WorldWideScience

Sample records for animal virus replication

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

    Institute of Scientific and Technical Information of China (English)

    Kristin L MacArthur; Catherine H Wu; George Y Wu

    2012-01-01

    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. A tale of two HSV-1 helicases: roles of phage and animal virus helicases in DNA replication and recombination.

    Science.gov (United States)

    Marintcheva, B; Weller, S K

    2001-01-01

    Helicases play essential roles in many important biological processes such as DNA replication, repair, recombination, transcription, splicing, and translation. Many bacteriophages and plant and animal viruses encode one or more helicases, and these enzymes have been shown to play many roles in their respective viral life cycles. In this review we concentrate primarily on the roles of helicases in DNA replication and recombination with special emphasis on the bacteriophages T4, T7, and A as model systems. We explore comparisons between these model systems and the herpesviruses--primarily herpes simplex virus. Bacteriophage utilize various pathways of recombination-dependent DNA replication during the replication of their genomes. In fact the study of recombination in the phage systems has greatly enhanced our understanding of the importance of recombination in the replication strategies of bacteria, yeast, and higher eukaryotes. The ability to "restart" the replication process after a replication fork has stalled or has become disrupted for other reasons is a critical feature in the replication of all organisms studied. Phage helicases and other recombination proteins play critical roles in the "restart" process. Parallels between DNA replication and recombination in phage and in the herpesviruses is explored. We and others have proposed that recombination plays an important role in the life cycle of the herpesviruses, and in this review, we discuss models for herpes simplex virus type 1 (HSV-1) DNA replication. HSV-1 encodes two helicases. UL9 binds specifically to the origins of replication and is believed to initiate HSV DNA replication by unwinding at the origin; the heterotrimeric helicase-primase complex, encoded by UL5, UL8, and UL52 genes, is believed to unwind duplex viral DNA at replication forks. Structure-function analyses of UL9 and the helicase-primase are discussed with attention to the roles these proteins might play during HSV replication. PMID

  3. 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.

  4. Hepatitis Delta Virus RNA Replication

    Directory of Open Access Journals (Sweden)

    Chung-Hsin Tseng

    2009-11-01

    Full Text Available Hepatitis delta virus (HDV is a distant relative of plant viroids in the animal world. Similar to plant viroids, HDV replicates its circular RNA genome using a double rolling-circle mechanism. Nevertheless, the production of hepatitis delta antigen (HDAg, which is indispensible for HDV replication, is a unique feature distinct from plant viroids, which do not encode any protein. Here the HDV RNA replication cycle is reviewed, with emphasis on the function of HDAg in modulating RNA replication and the nature of the enzyme involved.

  5. Hepatitis D Virus Replication.

    Science.gov (United States)

    Taylor, John M

    2015-11-01

    This work reviews specific related aspects of hepatitis delta virus (HDV) reproduction, including virion structure, the RNA genome, the mode of genome replication, the delta antigens, and the assembly of HDV using the envelope proteins of its helper virus, hepatitis B virus (HBV). These topics are considered with perspectives ranging from a history of discovery through to still-unsolved problems. HDV evolution, virus entry, and associated pathogenic potential and treatment of infections are considered in other articles in this collection. PMID:26525452

  6. Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand.

    Science.gov (United States)

    Steel, Olivia; Kraberger, Simona; Sikorski, Alyssa; Young, Laura M; Catchpole, Ryan J; Stevens, Aaron J; Ladley, Jenny J; Coray, Dorien S; Stainton, Daisy; Dayaram, Anisha; Julian, Laurel; van Bysterveldt, Katherine; Varsani, Arvind

    2016-09-01

    In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified. PMID:27211884

  7. Hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA,ε, as template, and depends on cellular chaperones;moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids.This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV),now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately,not be complemented by three-dimensional structural information on the involved components. However, at least for the s RNA element such information is emerging,raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal,will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

  8. The hepatitis delta virus: Replication and pathogenesis.

    Science.gov (United States)

    Sureau, Camille; Negro, Francesco

    2016-04-01

    Hepatitis delta virus (HDV) is a defective virus and a satellite of the hepatitis B virus (HBV). Its RNA genome is unique among animal viruses, but it shares common features with some plant viroids, including a replication mechanism that uses a host RNA polymerase. In infected cells, HDV genome replication and formation of a nucleocapsid-like ribonucleoprotein (RNP) are independent of HBV. But the RNP cannot exit, and therefore propagate, in the absence of HBV, as the latter supplies the propagation mechanism, from coating the HDV RNP with the HBV envelope proteins for cell egress to delivery of the HDV virions to the human hepatocyte target. HDV is therefore an obligate satellite of HBV; it infects humans either concomitantly with HBV or after HBV infection. HDV affects an estimated 15 to 20million individuals worldwide, and the clinical significance of HDV infection is more severe forms of viral hepatitis - acute or chronic -, and a higher risk of developing cirrhosis and hepatocellular carcinoma in comparison to HBV monoinfection. This review covers molecular aspects of HDV replication cycle, including its interaction with the helper HBV and the pathogenesis of infection in humans. PMID:27084031

  9. Reverse genetics with animal viruses

    International Nuclear Information System (INIS)

    important insights into viral pathogenesis. The roles played by many accessory proteins, including V, C and NS proteins of Paramyxoviridae and influenza viruses as interferon antagonists were studied in detail using infectious clones. Since interferon antagonists are important virulence factors, their identification and modification by knocking them out or reducing their expression should provide opportunities to generate safe attenuated vaccine strains. Like other members of Paramyxovirinae, NDV produces the accessory V protein from the P gene by a process called RNA editing. Introduction of mutation into the editing site resulted in reduction of the editing frequency and as a result, V was expressed at a 20-fold lower level than the wild type NDV and was highly attenuated in chicken embryos. Administration of the recombinant NDV with an editing site mutation to 18-day-old chicken embryos did not affect hatchability. Hatched chickens developed high levels of NDV specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine. The ability to manipulate the genomes of animal viruses has also important implications in designing and developing marker vaccines. Different approaches can be used in designing marker vaccines. One approach that we employed for generating a marked NDV was first localizing a conserved B-cell immunodominant epitope (IDE) on the nucleoprotein (NP) gene and then successfully recovering a recombinant NDV lacking the IDE by reverse genetics. In addition, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace the IDE. Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the IDE is not only dispensable for virus replication, but can also be replaced by foreign sequences. Chickens immunised with the hybrid recombinants produced specific antibodies

  10. The IFITMs Inhibit Zika Virus Replication.

    Science.gov (United States)

    Savidis, George; Perreira, Jill M; Portmann, Jocelyn M; Meraner, Paul; Guo, Zhiru; Green, Sharone; Brass, Abraham L

    2016-06-14

    Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses. PMID:27268505

  11. The IFITMs Inhibit Zika Virus Replication

    Directory of Open Access Journals (Sweden)

    George Savidis

    2016-06-01

    Full Text Available Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses.

  12. The IFITMs Inhibit Zika Virus Replication

    OpenAIRE

    George Savidis; Jill M. Perreira; Jocelyn M. Portmann; Paul Meraner; Zhiru Guo; Sharone Green; Abraham L. Brass

    2016-01-01

    Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs mig...

  13. Role of Host Reticulon Proteins in Rearranging Membranes for Positive-strand RNA Virus Replication

    OpenAIRE

    Diaz, Arturo; Ahlquist, Paul

    2012-01-01

    Positive-strand RNA [(+)RNA] viruses are responsible for numerous human, animal, and plant diseases. Because of the limiting coding capacity of (+)RNA viruses, their replication requires a complex orchestration of interactions between the viral genome, viral proteins and exploited host factors. To replicate their genomic RNAs, (+)RNA viruses induce membrane rearrangements that create membrane-linked RNA replication compartments. Along with substantial advances on the ultrastructure of the mem...

  14. 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.

  15. Animal Models of Dengue Virus Infection

    OpenAIRE

    Eva Harris; Simona Zompi

    2012-01-01

    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 deng...

  16. Replication strategy of human hepatitis B virus

    International Nuclear Information System (INIS)

    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. Human cytomegalovirus induces JC virus DNA replication in human fibroblasts.

    Science.gov (United States)

    Heilbronn, R; Albrecht, I; Stephan, S; Bürkle, A; zur Hausen, H

    1993-01-01

    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. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8248262

  18. Replication and transmission of influenza viruses in Japanese quail

    International Nuclear Information System (INIS)

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

  19. Autophagic machinery activated by dengue virus enhances virus replication

    International Nuclear Information System (INIS)

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

    NARCIS (Netherlands)

    Belov, G.A.; Kuppeveld, F.J.M. van

    2012-01-01

    Positive-strand RNA [(+)RNA] viruses show a significant degree of conservation of their mechanisms of replication. The universal requirement of (+)RNA viruses for cellular membranes for genome replication, and the formation of membranous replication organelles with similar architecture, suggest that

  1. 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; Røntved, C.M.; Uttenthal, Åse; Rønsholt, L.; Alexandersen, Søren

    2002-01-01

    Human respiratory syncytial virus is an important cause of severe respiratory disease in young children, the elderly, and in immunocompromised adults. Similarly, bovine respiratory syncytial virus (BRSV) is causing severe, sometimes fatal, respiratory disease in calves. Both viruses are pneumovirus...... 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...... 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...

  2. Phosphonoacetic Acid Inhibition of Frog Virus 3 Replication

    OpenAIRE

    Elliott, R. M.; Bateson, A.; Kelly, D C

    1980-01-01

    Phosphonoacetic acid at concentrations above 200 μg/ml inhibited the replication of frog virus 3 in BHK cells. The inhibition of viral DNA replication observed in these cells was reversible and correlated with the inhibition of the virus-induced DNA polymerase activity in an in vitro assay. The synthesis of frog virus 3-induced late or γ polypeptides was also inhibited by phosphonoacetic acid, although the early (α and β) polypeptides were unaffected.

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

    OpenAIRE

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

    1995-01-01

    The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) ...

  4. RNA recombination in animal and plant viruses.

    OpenAIRE

    Lai, M M

    1992-01-01

    An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses ...

  5. Gold Nanoparticles Impair Foot-and-Mouth Disease Virus Replication.

    Science.gov (United States)

    Rafiei, Solmaz; Rezatofighi, Seyedeh Elham; Roayaei Ardakani, Mohammad; Rastegarzadeh, Saadat

    2016-01-01

    In this study, we evaluated the antiviral activity of gold nanoparticles (AuNPs) against the foot-and-mouth disease virus (FMDV), that causes a contagious disease in cloven-hoofed animals. The anti-FMDV activity of AuNPs was assessed using plaque reduction assay. MTT assay was used for quantitatively measuring the cytopathic effect caused by the viral infection. The 50% cytotoxicity concentration of nanoparticles was measured and found to be 10.4 μg/ml. The virus yield reduction assay showed that AuNP have an approximately 4-fold virus titer reduction compared with controls. Plaque reduction assay showed that at non-cytotoxic concentrations, AuNPs do not show extracellular virucidal activity and inhibition of FMDV growth at the early stages of infection including attachment and penetration. Time-of-addition experiments revealed that AuNPs inhibited post-entry stages of viral replication concomitant with the onset of intracellular viral RNA synthesis; however, the mechanism of AuNPs against FMDV was unclear. PMID:26685261

  6. Restriction of Human Polyomavirus BK Virus DNA Replication in Murine Cells and Extracts

    OpenAIRE

    Mahon, C.; Liang, B.; Tikhanovich, I.; et al

    2009-01-01

    BK virus (BKV) causes persistent and asymptomatic infections in most humans and is the etiologic agent of polyomavirus-associated nephropathy (PVAN) and other pathologies. Unfortunately, there are no animal models with which to study activation of BKV replication in the human kidney and the accompanying PVAN. Here we report studies of the restriction of BKV replication in murine cells and extracts and the cause(s) of this restriction. Upon infection of murine cells, BKV expressed large T anti...

  7. Hepatitis B virus replication within the human spleen.

    OpenAIRE

    Di Bisceglie, A M; Hoofnagle, J H

    1990-01-01

    Southern blot analysis of splenic DNA from three patients with chronic hepatitis B revealed the presence of replicative intermediate forms of viral DNA. Hepatitis B viral RNA was detected in splenic RNA from two cases. Thus, hepatitis B virus appears to replicate in the human spleen, although at a lower level than in the liver.

  8. Interference with virus and bacteria replication by the tissue specific expression of antibodies and interfering molecules.

    Science.gov (United States)

    Enjuanes, L; Sola, I; Izeta, A; Sánchez-Morgado, J M; González, J M; Alonso, S; Escors, D; Sánchez, C M

    1999-01-01

    Historically, protection against virus infections has relied on the use of vaccines, but the induction of an immune response requires several days and in certain situations, like in newborn animals that may be infected at birth and die in a few days, there is not sufficient time to elicit a protective immune response. Immediate protection in new born could be provided either by vectors that express virus-interfering molecules in a tissue specific form, or by the production of animals expressing resistance to virus replication. The mucosal surface is the largest body surface susceptible to virus infection that can serve for virus entry. Then, it is of high interest to develop strategies to prevent infections of these areas. Virus growth can be interfered intracellularly, extracellularly or both. The antibodies neutralize virus intra- and extracellularly and their molecular biology is well known. In addition, antibodies efficiently neutralize viruses in the mucosal areas. The autonomy of antibody molecules in virus neutralization makes them functional in cells different from those that produce the antibodies and in the extracellular medium. These properties have identified antibodies as very useful molecules to be expressed by vectors or in transgenic animals to provide resistance to virus infection. A similar role could be played by antimicrobial peptides in the case of bacteria. Intracellular interference with virus growth (intracellular immunity) can be mediated by molecules of very different nature: (i) full length or single chain antibodies; (ii) mutant viral proteins that strongly interfere with the replication of the wild type virus (dominant-negative mutants); (iii) antisense RNA and ribozyme sequences; and (iv) the product of antiviral genes such as the Mx proteins. All these molecules inhibiting virus replication may be used to obtain transgenic animals with resistance to viral infection built in their genomes. We have developed two strategies to target

  9. Susceptibility of Rat-Derived Cells to Replication by Human Immunodeficiency Virus Type 1

    OpenAIRE

    Keppler, Oliver T.; Yonemoto, Wesley; Welte, Frank J.; Patton, Kathryn S.; Iacovides, Demetris; Atchison, Robert E.; Ngo, Tuan; Hirschberg, David L.; Roberto F Speck; Goldsmith, Mark A.

    2001-01-01

    Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early ...

  10. Triterpene Derivatives that Inhibit Human Immunodeficiency Virus Type 1 Replication

    OpenAIRE

    Dorr, Casey R.; Yemets, Sergiy; Kolomitsyna, Oksana; Krasutsky, Pavel; Louis M. Mansky

    2011-01-01

    Triterpene derivatives were analyzed for anti-HIV-1 activity and for cellular toxicity. Betulinic aldehyde, betulinic nitrile, and morolic acid derivatives were identified to have anti-HIV-1 activity. These derivatives inhibit a late step in virus replication, likely virus maturation.

  11. 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...

  12. Animal viruses, radiation, repair mechanisms, and cancer

    International Nuclear Information System (INIS)

    Effects of radiation on animal viruses are reviewed in terms of repair mechanisms and their possible relationship to virus transformation and carcinogenesis. Repair mechanisms are reviewed under the headings; multiplicity reactivation, photoreactivation, host-cell reactivation (dark repair), and uv reactivation. Other topics discussed are: transformation of cells in vitro by irradiated viruses, oncogenicity of virus-transformed cells in vivo, and possible role of repair mechanisms in virus transformation and oncogenesis

  13. Animal Models of Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Jiang, Zhiwu; Gu, Liming; Chen, Yanxia

    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. PMID:27525278

  15. Small rodent models of hepatitis B and C virus replication and pathogenesis

    Institute of Scientific and Technical Information of China (English)

    Mark A.Feitelson; Alla Arzumanyan; Marcia M.Clayton

    2012-01-01

    The narrow host range of infection supporting the long-term propagation of hepatitis B and C viruses is a major limitation that has prevented a more thorough understanding of persistent infection and the pathogenesis of chronic liver disease (CLD).With hepatitis B virus (HBV),this has been partially overcome by the discovery and characterization of HBV-like viruses in wild animals.With hepatitis C virus (HCV),related Fiaviviruses have been used as surrogate systems for such studies.Independent work has developed various mouse strains for the transplantation of human hepatocytes,which are then susceptible to infection with HBV and HCV.Other laboratories have developed transgenic mice that express virus gene products and/or support virus replication.Some HBV transgenic mouse models develop fulminant hepatitis,acute hepatitis,or CLD following adoptive transfer,while others spontaneously develop hepatocellular carcinoma (HCC),as in human infections.Among HCV transgenic mice,most develop no disease,but acute hepatitis has been observed in one model,while HCC appears in another.Other mouse models include the introduction of xenographs that replicate HBV or HCV.Although mice are not susceptible to these viruses,their ability to support virus replication and to develop liver disease characteristic of human infections,provides new opportunities to study pathogenesis and develop novel therapeutics.

