novel H7N9-subtyped virus has been causing epizootics in China with lethality rates around 20%. To better understand the replication kinetics of these viruses, we combined several extensive viral kinetics experiments with mathematical modelling of in vitro infections in human A549 cells. We extracted fundamental replication parameters revealing that, while both the H5N1 and H7N9 viruses replicate faster and to higher titers than two low-pathogenicity H1N1 strains, they accomplish this via different mechanisms. While the H7N9 virions exhibit a faster rate of infection, the H5N1 virions are produced at a higher rate. Of the two H1N1 strains studied, the 2009 pandemic H1N1 strain exhibits the longest eclipse phase, possibly indicative of a less effective neuraminidase activity, but causes infection more rapidly than the seasonal strain. This explains, in part, the pandemic strain’s generally slower growth kinetics and permissiveness to accept mutations causing neuraminidase inhibitor resistance without significant loss in fitness. Our results highlight differential growth properties of H1N1, H5N1 and H7N9 influenza viruses.

  3. Replication and adaptive mutations of low pathogenic avian influenza viruses in tracheal organ cultures of different avian species.

    Directory of Open Access Journals (Sweden)

    Henning Petersen

    Full Text Available Transmission of avian influenza viruses (AIV between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants.

  4. Replication of human syncytium-forming virus in human cells: effect of certain biological factors and selective chemicals.

    Science.gov (United States)

    Loh, P C; Ang, K S

    1981-01-01

    The growth characteristics of the human syncytium-forming virus (HSFV) were examined in several human cell lines of normal and malignant origins and composing of either fibroblastic or epithelial-like cells. Virus production occurred only in the fibroblastic diploid cell lines: HEF (human embryonic cells, Flow #5,000) and HFDL #645 (human fetal diploid lung), but not in the epithelial-like heteroploid cell lines: RA (a continuous line of human amnion), #999 (human bone marrow), and KB (carcinoma of the nasopharynx). While the single-cycle growth pattern of the virus in HEF and HFDL #645 cell lines were essentially similar, the virus yield per cell was greater in the HFDL #645 cells. Furthermore, the physiological state of the cell had a marked effect on virus production. Subconfluent actively growing HFDL #645 cells produced higher yields of virus than density-inhibited confluent HFDL #645 cell cultures. The replication of HSFV was inhibited by actinomycin D at concentrations that did not interfere with poliovirus replication (0.001 to 0.01 microgram/ml). Pretreatment and posttreatments of infected cell cultures with either the polycation polybrene (hexadimethrine bromide) or the synthetic glucocorticosteroid dexamethasone did not enhance HSFV production.

  5. HERP Binds TBK1 To Activate Innate Immunity and Repress Virus Replication in Response to Endoplasmic Reticulum Stress.

    Science.gov (United States)

    Ge, Maolin; Luo, Zhen; Qiao, Zhi; Zhou, Yao; Cheng, Xin; Geng, Qibin; Cai, Yanyan; Wan, Pin; Xiong, Ying; Liu, Fang; Wu, Kailang; Liu, Yingle; Wu, Jianguo

    2017-11-01

    Host innate immunity is crucial for cellular responses against viral infection sensed by distinct pattern recognition receptors and endoplasmic reticulum (ER) stress. Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and neurological diseases. However, the exact mechanism underlying the link between ER stress induced by EV71 infection and host innate immunity is largely unknown. In this study, we demonstrated that EV71 infection induces the homocysteine-induced ER protein (HERP), a modulator of the ER stress response which is dependent on the participation of MAVS. Virus-induced HERP subsequently stimulates host innate immunity to repress viral replication by promoting type-I IFNs (IFN-α and IFN-β) and type-III IFN (IFN-λ1) expression. Through interacting with TANK-binding kinase 1, HERP amplifies the MAVS signaling and facilitates the phosphorylation and nuclear translocation of IFN regulatory factor 3 and NF-κB to enhance the expression of IFNs, which leads to a broad inhibition of the replication of RNA viruses, including EV71, Sendai virus, influenza A virus, and vesicular stomatitis virus. Therefore, we demonstrated that HERP plays an important role in the regulation of host innate immunity in response to ER stress during the infection of RNA viruses. These findings provide new insights into the mechanism underlying the replication of RNA viruses and the production of IFNs, and also demonstrate a new role of HERP in the regulation of host innate immunity in response to viral infection. Copyright © 2017 by The American Association of Immunologists, Inc.