  16. 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.

  17. VIRUS: A hugely replicated integral field spectrograph for HETDEX

    Science.gov (United States)

    Hill, Gary J.; MacQueen, Phillip J.; Palunas, Povilas; Kelz, Andreas; Roth, Martin M.; Gebhardt, Karl; Grupp, Frank

    2006-06-01

    We present the visible integral-field replicable unit spectrograph (VIRUS), the basis of the Hobby-Eberly telescope dark energy experiment (HETDEX); a survey of a 5 Gpc 3 volume at 1.8 < z < 3.7 that will constrain the evolution of dark energy. VIRUS consists of 145 copies of a simple unit spectrograph, deployed on the HET. Industrial replication will allow VIRUS to be built quickly, at considerable cost-savings, with substantial risk-mitigation, compared to conventional instruments. VIRUS will cover 30 sq. arcmin per observation and detect 14 million resolution elements per exposure, an order of magnitude larger than existing instruments. VIRUS can complete HETDEX in about 100 nights observing.

  18. Prostaglandin A1 Inhibits Replication of Classical Swine Fever Virus

    OpenAIRE

    Tânia Rosária Pereira Freitas; Lucio Ayres Caldas; Moacyr Alcoforado Rebello

    1998-01-01

    Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 µg/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus d...

  19. Prostaglandin A1 Inhibits Replication of Classical Swine Fever Virus

    Directory of Open Access Journals (Sweden)

    Tânia Rosária Pereira Freitas

    1998-11-01

    Full Text Available Prostaglandins (Pgs have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1 on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 µg/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

    OpenAIRE

    Iftode, C.; Borowiec, J A

    1997-01-01

    The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin wit...

  2. Sites of replication of bovine respiratory syncytial virus in naturally infected calves as determined by in situ hybridization

    DEFF Research Database (Denmark)

    Viuff, B.; Uttenthal, Åse; Tegtmeier, C.; Alexandersen, Søren

    1996-01-01

    Replication of bovine respiratory syncytial virus (BRSV) was studied in three naturally infected calves by in situ hybridization using strand-specific RNA probes. One of the calves was a 5-month-old Friesian, the other two calves were a 3-month-old and a 2-week-old Jersey. Two Jersey calves, 3...... months and 3 weeks of age, served as controls. Replication of BRSV took place in the luminal lining of the respiratory tract. In one of the BRSV infected animals (calf No. 1), replication was especially seen in the bronchi, whereas in the two other animals (calf Nos. 2 and 3) replication of BRSV was...

  3. Application of hepatitis B virus replication mouse model

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model's value ...

  4. Inhibition of Monkeypox virus replication by RNA interference

    Directory of Open Access Journals (Sweden)

    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.

  5. Origin of the membrane compartment for cowpea mosaic virus replication

    NARCIS (Netherlands)

    Carette, J.E.

    2002-01-01

    Replication of many positive-stranded RNA viruses takes place in association with intracellular membranes. Often these membranes are induced upon infection by vesiculation or rearrangement of membranes from different organelles including the early and late endomembrane system. Upon infection of cowp

  6. Evidence for Protection against Chronic Hepatitis C Virus Infection in Chimpanzees by Immunization with Replicating Recombinant Vaccinia Virus▿

    OpenAIRE

    Youn, Jin-Won; Hu, Yu-Wen; Tricoche, Nancy; Pfahler, Wolfram; Shata, Mohamed Tarek; Dreux, Marlene; Cosset, François-Loic; Folgori, Antonella; Lee, Dong-Hun; Brotman, Betsy; Prince, Alfred M.

    2008-01-01

    Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection. All four immunized animals resolved...

  7. Replication competent Semliki Forest virus prolongs survival in experimental lung cancer.

    Science.gov (United States)

    Määttä, Ann-Marie; Mäkinen, Kimmo; Ketola, Anna; Liimatainen, Timo; Yongabi, Felicitas Newu; Vähä-Koskela, Markus; Pirinen, Risto; Rautsi, Outi; Pellinen, Riikka; Hinkkanen, Ari; Wahlfors, Jarmo

    2008-10-01

    We evaluated the therapeutic potential of the replication competent vector VA7-EGFP, which is based on the avirulent Semliki Forest virus (SFV) strain A7 (74) carrying the EGFP marker gene in an orthotopic lung cancer tumor model in nude mice. We have previously shown that this oncolytic vector destroys tumor cells efficiently in vitro and in vivo (in subcutaneous tumor model). Tumor growth in animals with orthotopically implanted adenocarcinoma cells (A549) were monitored during the study with small animal CT. We show that locally administered virotherapy with VA7-EGFP increased survival rate in experimental lung cancer significantly (p < 0.001) comparable to results obtained with the second generation conditionally replicating adenoviral vector Ad5-Delta24TK-GFP, used for comparison. The limited efficacy in systemically administered oncolytic viruses is the essential problem in oncolytic virotherapy and also in this study we were not able to elicit significant response with systemic administration route. Despite the fact that tumor microenvironment in orthotopic lung cancer is more optimal, viruses failed to home to the tumors and were unable to initiate efficient intratumoral replication. Clearly, the efficacy of virotherapy is influenced by many factors such as the route of virus administration, immunological and physiological barriers and cancer cell-specific features (IFN-responsiveness). PMID:18651570

  8. 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

  9. 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.

  10. Replication-incompetent herpesvirus vector delivery of an interferon alpha gene inhibits human immunodeficiency virus replication in human monocytes.

    OpenAIRE

    Weir, J P; Elkins, K L

    1993-01-01

    Human monocytes and macrophages are nondividing cells that serve as a major reservoir for human immunodeficiency virus (HIV) at all stages of infection. To investigate viral-mediated gene delivery as a means of inhibiting HIV replication in human monocytes, a replication-incompetent herpes simplex virus vector was developed that expressed human interferon alpha. Monocytes infected with this herpes simplex virus vector and then challenged with HIV showed dramatically reduced cytopathic effects...

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen which causes huge economic damage globally in the swine industry. Current vaccination strategies provide only limited protection against PRRSV infection. Viperin is an interferon (IFN) stimulated protein that inhibits some virus infections via IFN-dependent or IFN-independent pathways. However, the role of viperin in PRRSV infection is not well understood. In this study, we cloned the full-length monkey viperin (mViperin) complementary DNA (cDNA) from IFN-α-treated African green monkey Marc-145 cells. It was found that the mViperin is up-regulated following PRRSV infection in Marc-145 cells along with elevated IRF-1 gene levels. IFN-α induced mViperin expression in a dose- and time-dependent manner and strongly inhibits PRRSV replication in Marc-145 cells. Overexpression of mViperin suppresses PRRSV replication by blocking the early steps of PRRSV entry and genome replication and translation but not inhibiting assembly and release. And mViperin co-localized with PRRSV GP5 and N protein, but only interacted with N protein in distinct cytoplasmic loci. Furthermore, it was found that the 13–16 amino acids of mViperin were essential for inhibiting PRRSV replication, by disrupting the distribution of mViperin protein from the granular distribution to a homogeneous distribution in the cytoplasm. These results could be helpful in the future development of novel antiviral therapies against PRRSV infection. PMID:27232627

  13. Kinetics of reparative DNA replication induced in CBA and C57BL/6 mouse spleen cells by urethane and influenza virus

    International Nuclear Information System (INIS)

    The authors investigated the reparative replication of DNA, induced by urethane and influenza virus in C57BL/6 and CBA mice which have marked differences in tumor formation. Reparative DNA replication, induced by urethane and influenza virus was investigated by a liquid scintillation method based on measuring incorporation of 3H-thymidine into the total mass of mouse spleen cells, when replicative DNA synthesis was inhibited by hydroxyurea. The intensity of reparative DNA replication was judged from the value of the stimulation index which is the ratio of the radioactivity in the spleen cells of the experimental animals to the corresponding parameters in cells of intact animals. The phenomenon of the stimulating and inhibitory effects of influenza virus on reparative DNA replication in cells depending on their genotype and sensitivity to virus is established

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

    OpenAIRE

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

    2009-01-01

    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 minim...

  15. Persistent Sin Nombre Virus Infection in the Deer Mouse (Peromyscus maniculatus) Model: Sites of Replication and Strand-Specific Expression

    OpenAIRE

    Botten, Jason; Mirowsky, Katy; Kusewitt, Donna; Ye, Chunyan; Gottlieb, Keith; Prescott, Joseph; Hjelle, Brian

    2003-01-01

    To address Sin Nombre (SN) virus persistence in deer mice, we sacrificed experimentally infected deer mice at eight time points from day 21 to day 217 postinoculation (p.i.) and examined their tissues for viral nucleocapsid (N) antigen expression and both negative-strand (genomic) and positive-strand (replicative/mRNA) viral S segment RNA titers. All the animals that we inoculated developed persistent infections, and SN virus could be isolated from tissues throughout the course of infection. ...

  16. VIRUS: a massively replicated integral-field spectrograph for HET

    Science.gov (United States)

    Hill, Gary J.; MacQueen, Phillip J.; Tufts, Joseph R.; Kelz, Andreas; Roth, Martin M.; Altmann, Werner; Segura, Pedro; Gebhardt, Karl; Palunas, Povilas

    2006-06-01

    We present the design of, and the science drivers for, the Visible Integral-field Replicable Unit Spectrograph (VIRUS). This instrument is made up of 145 individually small and simple spectrographs, each fed by a fiber integral field unit. The total VIRUS-145 instrument covers ~30 sq. arcminutes per observation, providing integral field spectroscopy from 340 to 570 nm, simultaneously, of 35,670 spatial elements, each 1 sq. arcsecond on the sky. This corresponds to 15 million resolution elements per exposure. VIRUS-145 will be mounted on the Hobby-Eberly Telescope and fed by a new wide-field corrector with 22 arcminutes diameter field of view. VIRUS represents a new approach to spectrograph design, offering the science multiplex advantage of huge sky coverage for an integral field spectrograph, coupled with the engineering multiplex advantage of >100 spectrographs making up a whole. VIRUS is designed for the Hobby-Eberly Telescope Dark Energy Experiment (HETDEX) which will use baryonic acoustic oscillations imprinted on the large-scale distribution of Lyman-α emitting galaxies to provide unique constraints on the expansion history of the universe that can constrain the properties of dark energy.

  17. Replication Cycle and Molecular Biology of the West Nile Virus

    Directory of Open Access Journals (Sweden)

    Margo A. Brinton

    2013-12-01

    Full Text Available West Nile virus (WNV is a member of the genus Flavivirus in the family Flaviviridae. Flaviviruses replicate in the cytoplasm of infected cells and modify the host cell environment. Although much has been learned about virion structure and virion-endosomal membrane fusion, the cell receptor(s used have not been definitively identified and little is known about the early stages of the virus replication cycle. Members of the genus Flavivirus differ from members of the two other genera of the family by the lack of a genomic internal ribosomal entry sequence and the creation of invaginations in the ER membrane rather than double-membrane vesicles that are used as the sites of exponential genome synthesis. The WNV genome 3' and 5' sequences that form the long distance RNA-RNA interaction required for minus strand initiation have been identified and contact sites on the 5' RNA stem loop for NS5 have been mapped. Structures obtained for many of the viral proteins have provided information relevant to their functions. Viral nonstructural protein interactions are complex and some may occur only in infected cells. Although interactions between many cellular proteins and virus components have been identified, the functions of most of these interactions have not been delineated.

  18. Establishment and primary application of a mouse model with hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To establish a rapid and convenient animal model with hepatitis B virus (HBV) replication. METHODS: A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen (HBeAg) was detected by enzymelinked immunosorbent assay (ELISA). Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid (polyIC) or phosphate-buffered saline (PBS). RESULTS: After hydrodynamicin vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS. CONCLUSION: A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.

  19. RNA interference-mediated inhibition of Hepatitis B Virus replication

    Institute of Scientific and Technical Information of China (English)

    TANG Ni; ZHANG Bingqiang; YAN Ge; PU Dan; GAO Xiaolin; Tong-Chuan He; HUANG Ailong

    2004-01-01

    Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium. Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore, our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.

  20. Fowl plague virus replication in mammalian cell-avian erythrocyte heterokaryons: studies concerning the actinomycin D and ultra-violet lig sensitive phase in influenza virus replication

    International Nuclear Information System (INIS)

    The replication of fowl plague virus in BHK and L cells specifically blocked prior to infection with inhibitors of influenza virus replication (actinomycin D and ultraviolet light irradiation) has been studied by the introduction of a metabolically dormant avian erythrocyte nucleus. This permits the synthesis of just the influenza virus nucleoprotein in actinomycin D (but not ultraviolet light) blocked cells. The NP antigen is first detected in the avian erythrocyte nucleus and subsequently in the heterokaryon cytoplasm

  1. 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.

  2. Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

    Science.gov (United States)

    Liang, Libin; Deng, Guohua; Shi, Jianzhong; Wang, Shuai; Zhang, Qianyi; Kong, Huihui; Gu, Chunyang; Guan, Yuntao; Suzuki, Yasuo; Li, Yanbing; Jiang, Yongping; Tian, Guobin; Liu, Liling

    2015-01-01

    ABSTRACT H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs. IMPORTANCE Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between

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

    Directory of Open Access Journals (Sweden)

    Sibylle Schneider-Schaulies

    2011-01-01

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

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

    OpenAIRE

    Papaneri, Amy B.; Wirblich, Christoph; Cann, Jennifer A.; Cooper, Kurt; Peter B. Jahrling; 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 ...

  5. Lessons Learned from Mouse Mammary Tumor Virus in Animal Models.

    Science.gov (United States)

    Dudley, Jaquelin P; Golovkina, Tatyana V; Ross, Susan R

    2016-03-31

    Mouse mammary tumor virus (MMTV), which was discovered as a milk-transmitted, infectious, cancer-inducing agent in the 1930s, has been used as an animal model for the study of retroviral infection and transmission, antiviral immune responses, and breast cancer and lymphoma biology. The main target cells for MMTV infection in vivo are cells of the immune system and mammary epithelial cells. Although the host mounts an immune response to the virus, MMTV has evolved multiple means of evading this response. MMTV causes mammary tumors when the provirus integrates into the mammary epithelial and lymphoid cell genome during viral replication and thereby activates cellular oncogene expression. Thus, tumor induction is a by-product of the infection cycle. A number of important oncogenes have been discovered by carrying out MMTV integration site analysis, some of which may play a role in human breast cancer. PMID:27034391

  6. Suramin inhibits chikungunya virus replication through multiple mechanisms.

    Science.gov (United States)

    Albulescu, Irina C; van Hoolwerff, Marcella; Wolters, Laura A; Bottaro, Elisabetta; Nastruzzi, Claudio; Yang, Shih Chi; Tsay, Shwu-Chen; Hwu, Jih Ru; Snijder, Eric J; van Hemert, Martijn J

    2015-09-01

    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe and often persistent arthritis. In recent years, millions of people have been infected with this virus for which registered antivirals are still lacking. Using our recently established in vitro assay, we discovered that the approved anti-parasitic drug suramin inhibits CHIKV RNA synthesis (IC50 of ∼5μM). The compound inhibited replication of various CHIKV isolates in cell culture with an EC50 of ∼80μM (CC50>5mM) and was also active against Sindbis virus and Semliki Forest virus. In vitro studies hinted that suramin interferes with (re)initiation of RNA synthesis, whereas time-of-addition studies suggested it to also interfere with a post-attachment early step in infection, possibly entry. CHIKV (nsP4) mutants resistant against favipiravir or ribavirin, which target the viral RNA polymerase, did not exhibit cross-resistance to suramin, suggesting a different mode of action. The assessment of the activity of a variety of suramin-related compounds in cell culture and the in vitro assay for RNA synthesis provided more insight into the moieties required for antiviral activity. The antiviral effect of suramin-containing liposomes was also analyzed. Its approved status makes it worthwhile to explore the use of suramin to prevent and/or treat CHIKV infections. PMID:26112648

  7. Single-cycle replicable Rift Valley fever virus mutants as safe vaccine candidates.

    Science.gov (United States)

    Terasaki, Kaori; Tercero, Breanna R; Makino, Shinji

    2016-05-01

    Rift Valley fever virus (RVFV) is an arbovirus circulating between ruminants and mosquitoes to maintain its enzootic cycle. Humans are infected with RVFV through mosquito bites or direct contact with materials of infected animals. The virus causes Rift Valley fever (RVF), which was first recognized in the Great Rift Valley of Kenya in 1931. RVF is characterized by a febrile illness resulting in a high rate of abortions in ruminants and an acute febrile illness, followed by fatal hemorrhagic fever and encephalitis in humans. Initially, the virus was restricted to the eastern region of Africa, but the disease has now spread to southern and western Africa, as well as outside of the African continent, e.g., Madagascar, Saudi Arabia and Yemen. There is a serious concern that the virus may spread to other areas, such as North America and Europe. As vaccination is an effective tool to control RVFV epidemics, formalin-inactivated vaccines and live-attenuated RVFV vaccines have been used in endemic areas. The formalin-inactivated vaccines require boosters for effective protection, whereas the live-attenuated vaccines enable the induction of protective immunity by a single vaccination. However, the use of live-attenuated RVFV vaccines for large human populations having a varied health status is of concern, because of these vaccines' residual neuro-invasiveness and neurovirulence. Recently, novel vaccine candidates have been developed using replication-defective RVFV that can undergo only a single round of replication in infected cells. The single-cycle replicable RVFV does not cause systemic infection in immunized hosts, but enables the conferring of protective immunity. This review summarizes the properties of various RVFV vaccines and recent progress on the development of the single-cycle replicable RVFV vaccines. PMID:26022573

  8. Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells.