  6. The cis-acting replication signal at the 3' end of Flock House virus RNA2 is RNA3-dependent

    International Nuclear Information System (INIS)

    Albarino, Cesar G.; Eckerle, Lance D.; Ball, L. Andrew

    2003-01-01

    The nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3' 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3' termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3

  7. Hepatitis C virus replicative double-stranded RNA is a potent interferon inducer that triggers interferon production through MDA5.

    Science.gov (United States)

    Du, Xiaoting; Pan, Tingting; Xu, Jun; Zhang, Yang; Song, Wuhui; Yi, Zhigang; Yuan, Zhenghong

    2016-11-01

    The cytoplasmic RNA sensors, retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, play crucial roles in innate sensing of hepatitis C virus (HCV). However, the exact identity of the IFN inducer generated during HCV infection is poorly understood. To identify the IFN inducer, we extracted the RNAs from HCV-replicating cells and introduced these into IFN signalling-competent cells to examine IFN production. RNAs isolated from HCV-replicating cells triggered robust IFN-β and IFN-λ production in Huh7 cells in a viral replication-dependent manner, preferentially through the melanoma differentiation-associated gene 5 but not through the retinoic acid-inducible gene I-mediated pathway. The IFN-inducing capacity of HCV RNA survived after calf intestinal alkaline phosphatase and ssRNA-specific S1 nuclease treatment, but was completely eliminated by dsRNA-specific RNase III digestion, suggesting that viral replicative dsRNA is an IFN inducer. Furthermore, HCV viral RNA extracted from replicating cells was sensitive to 5'-monophosphate-dependent 5'→3' exonuclease (TER) digestion, suggesting that the HCV genome lacks a 5'-triphosphate or -diphosphate. In semi-permeabilized cells, the HCV IFN inducer primarily resided in an enclosed membranous structure that protects the IFN inducer from RNase digestion. Taken together, we identified HCV replicative dsRNA as a viral IFN inducer enclosed within the viral replication factory.

  8. Cellular Cholesterol Facilitates the Postentry Replication Cycle of Herpes Simplex Virus 1.

    Science.gov (United States)

    Wudiri, George A; Nicola, Anthony V

    2017-07-15

    Cholesterol is an essential component of cell membranes and is required for herpes simplex virus 1 (HSV-1) entry (1-3). Treatment of HSV-1-infected Vero cells with methyl beta-cyclodextrin from 2 to 9 h postentry reduced plaque numbers. Transport of incoming viral capsids to the nuclear periphery was unaffected by the cholesterol reduction, suggesting that cell cholesterol is important for the HSV-1 replicative cycle at a stage(s) beyond entry, after the arrival of capsids at the nucleus. The synthesis and release of infectious HSV-1 and cell-to-cell spread of infection were all impaired in cholesterol-reduced cells. Propagation of HSV-1 on DHCR24 -/- fibroblasts, which lack the desmosterol-to-cholesterol conversion enzyme, resulted in the generation of infectious extracellular virions (HSV des ) that lack cholesterol and likely contain desmosterol. The specific infectivities (PFU per viral genome) of HSV chol and HSV des were similar, suggesting cholesterol and desmosterol in the HSV envelope support similar levels of infectivity. However, infected DHCR24 -/- fibroblasts released ∼1 log less infectious HSV des and ∼1.5 log fewer particles than release of cholesterol-containing particles (HSV chol ) from parental fibroblasts, suggesting that the hydrocarbon tail of cholesterol facilitates viral synthesis. Together, the results suggest multiple roles for cholesterol in the HSV-1 replicative cycle. IMPORTANCE HSV-1 infections are associated with a wide range of clinical manifestations that are of public health importance. Cholesterol is a key player in the complex interaction between viral and cellular factors that allows HSV-1 to enter host cells and establish infection. Previous reports have demonstrated a role for cellular cholesterol in the entry of HSV-1 into target cells. Here, we employed both chemical treatment and cells that were genetically defined to synthesize only desmosterol to demonstrate that cholesterol is important at stages following the

  9. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    Science.gov (United States)

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  10. Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1.

    Science.gov (United States)

    Franzoso, Francesca D; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F; Sutter, Sereina O; Tobler, Kurt; Vogt, Bernd; Greber, Urs F; Büning, Hildegard; Ackermann, Mathias; Fraefel, Cornel

    2017-08-01

    Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G 2 -phase cells, while HSV-1 DNA replication is restricted to G 1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G 2 -phase cells, suggesting that the preference for S/G 2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G 2 -phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time

  11. A Scorpion Defensin BmKDfsin4 Inhibits Hepatitis B Virus Replication in Vitro

    Directory of Open Access Journals (Sweden)

    Zhengyang Zeng

    2016-04-01

    Full Text Available Hepatitis B virus (HBV infection is a major worldwide health problem which can cause acute and chronic hepatitis and can significantly increase the risk of liver cirrhosis and primary hepatocellular carcinoma (HCC. Nowadays, clinical therapies of HBV infection still mainly rely on nucleotide analogs and interferons, the usage of which is limited by drug-resistant mutation or side effects. Defensins had been reported to effectively inhibit the proliferation of bacteria, fungi, parasites and viruses. Here, we screened the anti-HBV activity of 25 scorpion-derived peptides most recently characterized by our group. Through evaluating anti-HBV activity and cytotoxicity, we found that BmKDfsin4, a scorpion defensin with antibacterial and Kv1.3-blocking activities, has a comparable high inhibitory rate of both HBeAg and HBsAg in HepG2.2.15 culture medium and low cytotoxicity to HepG2.2.15. Then, our experimental results further showed that BmKDfsin4 can dose-dependently decrease the production of HBV DNA and HBV viral proteins in both culture medium and cell lysate. Interestingly, BmKDfsin4 exerted high serum stability. Together, this study indicates that the scorpion defensin BmKDfsin4 also has inhibitory activity against HBV replication along with its antibacterial and potassium ion channel Kv1.3-blocking activities, which shows that BmKDfsin4 is a uniquely multifunctional defensin molecule. Our work also provides a good molecule material which will be used to investigate the link or relationship of its antiviral, antibacterial and ion channel–modulating activities in the future.

  12. Potential for Low-Pathogenic Avian H7 Influenza A Viruses To Replicate and Cause Disease in a Mammalian Model.

    Science.gov (United States)

    Zanin, Mark; Koçer, Zeynep A; Poulson, Rebecca L; Gabbard, Jon D; Howerth, Elizabeth W; Jones, Cheryl A; Friedman, Kimberly; Seiler, Jon; Danner, Angela; Kercher, Lisa; McBride, Ryan; Paulson, James C; Wentworth, David E; Krauss, Scott; Tompkins, Stephen M; Stallknecht, David E; Webster, Robert G