    Science.gov (United States)

    Frensing, Timo; Kupke, Sascha Y; Bachmann, Mandy; Fritzsche, Susanne; Gallo-Ramirez, Lili E; Reichl, Udo

    2016-08-01

    Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination. These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney (MDCK) cells. The cell cycle stage of MDCK cells had no impact during early infection. Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination (HA) titer. The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps. Moreover, our data suggest that in order to achieve higher cell-specific yields, a prolonged phase of viral RNA synthesis and/or a more efficient release of influenza virus particles is required. PMID:27129532

  9. Transmission of Avian Influenza A Viruses Between Animals and People

    Science.gov (United States)

    ... Newsletters Transmission of Avian Influenza A Viruses Between Animals and People Language: English Español Recommend on ... Compartir Influenza A viruses have infected many different animals, including ducks, chickens, pigs, whales, horses, and seals. ...

  10. Cellular chaperonin CCTγ contributes to rabies virus replication during infection.

    Science.gov (United States)

    Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; Ye, Chengjin; Ruan, Xizhen; Zhou, Jiyong

    2013-07-01

    Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. PMID:23637400

  11. 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.

  12. Targeted disruption of influenza A virus hemagglutinin in genetically modified mice reduces viral replication and improves disease outcome.

    Science.gov (United States)

    Wang, Song; Chen, Chao; Yang, Zhou; Chi, Xiaojuan; Zhang, Jing; Chen, Ji-Long

    2016-01-01

    Influenza A virus can cause acute respiratory infection in animals and humans around the globe, and is still a major threat to animal husbandry and public health. Due to antigenic drift and antigenic shift of the virus, development of novel anti-influenza strategies has become an urgent task. Here we generated transgenic (TG) mice stably expressing a short-hairpin RNA specifically targeting hemagglutinin (HA) of influenza A virus, and investigated the susceptibility of the mice to influenza virus infection. We found that HA expression was dramatically disrupted in TG mice infected with WSN or PR8 virus. Importantly, the animals showed reduced virus production in lungs, slower weight loss, attenuated acute organ injury and consequently increased survival rates as compared to wild type (WT) mice after the viral infection. Moreover, TG mice exhibited a normal level of white blood cells following the virus infection, whereas the number of these cells was significantly decreased in WT mice with same challenge. Together, these experiments demonstrate that the TG mice are less permissive for influenza virus replication, and suggest that shRNA-based efficient disruption of viral gene expression in animals may be a useful strategy for prevention and control of a viral zoonosis. PMID:27033724

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

    Science.gov (United States)

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

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

    OpenAIRE

    Eduardo Furtado Flores; Rudi Weiblen; Juliana Felipetto Cargnelutti; Fernando Viçosa Bauermann; Fernando Rosado Spilki; Enio Mori; Ana Cláudia Franco

    2013-01-01

    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 transmit...

  15. The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

    OpenAIRE

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

    2013-01-01

    Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at posit...

  16. Foot-and-Mouth Disease Virus Utilizes an Autophagic Pathway During Viral Replication

    Science.gov (United States)

    Infection with positive-strand RNA viruses results in the rearrangement of intracellular membranes into viral replication complexes (VRC) which are the sites of viral RNA replication. Cellular autophagy has been proposed to be a mechanism of VRC formation for a number of positive-stranded RNA viruse...

  17. 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...

  18. 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.

  19. Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Critical for Ebola Virus Replication in Nonhuman Primates▿

    OpenAIRE

    Neumann, Gabriele; Geisbert, Thomas W.; Ebihara, Hideki; Geisbert, Joan B.; Daddario-DiCaprio, Kathleen M.; Feldmann, Heinz; Kawaoka, Yoshihiro

    2007-01-01

    Enveloped viruses often require cleavage of a surface glycoprotein by a cellular endoprotease such as furin for infectivity and virulence. Previously, we showed that Ebola virus glycoprotein does not require the furin cleavage motif for virus replication in cell culture. Here, we show that there are no appreciable differences in disease progression, hematology, serum biochemistry, virus titers, or lethality in nonhuman primates infected with an Ebola virus lacking the furin recognition sequen...

  20. N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

    OpenAIRE

    Patton, J T; Davis, N L; Wertz, G W

    1984-01-01

    Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent retic...

  1. Animal Models of Varicella Zoster Virus Infection

    Directory of Open Access Journals (Sweden)

    Ilhem Messaoudi

    2013-05-01

    Full Text Available Primary infection with varicella zoster virus (VZV results in varicella (chickenpox followed by the establishment of latency in sensory ganglia. Declining T cell immunity due to aging or immune suppressive treatments can lead to VZV reactivation and the development of herpes zoster (HZ, shingles. HZ is often associated with significant morbidity and occasionally mortality in elderly and immune compromised patients. There are currently two FDA-approved vaccines for the prevention of VZV: Varivax® (for varicella and Zostavax® (for HZ. Both vaccines contain the live-attenuated Oka strain of VZV. Although highly immunogenic, a two-dose regimen is required to achieve a 99% seroconversion rate. Zostavax vaccination reduces the incidence of HZ by 51% within a 3-year period, but a significant reduction in vaccine-induced immunity is observed within the first year after vaccination. Developing more efficacious vaccines and therapeutics requires a better understanding of the host response to VZV. These studies have been hampered by the scarcity of animal models that recapitulate all aspects of VZV infections in humans. In this review, we describe different animal models of VZV infection as well as an alternative animal model that leverages the infection of Old World macaques with the highly related simian varicella virus (SVV and discuss their contributions to our understanding of pathogenesis and immunity during VZV infection.

  2. A mathematical model of rna3 recruitment in the replication cycle of brome mosaic virus

    OpenAIRE

    Huffman, Tori; Link, Kathryn; Nardini, John; Poag, Laura; Flores, Kevin; Banks, H. T.; Blasco, Bernat; Jungfleisch, Jennifer, 1986-; D??ez Ant??n, Juana, 1962-

    2014-01-01

    Positive-strand RNA viruses, such as the brome mosaic virus (BMV) and hepatitis C virus, utilize a replication cycle which involves the recruitment of RNA genomes from the cellular translation machinery to the viral replication complexes. Here, we coupled mathematical modeling with a statistical inverse problem methodology to better understand this crucial recruitment process. We developed a discrete-delay differential equation model that describes the production of BMV protein 1a and BMV RNA...

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

    OpenAIRE

    Raquel Elvira Ocazionez; Rocio Meneses; Flor Ángela Torres; Elena Stashenko

    2010-01-01

    The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37ºC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was...

  4. Epstein-Barr virus stimulates torque teno virus replication: a possible relationship to multiple sclerosis.

    Science.gov (United States)

    Borkosky, Silvia S; Whitley, Corinna; Kopp-Schneider, Annette; zur Hausen, Harald; de Villiers, Ethel-Michele

    2012-01-01

    Viral infections have been implicated in the pathogenesis of multiple sclerosis. Epstein-Barr virus (EBV) has frequently been investigated as a possible candidate and torque teno virus (TTV) has also been discussed in this context. Nevertheless, mechanistic aspects remain unresolved. We report viral replication, as measured by genome amplification, as well as quantitative PCR of two TTV-HD14 isolates isolated from multiple sclerosis brain in a series of EBV-positive and -negative lymphoblastoid and Burkitt's lymphoma cell lines. Our results demonstrate the replication of both transfected TTV genomes up to day 21 post transfection in all the evaluated cell lines. Quantitative amplification indicates statistically significant enhanced TTV replication in the EBV-positive cell lines, including the EBV-converted BJAB line, in comparison to the EBV-negative Burkitt's lymphoma cell line BJAB. This suggests a helper effect of EBV infections in the replication of TTV. The present study provides information on a possible interaction of EBV and TTV in the etiology and progression of multiple sclerosis. PMID:22384166

  5. 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.

  6. Rhesus monkey rhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication

    International Nuclear Information System (INIS)

    Rhesus monkey rhadinovirus (RRV) is a γ-2-herpesvirus that is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Lack of an efficient culture system to grow high titers of virus, and the lack of an in vivo animal model system, has hampered the study of KSHV replication and pathogenesis. RRV is capable of replicating to high titers on fibroblasts, thus facilitating the construction of recombinant rhadinoviruses. In addition, the ability to experimentally infect naieve rhesus macaques with RRV makes it an excellent model system to study γ-herpesvirus replication. Our study describes, for the first time, the construction of a GFP-expressing RRV recombinant virus using a traditional homologous recombination strategy. We have also developed two new methods for determining viral titers of RRV including a traditional viral plaque assay and a quantitative real-time PCR assay. We have compared the replication of wild-type RRV with that of the RRV-GFP recombinant virus in one-step growth curves. We have also measured the sensitivity of RRV to a small panel of antiviral drugs. The development of both the recombination strategy and the viral quantitation assays for RRV will lay the foundation for future studies to evaluate the contribution of individual genes to viral replication both in vitro and in vivo

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

    Indian Academy of Sciences (India)

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

    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% 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% reduction in viral plaque counts and 94% inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68–73% and 87–88% 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.

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

    International Nuclear Information System (INIS)

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

  9. 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.

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

    International Nuclear Information System (INIS)

    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.

  11. AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L; Ecroyd, Heath; Tritsch, Sarah R; Bavari, Sina; Reid, St Patrick; Proniuk, Stefan; Zukiwski, Alexander; Jacob, Abraham; Sepúlveda, Claudia S; Giovannoni, Federico; García, Cybele C; Damonte, Elsa; González-Gallego, Javier; Tuñón, María J; Dent, Paul

    2016-10-01

    We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc. PMID:27187154

  12. Replication and virulence of pseudorabies virus mutants lacking glycoprotein gX.

    OpenAIRE

    Thomsen, D R; Marchioli, C C; Yancey, R J; Post, L E

    1987-01-01

    Pseudorabies virus (PRV) glycoprotein gX accumulates in the medium of infected cells. In an attempt to study the function of gX, two viruses were constructed that lacked a functional gX gene. One virus, PRV delta GX1, was derived by insertion of the herpes simplex virus thymidine kinase gene into the gX-coding region. The other virus, PRV delta GXTK-, was derived by subsequent deletion of the inserted herpes simplex virus thymidine kinase gene. Both viruses replicated in cell cultures but pro...

  13. 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

    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 that a...... active-site tyrosine and the 5' end of the DNA, releasing a 3' DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely...... 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...

  14. Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication

    OpenAIRE

    Dechtawewat, Thanyaporn; Songprakhon, Pucharee; Limjindaporn, Thawornchai; Puttikhunt, Chunya; Kasinrerk, Watchara; Saitornuang, Sawanan; Yenchitsomanus, Pa-Thai; Noisakran, Sansanee

    2015-01-01

    Background Host and viral proteins are involved in dengue virus (DENV) replication. Heterogeneous ribonucleoprotein (hnRNP) C1/C2 are abundant host cellular proteins that exhibit RNA binding activity and play important roles in the replication of positive-strand RNA viruses such as poliovirus and hepatitis C virus. hnRNP C1/C2 have previously been shown to interact with vimentin and viral NS1 in DENV-infected cells; however, their functional role in DENV replication is not clearly understood....

  15. SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication

    Science.gov (United States)

    Su, Chan-I; Tseng, Chung-Hsin

    2016-01-01

    ABSTRACT Small ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular gene Ubc9, which encodes the sole E2-conjugating enzyme required for SUMOylation. By in vivo and in vitro SUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication. IMPORTANCE SUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a “double indemnity” of NS5 to support DENV replication. Without SUMOylation, NS5 fails to

  16. Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing.

    Science.gov (United States)

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

    2003-10-01

    Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L(11) and L(11)A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L(11)A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed. PMID:14512550

  17. In Vivo Accumulation of Cyclin A and Cellular Replication Factors in Autonomous Parvovirus Minute Virus of Mice-Associated Replication Bodies

    OpenAIRE

    Bashir, Tarig; Rommelaere, Jean; Cziepluch, Celina

    2001-01-01

    Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases α and δ in these bodies suggests that MVM utilizes partially preformed cellular replication compl...

  18. Age-dependent resistance to Porcine reproductive and respiratory syndrome virus replication in swine

    Directory of Open Access Journals (Sweden)

    Bautista Elida M

    2009-10-01

    Full Text Available Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV causes a prolonged, economically devastating infection in pigs, and immune resistance to infection appears variable. Since the porcine adaptive immune system is not fully competent at birth, we hypothesized that age influences the dynamics of PRRSV infection. Thus, young piglets, growing 16-20-week-old finisher pigs, and mature third parity sows were infected with virulent or attenuated PRRSV, and the dynamics of viral infection, disease, and immune response were monitored over time. Results Virulent PRRSV infection and disease were markedly more severe and prolonged in young piglets than in finishers or sows. Attenuated PRRSV in piglets also produced a prolonged viremia that was delayed and reduced in magnitude, and in finishers and sows, about half the animals showed no viremia. Despite marked differences in infection, antibody responses were observed in all animals irrespective of age, with older pigs tending to seroconvert sooner and achieve higher antibody levels than 3-week-old animals. Interferon γ (IFN γ secreting peripheral blood mononuclear cells were more abundant in sows but not specifically increased by PRRSV infection in any age group, and interleukin-10 (IL-10 levels in blood were not correlated with PRRSV infection status. Conclusion These findings show that animal age, perhaps due to increased innate immune resistance, strongly influences the outcome of acute PRRSV infection, whereas an antibody response is triggered at a low threshold of infection that is independent of age. Prolonged infection was not due to IL-10-mediated immunosuppression, and PRRSV did not elicit a specific IFN γ response, especially in non-adult animals. Equivalent antibody responses were elicited in response to virulent and attenuated viruses, indicating that the antigenic mass necessary for an immune response is produced at a low level of infection, and is not predicted by

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

    Science.gov (United States)

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

    2007-04-25

    The myxoma virus M063R gene product exhibits some sequence similarity to the poxvirus host range gene, C7L, of vaccinia virus. To address the potential host range function of the M063R gene product in rabbits, a deletion mutant of myxoma virus (vMyx63KO) was generated and characterized. vMyx63KO replicated to normal titre levels and produced foci that were indistinguishable from those produced by MV in vitro in a monkey kidney cell line (BGMK) that are permissive for wild type MV. However, vMyx63KO failed to replicate in all rabbit cell lines tested, including both primary and established cells lines, as well as cells derived from a variety of tissues. M063R expression was not required for myxoma virus binding, entry or early gene expression, whereas DNA replication was aborted and late genes were not expressed in vMyx63KO infected rabbit cells. Thus, the replication block for vMyx63KO in rabbit cells preceded the stage of late gene expression and DNA replication. Finally, an in vivo pathogenesis study indicated that vMyx63KO failed to cause any signs of classic myxomatosis in infected rabbits, but functioned as a non-replicating vaccine and provided protection for subsequent challenge by wild type myxoma virus. Altogether, these observations demonstrate that M063R plays a critical role in determining the host specificity of myxoma virus in rabbit cells. PMID:17184804

  20. Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex.