    2017-02-01

    H7 subtype influenza A viruses are widely distributed and have been responsible for human infections and numerous outbreaks in poultry with significant impact. Despite this, the disease-causing potential of the precursor low-pathogenic (LP) H7 viruses from the wild bird reservoir has not been investigated. Our objective was to assess the disease-causing potential of 30 LP H7 viruses isolated from wild avian species in the United States and Canada using the DBA/2J mouse model. Without prior mammalian adaptation, the majority of viruses, 27 (90%), caused mortality in mice. Of these, 17 (56.7%) caused 100% mortality and 24 were of pathogenicity similar to that of A/Anhui/1/2013 (H7N9), which is highly pathogenic in mice. Viruses of duck origin were more pathogenic than those of shorebird origin, as 13 of 18 (72.2%) duck origin viruses caused 100% mortality while 4 of 12 (33.3%) shorebird origin viruses caused 100% mortality, despite there being no difference in mean lung viral titers between the groups. Replication beyond the respiratory tract was also evident, particularly in the heart and brain. Of the 16 viruses studied for fecal shedding, 11 were detected in fecal samples. These viruses exhibited a strong preference for avian-type α2,3-linked sialic acids; however, binding to mammalian-type α2,6-linked sialic acids was also detected. These findings indicate that LP avian H7 influenza A viruses are able to infect and cause disease in mammals without prior adaptation and therefore pose a potential public health risk. Low-pathogenic (LP) avian H7 influenza A viruses are widely distributed in the avian reservoir and are the precursors of numerous outbreaks of highly pathogenic avian influenza viruses in commercial poultry farms. However, unlike highly pathogenic H7 viruses, the disease-causing potential of LP H7 viruses from the wild bird reservoir has not been investigated. To address this, we studied 30 LP avian H7 viruses isolated from wild avian species in the

  13. Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

    Directory of Open Access Journals (Sweden)

    Anne Frentzen

    2011-04-01

    Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

  14. A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus.

    Directory of Open Access Journals (Sweden)

    Yoshimi Tsuda

    2011-08-01

    Full Text Available Human outbreaks of Ebola virus (EBOV are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV vector expressing a CD8+ T cell epitope from the nucleoprotein (NP of Zaire ebolavirus (ZEBOV (MCMV/ZEBOV-NP(CTL. MCMV/ZEBOV-NP(CTL induced high levels of long-lasting (>8 months CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.

  15. A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus.

    Science.gov (United States)

    Tsuda, Yoshimi; Caposio, Patrizia; Parkins, Christopher J; Botto, Sara; Messaoudi, Ilhem; Cicin-Sain, Luka; Feldmann, Heinz; Jarvis, Michael A

    2011-08-01

    Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes. We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)). MCMV/ZEBOV-NP(CTL) induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection. This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.

  16. Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

    Science.gov (United States)

    Savarino, Andrea; Pistello, Mauro; D'Ostilio, Daniela; Zabogli, Elisa; Taglia, Fabiana; Mancini, Fabiola; Ferro, Stefania; Matteucci, Donatella; De Luca, Laura; Barreca, Maria Letizia; Ciervo, Alessandra; Chimirri, Alba; Ciccozzi, Massimo; Bendinelli, Mauro

    2007-01-01

    Background Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in

  17. Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2007-10-01

    Full Text Available Abstract Background Treatment of feline immunodeficiency virus (FIV infection has been hampered by the absence of a specific combination antiretroviral treatment (ART. Integrase strand transfer inhibitors (INSTIs are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD. Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound

  18. The rolling-circle melting-pot model for porcine circovirus DNA replication

    Science.gov (United States)

    A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

  19. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.

    Directory of Open Access Journals (Sweden)

    Thomas Pietschmann

    2009-06-01

    Full Text Available With the advent of subgenomic hepatitis C virus (HCV replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs, previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A, but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I as well as NS5A (S2204R, whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

  20. AR-12 suppresses dengue virus replication by down-regulation of PI3K/AKT and GRP78.

    Science.gov (United States)

    Chen, Hsin-Hsin; Chen, Chien-Chin; Lin, Yee-Shin; Chang, Po-Chun; Lu, Zi-Yi; Lin, Chiou-Feng; Chen, Chia-Ling; Chang, Chih-Peng

    2017-06-01

    Dengue virus (DENV) infection has become a public health issue of worldwide concern and is a serious health problem in Taiwan, yet there are no approved effective antiviral drugs to treat DENV. The replication of DENV requires both viral and cellular factors. Targeting host factors may provide a potential antiviral strategy. It has been known that up-regulation of PI3K/AKT signaling and GRP78 by DENV infection supports its replication. AR-12, a celecoxib derivative with no inhibiting activity on cyclooxygenase, shows potent inhibitory activities on both PI3K/AKT signaling and GRP78 expression levels, and recently has been found to block the replication of several hemorrhagic fever viruses. However the efficacy of AR-12 in treating DENV infection is still unclear. Here, we provide evidence to show that AR-12 is able to suppress DENV replication before or after virus infection in cell culture and mice. The antiviral activities of AR-12 are positive against infection of the four different DENV serotypes. AR-12 significantly down-regulates the PI3K/AKT activity and GRP78 expression in DENV infected cells whereas AKT and GRP78 rescue are able to attenuate anti-DENV effect of AR-12. Using a DENV-infected suckling mice model, we further demonstrate that treatment of AR-12 before or after DENV infection reduces virus replication and mice mortality. In conclusion, we uncover the potential efficacy of AR-12 as a novel drug for treating dengue. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. A replication analysis of foot-and-mouth disease virus in swine lymphoid tissue might indicate a putative carrier stage in pigs

    Directory of Open Access Journals (Sweden)

    Rodríguez-Calvo Teresa

    2011-02-01

    Full Text Available Abstract Foot-and-mouth disease virus (FMVD, one of the most contagious viruses of cloven-hoofed animals, may cause a prolonged, asymptomatic but persistent infection in ruminants, named the "carrier state". However, it remains an open question whether this carrier state occurs in pigs. Here we present quantitative analyses of the duration of FMDV RNA and infectivity in lymphoid and epithelial tissues in experimentally infected pigs with FMDV C-S8c1. The data indicated that although FMDV RNA remained in blood until day 14 post-infection (pi, viremia was cleared by day 7 pi. However, all tissues tested were positive for FMDV until day 14-17 pi. Interestingly, the specific infectivity of FMDV in these tissues was in some cases even higher than the FMDV C-S8c1. We therefore propose that a "pseudopersistent state" may occur in pigs in which virus replicates in lymphoid tissues for a prolonged period of time, thereby representing a potential source of virus.

  2. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    International Nuclear Information System (INIS)

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S.; Arbuthnot, Patrick

    2009-01-01

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  3. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

    2009-11-20

    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  4. Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Ciomperlik, Jessica J. [Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 (United States); Basta, Holly A. [Department of Biology, Rocky Mountain College, Billings, MT (United States); Palmenberg, Ann C., E-mail: acpalmen@wisc.edu [Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 (United States)

    2016-01-15

    Cardiovirus Leader proteins (L{sub X}) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus L{sub M} structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus L{sub E}, and also larger complexes with L{sub E}:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant L{sub S} and L{sub T} from Theiloviruses. When mutations were introduced to alter the L{sub E} zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on L{sub E} must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by L{sub E}.

  5. Intrinsic Stability of Episomal Circles Formed during Human Immunodeficiency Virus Type 1 Replication

    Science.gov (United States)

    Pierson, TheodoreC.; Kieffer, Tara L.; Ruff, Christian T.; Buck, Christopher; Gange, Stephen J.; Siliciano, Robert F.

    2002-01-01

    The development of surrogate markers capable of detecting residual ongoing human immunodeficiency virus type 1 (HIV-1) replication in patients receiving highly active antiretroviral therapy is an important step in understanding viral dynamics and in developing new treatment strategies. In this study, we evaluated the utility of circular forms of the viral genome for the detection of recent infection of cells by HIV-1. We measured the fate of both one-long terminal repeat (1-LTR) and 2-LTR circles following in vitro infection of logarithmically growing CD4+ T cells under conditions in which cell death was not a significant contributing factor. Circular forms of the viral genome were found to be highly stable and to decrease in concentration only as a function of dilution resulting from cell division. We conclude that these DNA circles are not intrinsically unstable in all cell types and suggest that the utility of 2-LTR circle assays in measuring recent HIV-1 infection of susceptible cells in vivo needs to be reevaluated. PMID:11907256