    Science.gov (United States)

    Tian, Miaoying; Sasvari, Zsuzsanna; Gonzalez, Paulina Alatriste; Friso, Giulia; Rowland, Elden; Liu, Xiao-Min; van Wijk, Klaas J; Nagy, Peter D; Klessig, Daniel F

    2015-04-01

    Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA's direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (-)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication. PMID:25584724

  1. 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...

  2. Viral hemorrhagic fevers of animals caused by DNA viruses

    Science.gov (United States)

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

  3. Hepatitis B virus replication in acute glomerulonephritis with chronic active hepatitis.

    OpenAIRE

    Cadrobbi, P; Bortolotti, F; Zacchello, G.; Rinaldi, R; Armigliato, M; Realdi, G

    1985-01-01

    A 3 year old boy who had chronic active hepatitis type B with features of ongoing liver damage and active virus replication, developed acute membranous glomerulonephritis two years after the clinical onset of liver disease, when both hepatitis B e antigen and antibody were detectable in serum. After withdrawal of short term steroid treatment and resolution of hepatitis B virus replication, both glomerulonephritis and chronic hepatitis went into remission. Some months later hepatitis B surface...

  4. Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication.

    OpenAIRE

    Snapka, R M

    1986-01-01

    I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers ...

  5. Local Production of Tumor Necrosis Factor Encoded by Recombinant Vaccinia Virus is Effective in Controlling Viral Replication in vivo

    Science.gov (United States)

    Sambhi, Sharan K.; Kohonen-Corish, Maija R. J.; Ramshaw, Ian A.

    1991-05-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-α. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-α during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo.

  6. The "tobacco mosaic virus" 126-kDa protein associated with virus replication and movement suppresses RNA silencing

    Science.gov (United States)

    Systemic symptoms induced on "Nicotiana tabacum" cv. Xanthi by "Tobacco mosaic virus" (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins, proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing c...

  7. 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.

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

    International Nuclear Information System (INIS)

    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

  9. Genistein inhibits the replication of avian leucosis virus subgroup J in DF-1 cells.

    Science.gov (United States)

    Qian, Kun; Gao, Ai-jun; Zhu, Ming-yue; Shao, Hong-xia; Jin, Wen-jie; Ye, Jian-qiang; Qin, Ai-jian

    2014-11-01

    To investigate the antiviral effects of genistein on the replication of avian leukosis virus subgroup J (ALV-J) in DF-1 cells, the cells were treated with genistein at different time points and the antiviral effects were examined by using a variety of assays. We determined that genistein strongly inhibited viral gene expression and decreased the viral protein level in the cell supernatant and the cytoplasm without alerting virus receptor expression and viral attachment. We also observed that genistein was not found to interfere with virus entry, but significantly inhibited both viral gene transcriptions at 24h post infection and virus release, which indicate that genistein exerts its inhibitory effects on the late phase of ALV-J replicative cycle. These results demonstrate that genistein effectively block ALV-J replication by inhibiting virus transcription and release in DF-1 cells, which may be useful for therapeutic drug design. PMID:25197039

  10. 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.

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

    International Nuclear Information System (INIS)

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

  12. Genes and sequences involved in the replication of cowpea mosaic virus RNAs.

    NARCIS (Netherlands)

    Eggen, R.

    1989-01-01

    The aim of the studies described in this thesis was to gain more insight in the complex molecular mechanisms underlying the RNA replication of the cowpea mosaic virus genome. Previously the replication of CPMV RNA has been examined extensively with crude membrane fractions prepared from CPMV inf

  13. 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.

  14. 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......). Like the original RFB osteoma virus stock, viruses derived from the molecular RFB clones induced multiple osteomas in mice of the CBA/Ca strain. The cloned RFB viruses were indistinguishable by restriction enzyme analysis and by nucleotide sequence analysis of their long-terminal-repeat regions and...

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

    OpenAIRE

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

    2012-01-01

    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 a...

  16. Replication of human herpes virus 1 (HHV-1)as a ubiqui-tous virus:A mini review

    Institute of Scientific and Technical Information of China (English)

    Mohammad Derakhshan

    2008-01-01

    Human herpes viruses cause a range of human disorders including cold sores,roseola,genital warts and most importantly,tumours.These viruses cause chronic,latent and recurrent infections.Among them HHV-1 ,an alpha-herpesvirus could become latent after a primary infection,becoming reactivated after later provocation. Epidemically,they are found everywhere and are neurotropic.They also have a rapid and highly regulated rep-lication cycle and usually a broad host and cell range.This article summarizes and focuses on replication strat-egies of the virus.

  17. Guanylylation-competent replication proteins of Tomato mosaic virus are disulfide-linked.

    Science.gov (United States)

    Nishikiori, Masaki; Meshi, Tetsuo; Ishikawa, Masayuki

    2012-12-01

    The 130-kDa and 180-kDa replication proteins of Tomato mosaic virus (ToMV) covalently bind guanylate and transfer it to the 5' end of RNA to form a cap. We found that guanylylation-competent ToMV replication proteins are in membrane-bound, disulfide-linked complexes. Guanylylation-competent replication proteins of Brome mosaic virus and Cucumber mosaic virus behaved similarly. To investigate the roles of disulfide bonding in the functioning of ToMV replication proteins, each of the 19 cysteine residues in the 130-kDa protein was replaced by a serine residue. Interestingly, three mutant proteins (C179S, C186S and C581S) failed not only to be guanylylated, but also to bind to the replication template and membranes. These mutants could trans-complement viral RNA replication. Considering that ToMV replication proteins recognize the replication templates, bind membranes, and are guanylylated in the cytoplasm that provides a reducing condition, we discuss the roles of cysteine residues and disulfide bonds in ToMV RNA replication. PMID:23062762

  18. Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses.

    Science.gov (United States)

    Cheng, Li-Lin; Luk, Yan-Yeung; Murphy, Christopher J; Israel, Barbara A; Abbott, Nicholas L

    2005-12-01

    We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using

  19. 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.

  20. 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 evalua

  1. Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication

    Science.gov (United States)

    Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus, of the family Picornaviridae. Infection of cells with positive-strand RNA viruses results in a rearrangement of intracellular membranes into viral replication complexes. However, the origin of these membranes remains u...

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

    International Nuclear Information System (INIS)

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

    Science.gov (United States)

    Nair, Vidya P; Anang, Saumya; Subramani, Chandru; Madhvi, Abhilasha; Bakshi, Karishma; Srivastava, Akriti; Shalimar; Nayak, Baibaswata; Ct, Ranjith Kumar; Surjit, Milan

    2016-04-01

    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 model of the

  4. Identification and characterization of the role of c-terminal Src kinase in dengue virus replication.

    Science.gov (United States)

    Kumar, Rinki; Agrawal, Tanvi; Khan, Naseem Ahmed; Nakayama, Yuji; Medigeshi, Guruprasad R

    2016-01-01

    We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication. PMID:27457684

  5. 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.

  6. 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; Typsteen, Wim; Bosque, Alberto; Vanderkerckhove, Linos; Planelles, Vicente

    2016-02-01

    HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput. PMID:26171776

  7. Vaccinia virus replication is not affected by APOBEC3 family members

    Directory of Open Access Journals (Sweden)

    Sutter Gerd

    2006-10-01

    Full Text Available Abstract Background The APOBEC3G protein represents a novel innate defense mechanism against retroviral infection. It facilitates the deamination of the cytosine residues in the single stranded cDNA intermediate during early steps of retroviral infection. Most poxvirus genomes are relatively A/T-rich, which may indicate APOBEC3G-induced mutational pressure. In addition, poxviruses replicate exclusively in the cytoplasm where APOBEC3G is located. It was therefore tempting to analyze whether vaccinia virus replication is affected by APOBEC3G. Results The replication of vaccinia virus, a prototype poxvirus, was not, however, inhibited in APOBEC3G-expressing cells, nor did other members of the APOBEC3 family alter vaccinia virus replication. HIV counteracts APOBEC3G by inducing its degradation. However, Western blot analysis showed that the levels of APOBEC3G protein were not affected by vaccinia virus infection. Conclusion The data indicate that APOBEC3G is not a restriction factor for vaccinia virus replication nor is vaccinia virus able to degrade APOBEC3G.

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

    International Nuclear Information System (INIS)

    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

  9. 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

  10. 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.

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

    OpenAIRE

    Liu, Chengke; Richard S Nelson

    2013-01-01

    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,...

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

    Directory of Open Access Journals (Sweden)

    Denise Meyer

    Full Text Available 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.

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

    Science.gov (United States)

    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. PMID:26654952

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

    OpenAIRE

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-01-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity...

  15. Inhibition of influenza virus replication by targeting broad host cell pathways.

    Directory of Open Access Journals (Sweden)

    Isabelle Marois

    Full Text Available Antivirals that are currently used to treat influenza virus infections target components of the virus which can mutate rapidly. Consequently, there has been an increase in the number of resistant strains to one or many antivirals in recent years. Here we compared the antiviral effects of lysosomotropic alkalinizing agents (LAAs and calcium modulators (CMs, which interfere with crucial events in the influenza virus replication cycle, against avian, swine, and human viruses of different subtypes in MDCK cells. We observed that treatment with LAAs, CMs, or a combination of both, significantly inhibited viral replication. Moreover, the drugs were effective even when they were administered 8 h after infection. Finally, analysis of the expression of viral acidic polymerase (PA revealed that both drugs classes interfered with early events in the viral replication cycle. This study demonstrates that targeting broad host cellular pathways can be an efficient strategy to inhibit influenza replication. Furthermore, it provides an interesting avenue for drug development where resistance by the virus might be reduced since the virus is not targeted directly.

  16. RPB5-Mediating Protein Suppresses Hepatitis B Virus (HBV Transcription and Replication by Counteracting the Transcriptional Activation of Hepatitis B virus X Protein in HBV Replication Mouse Model

    Directory of Open Access Journals (Sweden)

    Zhou

    2015-09-01

    Full Text Available Background RPB5-Mediating protein (RMP is associated with the RNA polymerase II subunit RPB5. This protein functionally counteracts the transcriptional activation of Hepatitis B Virus X protein (HBx by competitively binding to the RPB5; however, the effects of RMP on Hepatitis B virus (HBV transcription and replication remain unknown. Objectives The purpose of this study was to investigate the effect of RMP on viral transcription and replication in vivo by using the hydrodynamic-based HBV replication mouse model. Materials and Methods Male balb/c mice were transfected with wild type (1.2 wt or the HBx minus HBV plasmids (1.2x (- with or without HBx and RMP, to establish an HBV replication mouse model by hydrodynamic injection through the tail vein. The HBV RNA and HBV DNA replication intermediates (RI were analyzed in the liver. Results RPB5-Mediating protein could inhibit HBV transcription and replication in groups transfected with the 1.2 wt and HBx. The inhibitory effect disappeared in the 1.2x (- groups, yet it reappeared in the groups co-transfected with 1.2x (- and HBx. An inhibitory effect was indicated at a low dose of RMP (0.3 ug, 0.5 ug and 0.7 ug compared to the control group and groups that had received high doses of RMP. Conclusions Our study demonstrated that a low dose of RMP could inhibit HBV transcription and replication, which is dependent on the appearance of HBx in vivo.

  17. Replication of Chinese hamster embryo cells transformed by temperature-sensitive T-antigen mutants of simian virus 40.

    OpenAIRE

    Robinson, C C; Swartzendruber, D E; Lehman, J M

    1980-01-01

    Chinese hamster embryo cells transformed by simian virus 40 temperature-sensitive T-antigen mutants replicated when confluent at 40.5 degrees C, regardless of the selection method, selection temperature, or virus strain used.

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

    Directory of Open Access Journals (Sweden)

    Nickolay Neznanov

    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.

  19. 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.

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

    International Nuclear Information System (INIS)

    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

  1. Mechanisms of Cellular Membrane Reorganization to Support Hepatitis C Virus Replication.

    Science.gov (United States)

    Wang, Hongliang; Tai, Andrew W

    2016-01-01

    Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication termed the membranous web (MW). Assembling replication factors at a membranous structure might facilitate the processes necessary for genome replication and packaging and shield viral components from host innate immune defenses. The biogenesis of the HCV MW is a complex process involving a concerted effort of HCV nonstructural proteins with a growing list of host factors. Although a comprehensive understanding of MW formation is still missing, a number of important viral and host determinants have been identified. This review will summarize the recent studies that have led to our current knowledge of the role of viral and host factors in the biogenesis of the MWs and discuss how HCV uses this specialized membrane structure for its replication. PMID:27213428

  2. Mechanisms of Cellular Membrane Reorganization to Support Hepatitis C Virus Replication

    Science.gov (United States)

    Wang, Hongliang; Tai, Andrew W.

    2016-01-01

    Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication termed the membranous web (MW). Assembling replication factors at a membranous structure might facilitate the processes necessary for genome replication and packaging and shield viral components from host innate immune defenses. The biogenesis of the HCV MW is a complex process involving a concerted effort of HCV nonstructural proteins with a growing list of host factors. Although a comprehensive understanding of MW formation is still missing, a number of important viral and host determinants have been identified. This review will summarize the recent studies that have led to our current knowledge of the role of viral and host factors in the biogenesis of the MWs and discuss how HCV uses this specialized membrane structure for its replication. PMID:27213428

  3. Interaction between human immunodeficiency virus and Toxoplasma gondii replication in dually infected monocytoid cells.

    OpenAIRE

    Welker, Y; Molina, J. M.; Poirot, C.; Ferchal, F; Decazes, J M; Lagrange, P.; Derouin, F.

    1993-01-01

    THP-1 monocytoid cells, either not infected or chronically infected with human immunodeficiency virus type 1 (HIV-1), were challenged with Toxoplasma gondii. Parasitic growth, as assessed by trophozoite counting and measurement of supernatant p30 membrane antigen, was similar in HIV-infected and noninfected THP-1 cells. Also, T. gondii did not affect HIV replication. These experiments therefore failed to demonstrate any interaction between HIV-1 and T. gondii replication in concurrently infec...

  4. Chikungunya Virus Replication in Salivary Glands of the Mosquito Aedes albopictus

    Directory of Open Access Journals (Sweden)

    Anubis Vega-Rúa

    2015-11-01

    Full Text Available Chikungunya virus (CHIKV is an emerging arbovirus transmitted to humans by mosquitoes such as Aedes albopictus. To be transmitted, CHIKV must replicate in the mosquito midgut, then disseminate in the hemocele and infect the salivary glands before being released in saliva. We have developed a standardized protocol to visualize viral particles in the mosquito salivary glands using transmission electron microscopy. Here we provide direct evidence for CHIKV replication and storage in Ae. albopictus salivary glands.

  5. 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

  6. Quantitative Analysis of Replication and Tropisms of Dengue Virus Type 2 in Aedes albopictus

    OpenAIRE

    Zhang, Meichun; Zheng, Xiaoying; Wu, Yu; Gan, Ming; He, Ai; Li, Zhuoya; Liu, Jing; Zhan, Ximei

    2010-01-01

    Dengue virus serotype 2 (DENV-2) RNA replication profiles and tropisms were studied by using quantitative RT-PCR (q-RTPCR) in intrathoracically infected Aedes albopictus. The virus RNA replication profiles were diverse in mosquito organs. In fat body, brain, salivary gland, and malpighian tubes, it peaked at 8, 23, 23, and 27 days post-infection, respectively, and then, all declined. In midgut, it increased all the time and had no trend of decline. In ovary, it had no apparent increase. Subse...

  7. In vitro inhibition of human influenza A virus replication by chloroquine

    OpenAIRE

    Loh Jin; Chew Janet; Ooi Eng; Chua Robert CS

    2006-01-01

    Abstract Chloroquine is a 9-aminoquinolone with well-known anti-malarial effects. It has biochemical properties that could be applied to inhibit viral replication. We report here that chloroquine is able to inhibit influenza A virus replication, in vitro, and the IC50s of chloroquine against influenza A viruses H1N1 and H3N2 are lower than the plasma concentrations reached during treatment of acute malaria. The potential of chloroquine to be added to the limited range of anti-influenza drugs ...

  8. In vitro inhibition of human influenza A virus replication by chloroquine

    Directory of Open Access Journals (Sweden)

    Loh Jin

    2006-05-01

    Full Text Available Abstract Chloroquine is a 9-aminoquinolone with well-known anti-malarial effects. It has biochemical properties that could be applied to inhibit viral replication. We report here that chloroquine is able to inhibit influenza A virus replication, in vitro, and the IC50s of chloroquine against influenza A viruses H1N1 and H3N2 are lower than the plasma concentrations reached during treatment of acute malaria. The potential of chloroquine to be added to the limited range of anti-influenza drugs should be explored further, particularly since antiviral drugs play a vital role in influenza pandemic preparedness.

  9. 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.

  10. Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA

    Directory of Open Access Journals (Sweden)

    Diamond Michael S

    2005-06-01

    Full Text Available Abstract Background West Nile virus is an emerging human pathogen for which specific antiviral therapy has not been developed. Recent studies have suggested that RNA interference (RNAi has therapeutic potential as a sequence specific inhibitor of viral infection. Here, we examine the ability of exogenous small interfering RNAs (siRNAs to block the replication of West Nile virus in human cells. Results WNV replication and infection was greatly reduced when siRNA were introduced by cytoplasmic-targeted transfection prior to but not after the establishment of viral replication. WNV appeared to evade rather than actively block the RNAi machinery, as sequence-specific reduction in protein expression of a heterologous transgene was still observed in WNV-infected cells. However, sequence-specific decreases in WNV RNA were observed in cells undergoing active viral replication when siRNA was transfected by an alternate method, electroporation. Conclusion Our results suggest that actively replicating WNV RNA may not be exposed to the cytoplasmic RNAi machinery. Thus, conventional lipid-based siRNA delivery systems may not be adequate for therapy against enveloped RNA viruses that replicate in specialized membrane compartments.

  11. Hsp90 inhibitors reduce influenza virus replication in cell culture

    International Nuclear Information System (INIS)

    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

  12. The inhibitory effect of essential oils on herpes simplex virus type-1 replication in vitro.

    Science.gov (United States)

    Minami, Masato; Kita, Masakazu; Nakaya, Takaaki; Yamamoto, Toshiro; Kuriyama, Hiroko; Imanishi, Jiro

    2003-01-01

    The antiviral effect of 12 essential oils on herpes simplex virus type-1 (HSV-1) replication was examined in vitro. The replication ability of HSV-1 was suppressed by incubation of HSV-1 with 1% essential oils at 4 C for 24 hr. Especially, lemongrass completely inhibited the viral replication even at a concentration of 0.1%, and its antiviral activity was dependent on the concentrations of the essential oil. When Vero cells were treated with the essential oil before or after viral adsorption, no antiviral activity was found, which suggests that the antiviral activity of essential oils including lemongrass may be due to the direct interaction with virions. PMID:14584615

  13. A Nucleotide Binding Motif in Hepatitis C Virus (HCV) NS4B Mediates HCV RNA Replication

    OpenAIRE

    Einav, Shirit; Elazar, Menashe; Danieli, Tsafi; Glenn, Jeffrey S.

    2004-01-01

    Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral s...

  14. Attenuation of Rabies Virus Replication and Virulence by Picornavirus Internal Ribosome Entry Site Elements▿

    OpenAIRE

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

    2008-01-01

    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 reporte...

  15. Drosophila RNAi screen identifies host genes important for influenza virus replication

    OpenAIRE

    Hao, Linhui; Sakurai, Akira; Watanabe, Tokiko; Sorensen, Ericka; Nidom, Chairul A.; Newton, Michael A.; Ahlquist, Paul; Kawaoka, Yoshihiro

    2008-01-01

    All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila1 that can be used to identify host gene...

  16. A Nucleotide Binding Motif in Hepatitis C Virus (HCV) NS4B Mediates HCV RNA Replication

    Science.gov (United States)

    Einav, Shirit; Elazar, Menashe; Danieli, Tsafi; Glenn, Jeffrey S.

    2004-01-01

    Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies. PMID:15452248

  17. 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. PMID:26748656

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

    Science.gov (United States)

    Ocazionez, Raquel Elvira; Meneses, Rocio; Torres, Flor Angela; Stashenko, Elena

    2010-05-01

    The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37 masculineC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 microg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 microg/mL and between 1.9-33.7 microg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell. PMID:20512244

  19. 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.

  20. Functionality of host proteins in Cucumber mosaic virus replication: GAPDH is obligatory to promote interaction between replication-associated proteins.

    Science.gov (United States)

    Chaturvedi, Sonali; Seo, Jang-Kyun; Rao, A L N

    2016-07-01

    Here, we evaluated the role of two host proteins, a Bromo domain containing RNA binding protein (BRP1) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in the replication of Cucumber mosaic virus (CMV). LC-MS/MS analysis of host/viral proteins pull down against BRP1 from CMV-infected plants co-infiltrated with BRP1-FLAG agroconstruct identified that BRP1 specifically interacts with a ten amino acid motif (843-SPQDVVPLVR-852) encompassing the helicase domain of replicase protein p1a. The interaction between BRP1 and p1a was subsequently confirmed using a BiFC assay. Among fourteen other host proteins identified to interact with BRP1 during CMV infection, six were found to block accumulation of viral progeny in Arabidopsis thaliana lines defective in each of these host proteins. Additional BiFC assays followed by trans-complementation assays identified that plant lines defective in the expression of GAPDH blocked CMV replication by interfering with p1a:p2a interaction. Distinct roles of BRP1 and GAPDH in the replication of CMV are discussed. PMID:27077230

  1. 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.

  2. Ferrets as a Novel Animal Model for Studying Human Respiratory Syncytial Virus Infections in Immunocompetent and Immunocompromised Hosts

    Science.gov (United States)

    Stittelaar, Koert J.; de Waal, Leon; van Amerongen, Geert; Veldhuis Kroeze, Edwin J.B.; Fraaij, Pieter L.A.; van Baalen, Carel A.; van Kampen, Jeroen J.A.; van der Vries, Erhard; Osterhaus, Albert D.M.E.; de Swart, Rik L.

    2016-01-01

    Human respiratory syncytial virus (HRSV) is an important cause of severe respiratory tract disease in immunocompromised patients. Animal models are indispensable for evaluating novel intervention strategies in this complex patient population. To complement existing models in rodents and non-human primates, we have evaluated the potential benefits of an HRSV infection model in ferrets (Mustela putorius furo). Nine- to 12-month-old HRSV-seronegative immunocompetent or immunocompromised ferrets were infected with a low-passage wild-type strain of HRSV subgroup A (105 TCID50) administered by intra-tracheal or intra-nasal inoculation. Immune suppression was achieved by bi-daily oral administration of tacrolimus, mycophenolate mofetil, and prednisolone. Throat and nose swabs were collected daily and animals were euthanized four, seven, or 21 days post-infection (DPI). Virus loads were determined by quantitative virus culture and qPCR. We observed efficient HRSV replication in both the upper and lower respiratory tract. In immunocompromised ferrets, virus loads reached higher levels and showed delayed clearance as compared to those in immunocompetent animals. Histopathological evaluation of animals euthanized 4 DPI demonstrated that the virus replicated in the respiratory epithelial cells of the trachea, bronchi, and bronchioles. These animal models can contribute to an assessment of the efficacy and safety of novel HRSV intervention strategies. PMID:27314379

  3. Ferrets as a Novel Animal Model for Studying Human Respiratory Syncytial Virus Infections in Immunocompetent and Immunocompromised Hosts

    Directory of Open Access Journals (Sweden)

    Koert J. Stittelaar

    2016-06-01

    Full Text Available Human respiratory syncytial virus (HRSV is an important cause of severe respiratory tract disease in immunocompromised patients. Animal models are indispensable for evaluating novel intervention strategies in this complex patient population. To complement existing models in rodents and non-human primates, we have evaluated the potential benefits of an HRSV infection model in ferrets (Mustela putorius furo. Nine- to 12-month-old HRSV-seronegative immunocompetent or immunocompromised ferrets were infected with a low-passage wild-type strain of HRSV subgroup A (105 TCID50 administered by intra-tracheal or intra-nasal inoculation. Immune suppression was achieved by bi-daily oral administration of tacrolimus, mycophenolate mofetil, and prednisolone. Throat and nose swabs were collected daily and animals were euthanized four, seven, or 21 days post-infection (DPI. Virus loads were determined by quantitative virus culture and qPCR. We observed efficient HRSV replication in both the upper and lower respiratory tract. In immunocompromised ferrets, virus loads reached higher levels and showed delayed clearance as compared to those in immunocompetent animals. Histopathological evaluation of animals euthanized 4 DPI demonstrated that the virus replicated in the respiratory epithelial cells of the trachea, bronchi, and bronchioles. These animal models can contribute to an assessment of the efficacy and safety of novel HRSV intervention strategies.

  4. Dengue Virus Reporter Replicon is a Valuable Tool for Antiviral Drug Discovery and Analysis of Virus Replication Mechanisms

    Science.gov (United States)

    Kato, Fumihiro; Hishiki, Takayuki

    2016-01-01

    Dengue, the most prevalent arthropod-borne viral disease, is caused by the dengue virus (DENV), a member of the Flaviviridae family, and is a considerable public health threat in over 100 countries, with 2.5 billion people living in high-risk areas. However, no specific antiviral drug or licensed vaccine currently targets DENV infection. The replicon system has all the factors needed for viral replication in cells. Since the development of replicon systems, transient and stable reporter replicons, as well as reporter viruses, have been used in the study of various virological aspects of DENV and in the identification of DENV inhibitors. In this review, we summarize the DENV reporter replicon system and its applications in high-throughput screening (HTS) for identification of anti-DENV inhibitors. We also describe the use of this system in elucidation of the mechanisms of virus replication and viral dynamics in vivo and in vitro. PMID:27164125

  5. Dengue Virus Reporter Replicon is a Valuable Tool for Antiviral Drug Discovery and Analysis of Virus Replication Mechanisms.

    Science.gov (United States)

    Kato, Fumihiro; Hishiki, Takayuki

    2016-01-01

    Dengue, the most prevalent arthropod-borne viral disease, is caused by the dengue virus (DENV), a member of the Flaviviridae family, and is a considerable public health threat in over 100 countries, with 2.5 billion people living in high-risk areas. However, no specific antiviral drug or licensed vaccine currently targets DENV infection. The replicon system has all the factors needed for viral replication in cells. Since the development of replicon systems, transient and stable reporter replicons, as well as reporter viruses, have been used in the study of various virological aspects of DENV and in the identification of DENV inhibitors. In this review, we summarize the DENV reporter replicon system and its applications in high-throughput screening (HTS) for identification of anti-DENV inhibitors. We also describe the use of this system in elucidation of the mechanisms of virus replication and viral dynamics in vivo and in vitro. PMID:27164125

  6. Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

    OpenAIRE

    Mena, Ignacio; Jambrina, Enrique; Albo, Carmen; Perales, Beatriz; Ortín, Juan; Arrese, Marta; Vallejo, Dolores; Portela, Agustín

    1999-01-01

    The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were hig...

  7. GB Virus C Proteins interfere with the Replication of HIV-1

    OpenAIRE

    Jung, Susan

    2010-01-01

    GB Virus C (GBV-C) was discovered in 1995 within the search for new hepatitis viruses. The human apathogenic enveloped RNA virus belongs to the family of the Flaviviridae and replicates primarily in lymphocytes. Transmission occurs mainly by sexual or parenteral exposure. In developed countries up to 6% of the healthy population and up to 40% of multiply exposed individuals, such as HIV patients, are viremic for GBV-C. Since the late 1990s, GBV-C has been investigated in the context of HIV. U...

  8. Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and pro-inflammatory dysregulation

    OpenAIRE

    Pengxiang Chang; Kuchipudi, Suresh V; Kenneth H. Mellits; Sujith Sebastian; Joe James; Jinhua Liu; Holly Shelton; Kin-Chow Chang

    2015-01-01

    Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) in...

  9. Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

    OpenAIRE

    Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; YE, CHENGJIN; Ruan, Xizhen; Zhou, Jiyong

    2013-01-01

    Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and ma...

  10. Induction of the Sulfolobus shibatae virus SSV1 DNA replication by mitomycin C

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The temperate virus SSV1 from the hyperthermophilic archaeon Sulfolobus shibatae provides a useful model system for the study of archaeal DNA replication. Southern hybridization showed that SSV1 existed primarily as a provirus in its host that was grown without shaking. Upon UV or mitomycin C induction, the cellular level of free SSV1 DNA increased drastically whereas that of integrated viral DNA remained unchanged. The results of mitomycin C induction were more reproducible than those of UV induction. We found that, when the cells that had been grown without shaking were shaken, the replication of SSV1 DNA was also induced. Based on our results, we developed a method for the induction of SSV1 DNA replication by mitomycin C. When the S. shibatae virus production was induced using this method, the cellular level of free SSV1 DNA started to increase 10 h after induction, and peaked after 12-15 h. A fully induced S. shibatae cell contained ~50 molecules of free SSV1 DNA. The development of this induction method and the description of the process of SSV1 DNA replication following induction are valuable to the analysis of the origin and mode of replication of the virus.

  11. 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. PMID:27039024

  12. Purified tomato spotted wilt virus particles support both genome replication and transcription

    NARCIS (Netherlands)

    Knippenberg, van I.; Goldbach, R.W.; Kormelink, R.

    2002-01-01

    Purified Tomato spotted wilt virus particles were shown to support either genome replication or transcription in vitro, depending on the conditions chosen. Transcriptional activity was observed only upon addition of rabbit reticulocyte lysate, indicating a dependence on translation. Under these cond

  13. Changes in H3 influenza A virus receptor specificity during replication in humans.

    Science.gov (United States)

    Ryan-Poirier, K; Suzuki, Y; Bean, W J; Kobasa, D; Takada, A; Ito, T; Kawaoka, Y

    1998-08-01

    Influenza A viruses of the H3 subtype caused the 1968 Hong Kong pandemic, the hemagglutinin (HA) gene being introduced into humans following a reassortment event with an avian virus. Receptor specificity and serum inhibitor sensitivity of the HA of influenza A viruses are linked to the host species. Human H3 viruses preferentially recognize N-acetyl sialic acid linked to galactose by alpha2,6 linkages (Neu5Acalpha2,6Gal) and are sensitive to serum inhibitors, whereas avian and equine viruses preferentially recognize Neu5Acalpha2,3Gal linkages and are resistant to serum inhibitors. We have examined the receptor specificity and serum inhibitor sensitivity of H3 human influenza A viruses from the time they were introduced into the human population to gain insight into the mechanism of viral molecular evolution and host tropism. All of the viruses were sensitive to neutralization and hemagglutination inhibition by horse serum. Early H3 viruses were resistant to pig and rabbit serum inhibitors. Viruses isolated after 1977 were uniformly sensitive to inhibition by pig and rabbit sera. The recognition of Neu5Acalpha2,3Gal or Neu5Acalpha2,6Gal linkages was not correlated with the serum sensitivity. These data showed that the receptor specificity of HA, measured as inhibitor sensitivity, has changed during replication in humans since its introduction from an avian virus. PMID:9783465

  14. Delayed Newcastle disease virus replication using RNA interference to target the nucleoprotein.

    Science.gov (United States)

    Hutcheson, Jessica M; Susta, Leonardo; Stice, Steven L; Afonso, Claudio L; West, Franklin D

    2015-07-01

    Each year millions of chickens die from Newcastle disease virus (NDV) worldwide leading to severe economic and food losses. Current vaccination campaigns have limitations especially in developing countries, due to elevated costs, need of trained personnel for effective vaccine administration, and functional cold chain network to maintain vaccine viability. These problems have led to heightened interest in producing new antiviral strategies, such as RNA interference (RNAi). RNAi methodology is capable of substantially decreasing viral replication at a cellular level, both in vitro and in vivo. In this study, we utilize microRNA (miRNA)-expressing constructs (a type of RNA interference) in an attempt to target and knockdown five NDV structural RNAs for nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), and large (L) protein genes. Immortalized chicken embryo fibroblast cells (DF-1) that transiently expressed miRNA targeting NP mRNA, showed increased resistance to NDV-induced cytopathic effects, as determined by cell count, relative to the same cells expressing miRNA against alternative NDV proteins. Upon infection with NDV, DF-1 cells constitutively expressing the NP miRNA construct had improved cell survival up to 48 h post infection (h.p.i) and decreased viral yield up to 24 h.p.i. These results suggest that overexpression of the NP miRNA in cells and perhaps live animal may provide resistance to NDV. PMID:26050911