  6. A novel inhibitor of dengue virus replication that targets the capsid protein.

    Science.gov (United States)

    Byrd, Chelsea M; Dai, Dongcheng; Grosenbach, Douglas W; Berhanu, Aklile; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Wineinger, Kristin A; Page, Jessica M; Harver, Chris; Stavale, Eric; Tyavanagimatt, Shanthakumar; Stone, Melialani A; Bartenschlager, Ralf; Scaturro, Pietro; Hruby, Dennis E; Jordan, Robert

    2013-01-01

    Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.

  7. Role of untranslated regions in regulation of gene expression, replication, and pathogenicity of Newcastle disease virus expressing green fluorescent protein.

    Science.gov (United States)

    Kim, Shin-Hee; Samal, Siba K

    2010-03-01

    To gain insight into the role of untranslated regions (UTRs) in regulation of foreign gene expression, replication, and pathogenicity of Newcastle disease virus (NDV), a green fluorescent protein (GFP) gene flanked by 5' and 3' UTRs of each NDV gene was individually expressed by recombinant NDVs. UTRs of each gene modulated GFP expression positively or negatively. In particular, UTRs of the M and F genes enhanced levels of GFP expression at the junction of the P and M genes without altering replication of NDV, suggesting that UTRs could be used for enhanced expression of a foreign gene by NDV.

  8. Efficient replication and expression of murine leukemia virus with major deletions in the enhancer region of U3

    DEFF Research Database (Denmark)

    Pedersen, K.; Lovmand, S.; Bonefeld-Jørgensen, Eva Cecilie

    1992-01-01

    of the viral RNA. Genetic tagging of the retrovirus with lacO facilitated the analysis. Among the individual mutated LTRs an over 100-fold difference in a transient expression assay was previously detected. This difference was not revealed in studies of viral replication in cell culture, where the expression......The effect of deletions within the enhancer region in the U3 part of the LTR derived from the murine retrovirus Akv was studied. The deletions were stably transmitted through normal virus replication as shown by sequence analysis of cloned polymerase chain reaction product of the cDNA copy...

  9. Compartmentalization of SIV Replication Within Secondary Lymphoid Tissues of Rhesus Macaques is Linked to Disease Stage and Inversely Related to Localization of Virus-Specific CTL1 2

    Science.gov (United States)

    Connick, Elizabeth; Folkvord, Joy M.; Lind, Katherine T.; Rakasz, Eva G.; Miles, Brodie; Wilson, Nancy A.; Santiago, Mario L.; Schmitt, Kimberly; Stephens, Edward B.; Kim, Hyeon O.; Wagstaff, Reece; Li, Shengbin; Abdelaal, Hadia M.; Kemp, Nathan; Watkins, David I.; MaWhinney, Samantha; Skinner, Pamela J.

    2014-01-01

    We previously demonstrated that HIV replication is concentrated in lymph node B cell follicles during chronic infection and that HIV-specific CTL fail to accumulate in large numbers at those sites. It is unknown whether these observations can be generalized to other secondary lymphoid tissues, or whether virus compartmentalization occurs in the absence of CTL. We evaluated these questions in SIVmac239-infected rhesus macaques by quantifying SIV RNA+ cells and SIV-specific CTL in situ in spleen, lymph nodes and intestinal tissues obtained at several stages of infection. During chronic asymptomatic infection prior to simian AIDS (SAIDS), SIV-producing cells were more concentrated in follicular compared to extrafollicular regions of secondary lymphoid tissues. At day 14 of infection, when CTL have minimal impact on virus replication, there was no compartmentalization of SIV-producing cells. Virus compartmentalization was diminished in animals with SAIDS, which often have low frequency CTL responses. SIV-specific CTL were consistently more concentrated within extrafollicular regions of lymph node and spleen in chronically infected animals regardless of epitope specificity. Frequencies of SIV-specific CTL within follicular and extrafollicular compartments predicted SIV RNA+ cells within these compartments in a mixed model. Few SIV-specific CTL expressed the follicular homing molecule CXCR5 in the absence of the extrafollicular retention molecule CCR7, possibly accounting for the paucity of follicular CTL. These findings bolster the hypothesis that B cell follicles are immune privileged sites and suggest that strategies to augment CTL in B cell follicles could lead to improved viral control and possibly a functional cure for HIV infection. PMID:25362178