  15. FAT10 Is Critical in Influenza A Virus Replication by Inhibiting Type I IFN.

    Science.gov (United States)

    Zhang, Yanli; Tang, Jun; Yang, Ning; Liu, Qiang; Zhang, Qingchao; Zhang, Yanxu; Li, Ning; Zhao, Yan; Li, Shunwang; Liu, Song; Zhou, Huandi; Li, Xiao; Tian, Mingyao; Deng, Jiejie; Xie, Peng; Sun, Yang; Lu, Huijun; Zhang, Michael Q; Jin, Ningyi; Jiang, Chengyu

    2016-08-01

    The H5N1 avian influenza virus causes severe disease and high mortality, making it a major public health concern worldwide. The virus uses the host cellular machinery for several steps of its life cycle. In this report, we observed overexpression of the ubiquitin-like protein FAT10 following live H5N1 virus infection in BALB/c mice and in the human respiratory epithelial cell lines A549 and BEAS-2B. Further experiments demonstrated that FAT10 increased H5N1 virus replication and decreased the viability of infected cells. Total RNA extracted from H5N1 virus-infected cells, but not other H5N1 viral components, upregulated FAT10, and this process was mediated by the retinoic acid-induced protein I-NF-κB signaling pathway. FAT10 knockdown in A549 cells upregulated type I IFN mRNA expression and enhanced STAT1 phosphorylation during live H5N1 virus infection. Taken together, our data suggest that FAT10 was upregulated via retinoic acid-induced protein I and NF-κB during H5N1 avian influenza virus infection. And the upregulated FAT10 promoted H5N1 viral replication by inhibiting type I IFN. PMID:27354218

  16. A nuclear export signal within the structural Gag protein is required for prototype foamy virus replication

    Directory of Open Access Journals (Sweden)

    Coiffic Audrey

    2011-01-01

    Full Text Available Abstract Background The Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV, Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytoplasm for capsid assembly and virus egress. The functional role of this nuclear stage as well as the molecular mechanism(s responsible for Gag nuclear export are not understood. Results We have identified a leptomycin B (LMB-sensitive nuclear export sequence (NES within the N-terminus of PFV Gag that is absolutely required for the completion of late stages of virus replication. Point mutations of conserved residues within this motif lead to nuclear redistribution of Gag, preventing subsequent virus egress. We have shown that a NES-defective PFV Gag acts as a dominant negative mutant by sequestrating its wild-type counterpart in the nucleus. Trans-complementation experiments with the heterologous NES of HIV-1 Rev allow the cytoplasmic redistribution of FV Gag, but fail to restore infectivity. Conclusions PFV Gag-Gag interactions are finely tuned in the cytoplasm to regulate their functions, capsid assembly, and virus release. In the nucleus, we have shown Gag-Gag interactions which could be involved in the nuclear export of Gag and viral RNA. We propose that nuclear export of unspliced and partially spliced PFV RNAs relies on two complementary mechanisms, which take place successively during the replication cycle.

  17. Use of a commercial enzyme immunoassay to monitor dengue virus replication in cultured cells

    Directory of Open Access Journals (Sweden)

    del Angel Rosa M

    2008-04-01

    Full Text Available Abstract Current methods for dengue virus quantitation are either time consuming, technically demanding or costly. As an alternative, the commercial enzyme immunoassay Platelia™ Dengue NS1 AG (Bio-Rad Laboratories was used to monitor semiquantitatively dengue virus replication in cultured cells. The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus. The amount of NS1 detected in the supernatants of infected cells was proportional to the initial MOI used and to the time of post infection harvest. This immunoassay was also able to detect the presence of NS1 in the supernatants of infected human macrophages. Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was readily monitored with the use of this assay. These results suggest that the Platelia™ Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells.

  18. Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

    Energy Technology Data Exchange (ETDEWEB)

    Yakobson, B.; Hrynko, T.A.; Peak, M.J.; Winocour, E.

    1989-03-01

    Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting a requirement for de novo protein synthesis.

  19. Endogenous hepatitis C virus homolog fragments in European rabbit and hare genomes replicate in cell culture.

    Directory of Open Access Journals (Sweden)

    Eliane Silva

    Full Text Available Endogenous retroviruses, non-retroviral RNA viruses and DNA viruses have been found in the mammalian genomes. The origin of Hepatitis C virus (HCV, the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans, remains unclear since its discovery. Here we show that fragments homologous to HCV structural and non-structural (NS proteins present in the European rabbit (Oryctolagus cuniculus and hare (Lepus europaeus genomes replicate in bovine cell cultures. The HCV genomic homolog fragments were demonstrated by RT-PCR, PCR, mass spectrometry, and replication in bovine cell cultures by immunofluorescence assay (IFA and immunogold electron microscopy (IEM using specific MAbs for HCV NS3, NS4A, and NS5 proteins. These findings may lead to novel research approaches on the HCV origin, genesis, evolution and diversity.

  20. 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. PMID:21827337

  1. Multiple proteases in foot-and-mouth disease virus replication.

    OpenAIRE

    Burroughs, J. N.; Sangar, D V; Clarke, B E; Rowlands, D J; Billiau, A; Collen, D

    1984-01-01

    Translation of foot-and-mouth disease virus RNA in a rabbit reticulocyte lysate for short time intervals resulted in the production of the peptides P20a , P16, and P88 (Lab, Lb, and P1) (R. R. Rueckert , Recommendations of the 3rd European Study Group on Molecular Biology of Picornavirus, Urbino , Italy, 1983). If further translation was prevented, the structural protein precursor P88 was not cleaved, even after prolonged incubation. This result indicates that the mechanism of the cleavage be...

  2. Naturally Occurring Animal Models of Human Hepatitis E Virus Infection

    OpenAIRE

    Yugo, Danielle M.; Cossaboom, Caitlin M.; Meng, Xiang-Jin

    2014-01-01

    Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus in the family Hepeviridae. Hepatitis E caused by HEV is a clinically important global disease. There are currently four well-characterized genotypes of HEV in mammalian species, although numerous novel strains of HEV likely belonging to either new genotypes or species have recently been identified from several other animal species. HEV genotypes 1 and 2 are limited to infection in humans, whereas genotypes 3 and 4 infect a...

  3. An avian leukosis virus subgroup J isolate with a Rous sarcoma virus-like 5'-LTR shows enhanced replication capability.

    Science.gov (United States)

    Gao, Yanni; Guan, Xiaolu; Liu, Yongzhen; Li, Xiaofei; Yun, Bingling; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Wang, Xiaomei; Gao, Yulong

    2015-01-01

    Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5'-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5'-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5'-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5'-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro. PMID:25274857

  4. Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene.

    Science.gov (United States)

    Lin, Amy H; Liu, Yanzheng; Burrascano, Cynthia; Cunanan, Kathrina; Logg, Christopher R; Robbins, Joan M; Kasahara, Noriyuki; Gruber, Harry; Ibañez, Carlos; Jolly, Douglas J

    2016-04-01

    We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required. PMID:26918465

  5. Influence of the Leader protein coding region of foot-and-mouth disease virus on virus replication

    DEFF Research Database (Denmark)

    Belsham, Graham

    2013-01-01

    The foot-and-mouth disease virus (FMDV) Leader (L) protein is produced in two forms, Lab and Lb, differing only at their amino-termini, due to the use of separate initiation codons, usually 84 nt apart. It has been shown previously, and confirmed here, that precise deletion of the Lab coding......, in the context of the virus lacking the Lb coding region, was also tolerated by the virus within BHK cells. However, precise loss of the Lb coding sequence alone blocked FMDV replication in primary bovine thyroid cells. Thus, the requirement for the Leader protein coding sequences is highly dependent...... on the nature and extent of the residual Leader protein sequences and on the host cell system used. FMDVs precisely lacking Lb and with the Lab initiation codon modified may represent safer seed viruses for vaccine production....

  6. Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of β-galactosidase.

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    Full Text Available Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV and murine hepatitis coronavirus (MHV.

  7. Coat protein activation of alfalfa mosaic virus replication is concentration dependent.

    Science.gov (United States)

    Guogas, Laura M; Laforest, Siana M; Gehrke, Lee

    2005-05-01

    Alfalfa mosaic virus (AMV) and ilarvirus RNAs are infectious only in the presence of the viral coat protein; therefore, an understanding of coat protein's function is important for defining viral replication mechanisms. Based on in vitro replication experiments, the conformational switch model states that AMV coat protein blocks minus-strand RNA synthesis (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999), while another report states that coat protein present in an inoculum is required to permit minus-strand synthesis (L. Neeleman and J. F. Bol, Virology 254:324-333, 1999). Here, we report on experiments that address these contrasting results with a goal of defining coat protein's function in the earliest stages of AMV replication. To detect coat-protein-activated AMV RNA replication, we designed and characterized a subgenomic luciferase reporter construct. We demonstrate that activation of viral RNA replication by coat protein is concentration dependent; that is, replication was strongly stimulated at low coat protein concentrations but decreased progressively at higher concentrations. Genomic RNA3 mutations preventing coat protein mRNA translation or disrupting coat protein's RNA binding domain diminished replication. The data indicate that RNA binding and an ongoing supply of coat protein are required to initiate replication on progeny genomic RNA transcripts. The data do not support the conformational switch model's claim that coat protein inhibits the initial stages of viral RNA replication. Replication activation may correlate with low local coat protein concentrations and low coat protein occupancy on the multiple binding sites present in the 3' untranslated regions of the viral RNAs. PMID:15827190

  8. Relationship between RNA polymerase II and efficiency of vaccinia virus replication

    International Nuclear Information System (INIS)

    It is clear from previous studies that host transcriptase or RNA polymerase II (pol II) has a role in poxvirus replication. To elucidate the participation of this enzyme further, in this study the authors examined several parameters related to pol II during the cycle of vaccinia virus infection in L-strain fibroblasts, HeLa cells, and L6H9 rat myoblasts. Nucleocytoplasmic transposition of pol II into virus factories and virions was assessed by immunofluorescence and immunoblotting by using anti-pol II immunoglobulin G. RNA polymerase activities were compared in nuclear extracts containing cured enzyme preparations. Rates of translation into cellular or viral polypeptides were ascertained by labeling with [35S]methionine. In L and HeLa cells, which produced vaccinia virus more abundantly, the rate of RNA polymerase and translation in controls and following infection were higher than in myoblasts. The data on synthesis and virus formation could be correlated with observations on transmigration of pol II, which was more efficient and complete in L and HeLa cells. The stimulus for pol II to leave the nucleus required the expression of both early and late viral functions. On the basis of current and past information, the authors suggest that mobilization of pol II depends on the efficiency of vaccinia virus replication and furthermore that control over vaccinia virus production by the host is related to the content or availability (or both) of pol II in different cell types

  9. HIV-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step

    International Nuclear Information System (INIS)

    The inhibitors of HIV-1 protease (PIs) have been designed to block the activity of the viral aspartyl-protease. However, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. Since the replication of many virus species requires the activity of host cell proteases, investigating the effects of PIs on the life cycle of viruses other than HIV would be of interest. Here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. These are unrelated enveloped RNA viruses infecting target cells upon endocytosis and intracellular fusion. The PI-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. These findings would open the way towards the therapeutic use of PIs against enveloped RNA viruses other than HIV.

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

    Directory of Open Access Journals (Sweden)

    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

  11. Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

    Directory of Open Access Journals (Sweden)

    Geiss Brian J

    2009-03-01

    Full Text Available Abstract Background Arthropod-borne viruses (arboviruses can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus, a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition. Results B2 protein expression from SINV (TE/3'2J inhibited the accumulation of non-specific small RNAs in Aedes aegypti mosquito cell culture and virus-specific small RNAs both in infected cell culture and Ae. aegypti mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2 compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in Ae. aegypti. Conclusion We show that RNAi is active in Ae. aegypti cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.

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

    International Nuclear Information System (INIS)

    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

  13. 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.

  14. Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs

    Directory of Open Access Journals (Sweden)

    Meng Qing-Wen

    2011-12-01

    Full Text Available Abstract Background Avian leukosis virus (ALV is a major infectious disease that impacts the poultry industry worldwide. Despite intensive efforts, no effective vaccine has been developed against ALV because of mutations that lead to resistant forms. Therefore, there is a dire need to develop antiviral agents for the treatment of ALV infections and RNA interference (RNAi is considered an effective antiviral strategy. Results In this study, the avian leukosis virus subgroup J (ALV-J proviral genome, including the gag genes, were treated as targets for RNAi. Four pairs of miRNA sequences were designed and synthesized that targeted different regions of the gag gene. The screened target (i.e., the gag genes was shown to effectively suppress the replication of ALV-J by 19.0-77.3%. To avoid the generation of escape variants during virus infection, expression vectors of multi-target miRNAs were constructed using the multi-target serial strategy (against different regions of the gag, pol, and env genes. Multi-target miRNAs were shown to play a synergistic role in the inhibition of ALV-J replication, with an inhibition efficiency of viral replication ranging from 85.0-91.2%. Conclusion The strategy of multi-target miRNAs might be an effective method for inhibiting ALV replication and the acquisition of resistant mutations.

  15. A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.

    Science.gov (United States)

    Miskin, J; Chipchase, D; Rohll, J; Beard, G; Wardell, T; Angell, D; Roehl, H; Jolly, D; Kingsman, S; Mitrophanous, K

    2006-02-01

    Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination. PMID:16208418

  16. 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. PMID:23629709

  17. Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

    Science.gov (United States)

    Chong, Weng Man; Hsu, Shih-Chin; Kao, Wei-Ting; Lo, Chieh-Wen; Lee, Kuan-Ying; Shao, Jheng-Syuan; Chen, Yi-Hung; Chang, Justin; Chen, Steve S-L; Yu, Ming-Jiun

    2016-02-19

    The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. PMID:26702051

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

    International Nuclear Information System (INIS)

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

  19. The intracellular dynamics of hepatitis B virus (HBV) replication with reproduced virion "re-cycling".

    Science.gov (United States)

    Nakabayashi, Jun

    2016-05-01

    Hepatitis B virus (HBV) is a causative agent of hepatitis. Clinical outcome of hepatitis type B depends on the viral titer observed in the peripheral blood of the patient. In the chronic hepatitis patient, production of HBV virion remains low level. On the other hand, the viral load prominently increases in fulminant hepatitis patient as compared with that in the chronic hepatitis patient. We previously proposed a mathematical model describing the intracellular dynamics of HBV replication. Our model clarified that there are two distinguishable replication patterns of HBV named "arrested" and "explosive" replication. In the arrested replication, the amount of virion newly reproduced from an infected cell remains low level, while the amount of virion extremely increases in the explosive replication. Viral load is drastically changed by slight alteration of expression ratio of 3.5kb RNA to 2.4kb mRNA of HBV. Though our model provided the switching mechanism determining the replication pattern of HBV, HBV dynamics is determined by not only the expression pattern of viral genes. In this study, "recycling" of HBV virion in the replication cycle is investigated as a new factor affecting the intracellular dynamics of HBV replication. A part of newly produced virion of HBV is reused as a core particle that is a resource of HBV replication. This recycling of HBV virion lowers the threshold for the explosive replication when waiting time for the next cycle of the replication is large. It is seemingly contradicting that prominent production of HBV is caused by large recycling rate and small release rate of HBV virion from infected cell to extracellular space. But the recycling of HBV virion can contribute to the positive feedback cycle of HBV replication for the explosive replication to accumulate the core particle as a resource of HBV replication in an infected cell. Accumulation of core particle in the infected cell can be risk factor for the exacerbation of hepatitis rather

  20. Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection.

    Science.gov (United States)

    Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N; Gestwicki, Jason E; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith

    2015-11-19

    Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals. PMID:26582131

  1. The Environmental Pollutant Cadmium Promotes Influenza Virus Replication in MDCK Cells by Altering Their Redox State

    Directory of Open Access Journals (Sweden)

    Lucia Nencioni

    2013-02-01

    Full Text Available Cadmium (Cd is a toxic heavy metal that is considered an environmental contaminant. Several sources of human exposure to Cd, including employment in primary metal industries, production of certain batteries, foods, soil and cigarette smoke, are known. Its inhalation has been related to different respiratory diseases and toxic effects, among which alterations of the physiological redox state in individuals exposed to the metal have been described. Host-cell redox changes characteristic of oxidative stress facilitate the progression of viral infection through different mechanisms. In this paper, we have demonstrated that pre-treatment with CdCl2 of MDCK cells increased influenza virus replication in a dose-dependent manner. This phenomenon was related to increased viral protein expression (about 40% compared with untreated cells. The concentration of CdCl2, able to raise the virus titer, also induced oxidative stress. The addition of two antioxidants, a glutathione (GSH derivative or the GSH precursor, N-acetyl-L-cysteine, to Cd pre-treated and infected cells restored the intracellular redox state and significantly inhibited viral replication. In conclusion, our data demonstrate that Cd-induced oxidative stress directly increases the ability of influenza virus to replicate in the host-cell, thus suggesting that exposure to heavy metals, such as this, could be a risk factor for individuals exposed to a greater extent to the contaminant, resulting in increased severity of virus-induced respiratory diseases.

  2. 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...... recombination within E. coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as...

  3. Human herpesvirus 6 inhibits human immunodeficiency virus type 1 replication in cell culture.

    OpenAIRE

    Levy, J A; Landay, A.; Lennette, E T

    1990-01-01

    The SF strain of human herpesvirus 6 (HHV-6SF) isolated from the saliva of a human immunodeficiency virus (HIV)-infected individual was shown to inhibit HIV type 1 (HIV-1) replication in both peripheral blood mononuclear cells and purified CD4+ lymphocytes. This suppression of HIV-1 replication led to decreased cytopathic effects of HIV-1 and prolonged survival of CD4+ cells in culture. Even low levels of HHV-6 added to peripheral blood mononuclear cells showed an inhibitory effect on HIV-1 r...

  4. 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.

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

    International Nuclear Information System (INIS)

    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

  6. 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

    2016-06-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

  7. 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

  8. 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

  9. Replicative intermediates of porcine circovirus in animal tissue cultured cells or in bacteria undergoing copy-release replication

    Science.gov (United States)

    Porcine circovirus (PCV) has been assumed to replicate its genome via the rolling-circle replication (RCR) mechanism because it encodes a Rep protein that contains several amino acid motifs commonly found in other RCR biological systems. Two proteins, Rep and Rep', are essential for PCV DNA replicat...

  10. 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.

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

    International Nuclear Information System (INIS)

    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

  12. 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.

  13. Animal models of human respiratory syncytial virus disease

    NARCIS (Netherlands)

    R.A. Bem; J.B. Domachowske; H.F. Rosenberg

    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 n

  14. Reverse genetics with animal viruses. NSV reverse genetics

    International Nuclear Information System (INIS)

    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)

  15. Inhibition of sandfly fever Sicilian virus (Phlebovirus) replication in vitro by antiviral compounds.

    Science.gov (United States)

    Crance, J M; Gratier, D; Guimet, J; Jouan, A

    1997-01-01

    Sandfly fever Sicilian virus (SFSV) was used in our laboratory to screen antiviral substances active toward viruses of the Bunyaviridae family. Antiviral activity was estimated by the reduction of the cytopathic effect of SFSV on infected Vero cells. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion. The specificity of action of each tested compound was estimated by the selectivity index (CD50/ED50). Selectivity indices of human recombinant interferon-alpha (IFN alpha) (Roferon and Introna), iota-, kappa- and lambda- carrageenans, fucoidan and 6-azauridine were much higher than that of ribavirin, the only antiviral substance which has been previously investigated for its inhibitory effects on Phlebovirus infections. Other compounds showed significant antiviral activity: glycyrrhizin, suramin sodium, dextran sulphate and pentosan polysulphate. All these compounds caused a concentration-dependent reduction in the virus yield. Ribavirin, 6-azauridine and IFN alpha have been shown to inhibit a late step of the virus replicative cycle, whereas glycyrrhizin and suramin sodium were active at an early step and the sulphated polysaccharides inhibited adsorption of SFSV on the cells. The antiviral compounds selected in this study as specific inhibitors of in vitro replication of SFSV are promising candidates for the chemotherapy of haemorrhagic fevers caused by viruses of the Bunyaviridae family. The combination of IFN alpha and ribavirin, which showed a synergistic antiviral effect, should be evaluated for the treatment of these infections. PMID:9403935

  16. Characterization of Virus host-interactions that regulate HIV-1 replication

    OpenAIRE

    Votteler, Jörg

    2009-01-01

    Retroviruses like the human immunodeficiency virus type 1 (HIV-1) are obligate cellular parasites that depend on host cell factors for their replication. The aim of this study was to characterize two specific cases of these numerous virus-host interactions. First, the previously identified interaction between the cellular peptidyl-prolyl cis/trans isomerase cyclophilin A (CypA) and the HIV-1 accessory protein Vpr was investigated on the molecular level. HIV-1 Vpr induces G2 cell-cycle arrest ...

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

    International Nuclear Information System (INIS)

    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/β-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.

  18. 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.

  19. Antiviral Activities of Different Interferon Types and Subtypes against Hepatitis E Virus Replication

    DEFF Research Database (Denmark)

    Todt, Daniel; François, Catherine; Anggakusuma, [Ukendt; Behrendt, Patrick; Engelmann, Michael; Knegendorf, Leonard; Vieyres, Gabrielle; Wedemeyer, Heiner; Hartmann, Rune; Pietschmann, Thomas; Duverlie, Gilles; Steinmann, Eike

    2016-01-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genusOrthohepevirusin the familyHepeviridae HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients, and type I...... interferon (IFN) has been evaluated in a few infected transplant patientsin vivo In this study, the antiviral effects of different exogenously administered interferons were investigated by using state-of-the-art subgenomic replicon and full-length HEV genome cell culture models. Hepatitis C virus (HCV......) subgenomic replicons based on the genotype 2a JFH1 isolate served as the reference. The experiments revealed that HEV RNA replication was inhibited by the application of all types of IFN, including IFN-α (type I), IFN-γ (type II), and IFN-λ3 (type III), but to a far lesser extent than HCV replication...

  20. Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues.

    Science.gov (United States)

    Cannon, P M; Wilson, W; Byles, E; Kingsman, S M; Kingsman, A J

    1994-08-01

    Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysis. Most of the mutant viruses also had normal Western blot (immunoblot) profiles, although three of the mutations resulted in reduced signals for IN relative to the wild type on the immunoblots and mutation of residue W235 completely abolished recognition of the protein by pooled sera from human immunodeficiency virus type 1-positive patients. Mutations that have previously been shown to abolish activity in in vitro studies produced noninfectious viruses. The substitution of W235 was notable in producing a noninfectious virus, despite previous reports of this residue being nonessential for IN activity in vitro (A.D. Leavitt, L. Shiue, and H.E. Varmus, J. Biol. Chem. 268:2113-2119, 1993). In addition, we have identified four highly conserved residues that can be mutated without any affect on viral replication in T-cell lines. PMID:8035478

  1. Replication and transmission of live attenuated infectious laryngotracheitis virus (ILTV) vaccines.

    Science.gov (United States)

    Rodríguez-Avila, Andrés; Oldoni, Ivomar; Riblet, Sylva; García, Maricarmen

    2007-12-01

    The aim of this study was to evaluate the replication of live attenuated infectious laryngotracheitis virus vaccines in selected tissues and their ability to transmit to contact-exposed birds. Four-week-old specific-pathogen-free chickens were eye drop-inoculated with tissue culture origin (TCO) and chicken embryo origin (CEO) vaccines. Contact-exposed chickens were housed in direct contact with eye drop-inoculated chickens from the first day postinoculation. Virus isolation and real-time polymerase chain reaction were used to detect the presence of live virus and viral DNA, respectively, in the trachea, trigeminal ganglia, eye conjunctiva, cecal tonsils, and cloaca from eye drop-inoculated and contact-exposed birds at days 2, 4, 5 to 10, 14, 18, 21, 24, and 28 postinoculation. No differences were observed in the ability of the TCO and CEO vaccines to replicate in the examined tissues. Both vaccines presented a localized replication in the eye conjunctiva and the trachea. Both vaccines were capable of transmitting to contact-exposed birds, attaining peaks of viral DNA as elevated as those observed in inoculated birds. The CEO vaccine replicated faster and reached higher viral genome copy number than the TCO vaccine in the conjunctiva and trachea of eye drop-inoculated and contact-exposed birds. The viral DNA from both vaccines migrated to the trigeminal ganglia during early stages of infection. Although the CEO and TCO vaccines were not recovered from the cecal tonsils and the cloaca, low levels of viral DNA were detected at these sites during the peak of viral replication in the upper respiratory tract. PMID:18251401

  2. CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

    OpenAIRE

    Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T.

    2016-01-01

    Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide...

  3. A Functional Role for ADAM10 in Human Immunodeficiency Virus Type-1 Replication

    OpenAIRE

    Rubin Donald H; Hodge Thomas W; Sheng Jinsong; Li Guangyu; Murray James L; Friedrich Brian M; O'Brien William A; Ferguson Monique R

    2011-01-01

    Abstract Background Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted ADAM10 (A Disintegrin and Metalloprotease 10) allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1. Results Silencing ADAM10 expression ...

  4. Visualization of the structures of the hepatitis C virus replication complex

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Shih-Ching [Graduate Institute of Molecular and Cellular Biology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Graduate Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan (China); Liou, Je-Wen [Graduate Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan (China); Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lin, Min-Ching [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Syu, Ciao-Ling [Graduate Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan (China); Lai, Meng-Jiun; Chen, Yi- Cheng [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun, E-mail: huichun@mail.tcu.edu.tw [Graduate Institute of Molecular and Cellular Biology, Tzu Chi University, Hualien, Taiwan (China); Graduate Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan (China); Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China)

    2011-01-07

    Research highlights: {yields} Lipid rafts are known to play an important role in virus entry and virus assembly of many viruses. {yields} However, HCV is the first example of the association of lipid raft with viral RNA replication. {yields} Our results in this manuscript demonstrate that purified HCV RCs with associated lipid raft membrane appeared as distinct particles of around 0.7 um under EM and AFM. {yields} Knockdown of proteins associated with lipid raft suppressed the HCV replication and reduced the number of these particles. {yields} To our knowledge, structures of HCV RCs were demonstrated at its first time in this manuscript. -- Abstract: Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.

  5. Visualization of the structures of the hepatitis C virus replication complex

    International Nuclear Information System (INIS)

    Research highlights: → Lipid rafts are known to play an important role in virus entry and virus assembly of many viruses. → However, HCV is the first example of the association of lipid raft with viral RNA replication. → Our results in this manuscript demonstrate that purified HCV RCs with associated lipid raft membrane appeared as distinct particles of around 0.7 um under EM and AFM. → Knockdown of proteins associated with lipid raft suppressed the HCV replication and reduced the number of these particles. → To our knowledge, structures of HCV RCs were demonstrated at its first time in this manuscript. -- Abstract: Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.

  6. Ginseng Protects Against Respiratory Syncytial Virus by Modulating Multiple Immune Cells and Inhibiting Viral Replication

    OpenAIRE

    Jong Seok Lee; Yu-Na Lee; Young-Tae Lee; Hye Suk Hwang; Ki-Hye Kim; Eun-Ju Ko; Min-Chul Kim; Sang-Moo Kang

    2015-01-01

    Ginseng has been used in humans for thousands of years but its effects on viral infection have not been well understood. We investigated the effects of red ginseng extract (RGE) on respiratory syncytial virus (RSV) infection using in vitro cell culture and in vivo mouse models. RGE partially protected human epithelial (HEp2) cells from RSV-induced cell death and viral replication. In addition, RGE significantly inhibited the production of RSV-induced pro-inflammatory cytokine (TNF-α) in murin...

  7. Macaques vaccinated with live-attenuated SIV control replication of heterologous virus

    OpenAIRE

    Reynolds, Matthew R.; Weiler, Andrea M.; Weisgrau, Kim L.; Piaskowski, Shari M.; Furlott, Jessica R.; Weinfurter, Jason T.; Kaizu, Masahiko; Soma, Taeko; León, Enrique J.; MacNair, Caitlin; Leaman, Dan P.; Zwick, Michael B.; Gostick, Emma; Musani, Solomon K.; Price, David A.

    2008-01-01

    An effective AIDS vaccine will need to protect against globally diverse isolates of HIV. To address this issue in macaques, we administered a live-attenuated simian immunodeficiency virus (SIV) vaccine and challenged with a highly pathogenic heterologous isolate. Vaccinees reduced viral replication by ∼2 logs between weeks 2–32 (P ≤ 0.049) postchallenge. Remarkably, vaccinees expressing MHC-I (MHC class I) alleles previously associated with viral control completely suppressed acute phase repl...

  8. 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

    OpenAIRE

    Hui Ma; Xing-Liang Zhao; Xue-Yan Wang; Xing-Wang Xie; Jin-Chao Han; Wen-Li Guan; Qin Wang; Lin Zhu; Xiao-Ben Pan; Lai Wei

    2013-01-01

    2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadeny...

  9. Recursion-based depletion of human immunodeficiency virus-specific naive CD4(+) T cells may facilitate persistent viral replication and chronic viraemia leading to acquired immunodeficiency syndrome.

    Science.gov (United States)

    Tsukamoto, Tetsuo; Yamamoto, Hiroyuki; Okada, Seiji; Matano, Tetsuro

    2016-09-01

    Although antiretroviral therapy has made human immunodeficiency virus (HIV) infection a controllable disease, it is still unclear how viral replication persists in untreated patients and causes CD4(+) T-cell depletion leading to acquired immunodeficiency syndrome (AIDS) in several years. Theorists tried to explain it with the diversity threshold theory in which accumulated mutations in the HIV genome make the virus so diverse that the immune system will no longer be able to recognize all the variants and fail to control the viraemia. Although the theory could apply to a number of cases, macaque AIDS models using simian immunodeficiency virus (SIV) have shown that failed viral control at the set point is not always associated with T-cell escape mutations. Moreover, even monkeys without a protective major histocompatibility complex (MHC) allele can contain replication of a super infected SIV following immunization with a live-attenuated SIV vaccine, while those animals are not capable of fighting primary SIV infection. Here we propose a recursion-based virus-specific naive CD4(+) T-cell depletion hypothesis through thinking on what may happen in individuals experiencing primary immunodeficiency virus infection. This could explain the mechanism for impairment of virus-specific immune response in the course of HIV infection. PMID:27515208

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

    International Nuclear Information System (INIS)

    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

  11. Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia.

    Directory of Open Access Journals (Sweden)

    Francesca D Frentiu

    2014-02-01

    Full Text Available INTRODUCTION: Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV, is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. METHODOLOGY/PRINCIPAL FINDINGS: Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

  12. 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.

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

    International Nuclear Information System (INIS)

    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

  14. Human heme oxygenase 1 is a potential host cell factor against dengue virus replication.

    Science.gov (United States)

    Tseng, Chin-Kai; Lin, Chun-Kuang; Wu, Yu-Hsuan; Chen, Yen-Hsu; Chen, Wei-Chun; Young, Kung-Chia; Lee, Jin-Ching

    2016-01-01

    Dengue virus (DENV) infection and replication induces oxidative stress, which further contributes to the progression and pathogenesis of the DENV infection. Modulation of host antioxidant molecules may be a useful strategy for interfering with DENV replication. In this study, we showed that induction or exogenous overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, effectively inhibited DENV replication in DENV-infected Huh-7 cells. This antiviral effect of HO-1 was attenuated by its inhibitor tin protoporphyrin (SnPP), suggesting that HO-1 was an important cellular factor against DENV replication. Biliverdin but not carbon monoxide and ferrous ions, which are products of the HO-1 on heme, mediated the HO-1-induced anti-DENV effect by non-competitively inhibiting DENV protease, with an inhibition constant (Ki) of 8.55 ± 0.38 μM. Moreover, HO-1 induction or its exogenous overexpression, rescued DENV-suppressed antiviral interferon response. Moreover, we showed that HO-1 induction by cobalt protoporphyrin (CoPP) and andrographolide, a natural product, as evidenced by a significant delay in the onset of disease and mortality, and virus load in the infected mice's brains. These findings clearly revealed that a drug or therapy that induced the HO-1 signal pathway was a promising strategy for treating DENV infection. PMID:27553177

  15. Evaluation of the conformational switch model for alfalfa mosaic virus RNA replication.

    Science.gov (United States)

    Petrillo, Jessica E; Rocheleau, Gail; Kelley-Clarke, Brenna; Gehrke, Lee

    2005-05-01

    Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3' untranslated regions of AMV and ilarvirus RNAs have the potential to fold into pseudoknots, we were unable to confirm that a proposed pseudoknot forms or has a functional role in regulating coat protein-RNA binding or viral RNA replication. Published work has suggested that the pseudoknot is part of a tRNA-like structure (TLS); however, we argue that the canonical sequence and functional features that define the TLS are absent. We suggest here that the absence of the TLS correlates directly with the distinctive requirement for coat protein to activate replication in these viruses. Experimental data are evidence that elevated magnesium concentrations proposed to stabilize the pseudoknot structure do not block coat protein binding. Additionally, covarying nucleotide changes proposed to reestablish pseudoknot pairings do not rescue replication. Furthermore, as described in the accompanying paper (L. M. Guogas, S. M. Laforest, and L. Gehrke, J. Virol. 79:5752-5761, 2005), coat protein is not, by definition, inhibitory to minus-strand RNA synthesis. Rather, the activation of viral RNA replication by coat protein is shown to be concentration dependent. We describe the 3' organization model as an alternate model of AMV replication that offers an improved fit to the available data. PMID:15827189

  16. The use of an E1-deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus.

    OpenAIRE

    Wang, Y.; Xiang, Z; Pasquini, S; Ertl, H. C.