  10. Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22.

    Science.gov (United States)

    Cheng, Fan; Ramos da Silva, Suzane; Huang, I-Chueh; Jung, Jae U; Gao, Shou-Jiang

    2018-02-15

    The recent outbreak of Zika virus (ZIKV), a reemerging flavivirus, and its associated neurological disorders, such as Guillain-Barré (GB) syndrome and microcephaly, have generated an urgent need to develop effective ZIKV vaccines and therapeutic agents. Here, we used human endothelial cells and astrocytes, both of which represent key cell types for ZIKV infection, to identify potential inhibitors of ZIKV replication. Because several pathways, including the AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) signaling pathways, have been reported to play important roles in flavivirus replication, we tested inhibitors and agonists of these pathways for their effects on ZIKV replication. We identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. PKI effectively suppressed the replication of ZIKV from both the African and Asian/American lineages with a high efficiency and minimal cytotoxicity. While ZIKV infection does not induce PKA activation, endogenous PKA activity is essential for supporting ZIKV replication. Interestingly, in addition to PKA, PKI also inhibited another unknown target(s) to block ZIKV replication. PKI inhibited ZIKV replication at the postentry stage by preferentially affecting negative-sense RNA synthesis as well as viral protein translation. Together, these results have identified a potential inhibitor of ZIKV replication which could be further explored for future therapeutic application. IMPORTANCE There is an urgent need to develop effective vaccines and therapeutic agents against Zika virus (ZIKV) infection, a reemerging flavivirus associated with neurological disorders, including Guillain-Barré (GB) syndrome and microcephaly. By screening for inhibitors of several cellular pathways, we have identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. We show that PKI effectively suppresses the replication of all ZIKV

  11. In vitro and in vivo inhibition of rabies virus replication by RNA interference

    Directory of Open Access Journals (Sweden)

    Ekaterina A. Durymanova Ono

    2013-09-01

    Full Text Available Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV (Mononegavirales: Rhabdoviridae: Lyssavirus. Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

  12. In vitro and in vivo inhibition of rabies virus replication by RNA interference.

    Science.gov (United States)

    Durymanova Ono, Ekaterina A; Iamamoto, Keila; Castilho, Juliana G; Carnieli, Pedro; de Novaes Oliveira, Rafael; Achkar, Samira M; Carrieri, Maria L; Kotait, Ivanete; Brandão, Paulo E

    2013-01-01

    Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

  13. Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

    Science.gov (United States)

    Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

    2013-01-01

    RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001

  14. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    OpenAIRE

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, J?rgen J.; Johne, Reimar

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Hu...

  15. Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

    Science.gov (United States)

    Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

    2016-10-01

    Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

  16. A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication

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    Li Yanhua

    2011-04-01

    Full Text Available Abstract Background It has been well documented that the 5' untranslated region (5' UTR of many positive-stranded RNA viruses contain key cis-acting regulatory sequences, as well as high-order structural elements. Little is known for such regulatory elements controlling porcine arterivirus replication. We investigated the roles of a conserved stem-loop 2 (SL2 that resides in the 5'UTR of the genome of a type II porcine reproductive and respiratory syndrome virus (PRRSV. Results We provided genetic evidences demonstrating that 1 the SL2 in type II PRRSV 5' UTR, N-SL2, could be structurally and functionally substituted by its counterpart in type I PRRSV, E-SL2; 2 the functionality of N-SL2 was dependent upon the G-C rich stem structure, while the ternary-loop size was irrelevant to RNA synthesis; 3 serial deletions showed that the stem integrity of N-SL2 was crucial for subgenomic mRNA synthesis; and 4 when extensive base-pairs in the stem region was deleted, an alternative N-SL2-like structure with different sequence was utilized for virus replication. Conclusion Taken together, we concluded that the phylogenetically conserved SL2 in the 5' UTR was crucial for PRRSV virus replication, subgenomic mRNA synthesis in particular.

  17. High levels of virus replication and an intense inflammatory response contribute to the severe pathology in lymphoid tissues caused by Newcastle disease virus genotype VIId.

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    Hu, Zenglei; Hu, Jiao; Hu, Shunlin; Song, Qingqing; Ding, Pingyun; Zhu, Jie; Liu, Xiaowen; Wang, Xiaoquan; Liu, Xiufan

    2015-03-01

    Some strains of Newcastle disease virus (NDV) genotype VIId cause more-severe tissue damage in lymphoid organs compared to other virulent strains. In this study, we aim to define the mechanism of this distinct pathological manifestation of genotype VII viruses. Pathology, virus replication, and the innate immune response in lymphoid tissues of chickens infected with two genotype VIId NDV strains (JS5/05 and JS3/05), genotype IX NDV F48E8 and genotype IV NDV Herts/33, were compared. Histopathologic examination showed that JS5/05 and JS3/05 produced more-severe lesions in the spleen and thymus, but these four virulent strains caused comparable mild lesions in the bursa. In addition, JS3/05 and JS5/05 replicated at significantly higher levels in the lymphatic organs than F48E8 and Herts/33. A microarray assay performed on the spleens of chickens infected with JS5/05 or Herts/33 revealed that JS5/05 elicited a more potent inflammatory response by increasing the number and expression levels of activated genes. Moreover, cytokine gene expression profiling showed that JS5/05 and JS3/05 induced a stronger cytokine response in lymphoid tissues compared to F48E8 and Herts/33. Taken together, our results indicate that the severe pathology in immune organs caused by genotype VIId NDV strains is associated with high levels of virus replication and an intense inflammatory response.

  18. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Deepika Bhullar

    Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

  19. Regulation of hepatitis C virus replication by nuclear translocation of nonstructural 5A protein and transcriptional activation of host genes.

    Science.gov (United States)

    Maqbool, Muhammad Ahmad; Imache, Mohamed R; Higgs, Martin R; Carmouse, Sophie; Pawlotsky, Jean-Michel; Lerat, Hervé

    2013-05-01

    Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is involved in regulating viral replication through its direct interaction with the HCV RNA-dependent RNA polymerase. NS5A also alters infected cell metabolism through complex interactions with numerous host cell proteins. NS5A has furthermore been suggested to act as a transcriptional activator, although the impact on viral replication is unclear. To study this, HCV NS5A variants were amplified from hepatic tissue from an HCV-infected patient, and their abilities to activate gene transcription were analyzed in a single-hybrid yeast (Saccharomyces cerevisiae) model. Different variants isolated from the same patient displayed different transactivational activities. When these variants were inserted into the HCV subgenomic replicon system, they demonstrated various levels of RNA replication, which correlated with their transactivational activities. We showed that the C-terminal fragment of NS5A was localized to the nucleus and that a functional NS5A nuclear localization signal and cellular caspase activity were required for this process. Furthermore, nuclear localization of NS5A was necessary for viral replication. Finally, we demonstrate that nuclear NS5A binds to host cell promoters of several genes previously identified as important for efficient HCV RNA replication, inducing their transcription. Taken together, these results demonstrate a new mechanism by which HCV modulates its cellular environment, thereby enhancing viral replication.

  20. Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Kaushik, Nidhi; Subramani, Chandru; Anang, Saumya; Muthumohan, Rajagopalan; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

    2017-11-01

    Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used