    1997-01-01

    An E1-deleted, replication-defective adenovirus recombinant of the human strain 5 expressing the rabies virus glycoprotein, termed Adrab.gp, was tested in young mice. Mice immunized at birth with the Adrab.gp construct developed antibodies to rabies virus and cytokine-secreting lymphocytes and were protected against subsequent challenge. Maternal immunity to rabies virus strongly interferes with vaccination of the offspring with a traditional inactivated rabies virus vaccine. The immune respo...

  17. The role of the replication of hepatitis B virus in the progress of liver fibrosis

    International Nuclear Information System (INIS)

    This article is to investigate the role of the replication of hepatitis B virus in the progress of liver fibrosis, 173 patients with hepatitis B virus were divided into six groups: chronic hepatitis B(CHB)-mild, -middle, -severe groups, Child-Pugh (CP)-A, -B, -C groups, and every group was again divided into two sub-groups according to HBV-DNA. The serum IL-6, IL-8, P III P, c-IV in the patient s were measured by RIA. The results were compared between HBV-DNA(+) and HBV-DNA(-) sub-groups. Except the case in CHB-mild and CP-C groups (P>0.05), the serum levels of IL-6, IL-8, P III P, c-IV were significantly higher in HBV-DNA positive sub-group than that in HBV-DNA negative sub-group (P<0.05), especially in CHB-severe and CP-A groups (P<0.01). The results suggest that hepatitis B virus may activate the hepatic starlike cell and make this progress continue , so the active replication of hepatitis B virus play an important role in the beginning stage and the progress stage of liver fibrosis

  18. Susceptibility of domestic animals to a pseudotype virus bearing RD-114 virus envelope protein.

    Science.gov (United States)

    Miyaho, Rie Nakaoka; Nakagawa, So; Hashimoto-Gotoh, Akira; Nakaya, Yuki; Shimode, Sayumi; Sakaguchi, Shoichi; Yoshikawa, Rokusuke; Takahashi, Mahoko Ueda; Miyazawa, Takayuki

    2015-08-10

    Retroviral vectors are used for gene transduction into cells and have been applied to gene therapy. Retroviral vectors using envelope protein (Env) of RD-114 virus, a feline endogenous retrovirus, have been used for gene transduction. In this study, we investigated the susceptibility to RD-114 Env-pseudotyped virus in twelve domestic animals including cattle, sheep, horse, pig, dog, cat, ferret, mink, rabbit, rat, mouse, and quail. Comparison of nucleotide sequences of ASCT2 (SLC1A5), a receptor of RD-114 virus, in 10 mammalian and 2 avian species revealed that insertion and deletion events at the region C of ASCT2 where RD-114 viral Env interacts occurred independently in the mouse and rat lineage and in the chicken and quail lineage. By the pseudotype virus infection assay, we found that RD-114 Env-pseudotyped virus could efficiently infect all cell lines except those from mouse and rat. Furthermore, we confirmed that bovine ASCT2 (bASCT2) functions as a receptor for RD-114 virus infection. We also investigated bASCT2 mRNA expression in cattle tissues and found that it is expressed in various tissues including lung, spleen and kidney. These results indicate that retrovirus vectors with RD-114 virus Env can be used for gene therapy in large domestic animals in addition to companion animals such as cat and dog. PMID:25936996

  19. Block to influenza virus replication in cells preirradiated with ultraviolet light

    International Nuclear Information System (INIS)

    Ultraviolet (uv) irradiation of CEF cells immediately before infection with influenza A (fowl plague) virus inhibited virus growth; no inhibition of the growth of a parainfluenza virus (Newcastle disease virus) could be detected in irradiated cells. The kinetics of inhibition after various doses of uv irradiation were multihit, with an extrapolation number of two. When irradiated cells were allowed to photoreactivate by exposure to visible light for 16 hr their capacity to support influenza virus replication was largely restored; this process was sensitive to caffeine, suggesting that it required DNA repair. In CEF cells exposed to 360 ergs/mm2 of uv radiation the rate of synthesis of host cellular RNA was reduced by more than 90%, and that of host cellular protein by 40 to 50%, as judged by incorporation of precursor molecules into an acid-insoluble form. When such irradiated cells were infected with influenza virus all the genome RNA segments were transcribed, but the overall concentration of virus-specific poly(A)-containing cRNA was reduced about 50-fold. Within this population of cRNA molecules, the RNAs coding for late proteins (HA, NA, and M) were reduced in amount relative to the other segments. The rates of synthesis of the M and HA proteins were specifically reduced in uv-irradiated cells, but the rates of synthesis of the P, NP, and NS proteins were only slightly reduced compared to normal cells. Immunofluorescent studies showed that, in uv-irradiated cells, NP migrated into the nucleus early after infection and later migrated out into the cytoplasm, as in normal cells. In contrast to normal cells, no specific immunofluorescence associated with M protein could be observed in uv-irradiated cells. It is concluded that uv-induced damage to host cellular DNA alters the pattern of RNA transcription in CEF cells infected with influenza virus, and that this results in a block to late protein synthesis which stops virus production

  20. Animal Models for Influenza Virus Pathogenesis and Transmission

    Directory of Open Access Journals (Sweden)

    Anice C. Lowen

    2010-07-01

    Full Text Available Influenza virus infection of humans results in a respiratory disease that ranges in severity from sub-clinical infection to primary viral pneumonia that can result in death. The clinical effects of infection vary with the exposure history, age and immune status of the host, and also the virulence of the influenza strain. In humans, the virus is transmitted through either aerosol or contact-based transfer of infectious respiratory secretions. As is evidenced by most zoonotic influenza virus infections, not all strains that can infect humans are able to transmit from person-to-person. Animal models of influenza are essential to research efforts aimed at understanding the viral and host factors that contribute to the disease and transmission outcomes of influenza virus infection in humans. These models furthermore allow the pre-clinical testing of antiviral drugs and vaccines aimed at reducing morbidity and mortality in the population through amelioration of the virulence or transmissibility of influenza viruses. Mice, ferrets, guinea pigs, cotton rats, hamsters and macaques have all been used to study influenza viruses and therapeutics targeting them. Each model presents unique advantages and disadvantages, which will be discussed herein.

  1. Cyclophilin E Functions as a Negative Regulator to Influenza Virus Replication by Impairing the Formation of the Viral Ribonucleoprotein Complex

    OpenAIRE

    Zengfu Wang; Xiaoling Liu; Zhendong Zhao; Chongfeng Xu; Ke Zhang; Caiwei Chen; Lei Sun; George F Gao; Xin Ye; Wenjun Liu

    2011-01-01

    BACKGROUND: The nucleoprotein (NP) of influenza A virus is a multifunctional protein that plays a critical role in the replication and transcription of the viral genome. Therefore, examining host factors that interact with NP may shed light on the mechanism of host restriction barriers and the tissue tropism of influenza A virus. Here, Cyclophilin E (CypE), a member of the peptidyl-propyl cis-trans isomerase (PPIase) family, was found to bind to NP and inhibit viral replication and transcript...

  2. 5-azacytidine and 5-azadeoxycytidine inhibit human immunodeficiency virus type 1 replication in vitro.

    Science.gov (United States)

    Bouchard, J; Walker, M C; Leclerc, J M; Lapointe, N; Beaulieu, R; Thibodeau, L

    1990-01-01

    Chemotherapeutic agents which affect the integration, stability, or inducibility of the human immunodeficiency virus (HIV) provirus would have considerable value in treating acquired immunodeficiency syndrome. Two nucleoside analogs of cytosine, 5-azacytidine and 5-azadeoxycytidine, which seem to have such value because of their capabilities to affect both the stability and the methylation patterns of the nucleic acids into which they are incorporated, were tested for their ability to inhibit the replication of HIV type 1 (HIV-1) in human CEM T cells in vitro. 5-Azadeoxycytidine (1 microM) completely inhibited HIV replication in CEM cells, by the criteria of reduced viral antigen expression and decreased supernatant reverse transcriptase activity, with little toxicity for the treated cells. 5-azacytidine (1 microM) also inhibited HIV replication, but less effectively. When added 2 or more h after CEM cells were infected with HIV-1, both 5-azacytosine derivatives were less effective than they were when added at the time of infection. Even 2 h of exposure to 5-azadeoxycytidine was sufficient for inhibition of HIV replication. Although long exposure to either analog at concentrations of 1 microM would result in pronounced cellular cytotoxicity, the the fact that short exposures to the same dose of drug inhibit HIV replication but are not toxic for the cells implies that cellular toxicity itself is not an important mechanism of the antiviral action of the analogs. PMID:1691617

  3. 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.

  4. Plasticity of DNA Replication Initiation in Epstein-Barr Virus Episomes

    Directory of Open Access Journals (Sweden)

    Norio Paolo

    2004-01-01

    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.

  5. 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.

  6. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-01-01

    Full Text Available Abstract Background To be transmitted by its mosquito vector, dengue virus (DENV must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. Results After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi. The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. Conclusion Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands differed in their response to DENV-2 infection.

  7. Active β-Catenin Signaling Is an Inhibitory Pathway for Human Immunodeficiency Virus Replication in Peripheral Blood Mononuclear Cells▿

    OpenAIRE

    Kumar, Anvita; Zloza, Andrew; Moon, Randall T.; Watts, Jeffrey; Tenorio, Allan R.; Al-Harthi, Lena

    2008-01-01

    The Wnt/β-catenin pathway is involved in cell functions governing development and disease. In modeling postentry restriction of human immunodeficiency virus (HIV) replication in astrocytes, we reported that part of this natural resistance to productive replication of HIV in astrocytes involved expression of proteins of the Wnt/β-catenin signaling pathway. We determined here whether induction of β-catenin signaling in peripheral blood mononuclear cells (PBMCs) can modulate HIV replication. Giv...

  8. Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity.

    Science.gov (United States)

    Lu, Gaofeng; Lomonosova, Elena; Cheng, Xiaohong; Moran, Eileen A; Meyers, Marvin J; Le Grice, Stuart F J; Thomas, Craig J; Jiang, Jian-kang; Meck, Christine; Hirsch, Danielle R; D'Erasmo, Michael P; Suyabatmaz, Duygu M; Murelli, Ryan P; Tavis, John E

    2015-02-01

    Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 μM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 μM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50]=25 to 79 μM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection. PMID:25451058

  9. RNASEK Is a V-ATPase-Associated Factor Required for Endocytosis and the Replication of Rhinovirus, Influenza A Virus, and Dengue Virus

    OpenAIRE

    Jill M. Perreira; Aaron M. Aker; George Savidis; Christopher R. Chin; William M. McDougall; Jocelyn M. Portmann; Paul Meraner; Miles C. Smith; Motiur Rahman; Richard E. Baker; Annick Gauthier; Michael Franti; Abraham L. Brass

    2015-01-01

    Human rhinovirus (HRV) causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase) proton pump. RNASEK is required for endocytosis, and its de...

  10. Marek's disease virus (MDV) ubiquitin-specific protease (USP) performs critical functions beyond its enzymatic activity during virus replication.

    Science.gov (United States)

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

    2013-03-15

    Marek's disease virus (MDV) encodes an ubiquitin-specific protease (USP) within its UL36 gene. USP is highly conserved among herpesviruses and was shown to be important for MDV replication and pathogenesis in MDV's natural host, the chicken. To further investigate the role of MDV USP, several recombinant (r) MDVs were generated and their in vitro phenotypes were evaluated using plaque size and growth kinetics assays. We discovered that the N-terminus of pUL36 is essential for MDV replication and could not be complemented by ectopic expression of MDV USP. In addition, we demonstrated that the region located between the conserved glutamine (Q85) and leucine (L106) residues comprising the active site cysteine (C98) is also essential for MDV replication. Based on the analyses of the rMDVs generated here, we concluded that MDV USP likely contributes to the structure and/or stability of pUL36 and affects replication and oncogenesis of MDV beyond its enzymatic activity. PMID:23399034

  11. Physical Factors Affecting in Vitro Replication of Foot and Mouth Disease Virus (Serotype “O”

    Directory of Open Access Journals (Sweden)

    Muhammad Taslim Ghori*, Khushi Muhammad and Masood Rabbani1

    2011-10-01

    Full Text Available Effect of physical factors (temperature, pH and UV light on replicating ability of “O” type of Foot and Mouth Disease (FMD virus on Baby Hamster Kidney (BHK cell line was determined. The freshly grown FMD virus containing 106 units of tissue culture infective dose (TCID50 was divided into aliquots. Each of the 9 virus aliquots was exposed to 37, 57 or 77C for 15, 30 or 45 minutes, respectively. Each of the 5 virus aliquots was mixed with MEM-199 maintenance medium having pH 3, 5, 7, 9, or 11. Similarly, each of the 3 aliquots having 1 mm depth of the medium was exposed to ultraviolet light (252.7 nm wavelength: one foot distance for 15, 30 or 45 minutes. Each of the virus aliquot exposed to either of the temperature, pH or ultraviolet light (UV for either of the interaction time was inoculated to 8 wells of the 96-well cell culture plate containing complete monolayer of BHK cell line. One row of 8 wells served as virus control and other row of 8 wells served as control for monolayer of the BHK-21 cell line. The plates were incubated at 37°C for 48 hours. It was observed that temperature of 57 and 77C inactivated the virus within 15 minutes. The virus when admixed in the MEM-199 maintenance medium having pH 3, 5, 9 or 11, of the medium inactivated the virus while pH 7 did not show any detrimental effect on its survival. The ultraviolet light for 15, 30 or 45 minutes showed undetectable effect on survival of the virus as either of the virus aliquot exposed to the UV light for either of the interaction time showed cytopathogenic effects (CPE. It was concluded that the temperature of 57°C or higher for 15 minutes, acidic pH (below 5 or basic pH (more than 9 may inactivate the FMD virus.

  12. 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

    2016-01-01

    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. PMID:27094903

  13. Crystallization and preliminary X-ray crystallographic analysis of a helicase-like domain from a tomato mosaic virus replication protein

    International Nuclear Information System (INIS)

    Crystals of the helicase domain from a tomato mosaic virus replication protein obtained using the hanging-drop vapour-diffusion method at 285 K diffracted X-rays to 2.05 Å resolution. They belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å. Tomato mosaic virus belongs to the genus Tobamovirus in the alphavirus-like superfamily of positive-strand RNA viruses. The alphavirus-like superfamily includes many plant and animal viruses of agronomical and clinical importance. These viruses encode replication-associated proteins that contain a putative superfamily 1 helicase domain. No three-dimensional structures for this domain have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of the 130K helicase domain are reported. Diffraction data were collected and processed to 2.05 and 1.75 Å resolution from native and selenomethionine-labelled crystals, respectively. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å

  14. Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication.

    Directory of Open Access Journals (Sweden)

    Zhen Wang

    Full Text Available Influenza A virus (IAV poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc and blasticidin resistance gene (Bsd were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication.

  15. Vector-mediated expression of interferon gamma inhibits replication of hepatitis B virus in vitro.

    Science.gov (United States)

    Kan, Q C; Li, D L; Yu, Z J

    2013-01-01

    Despite the existence of efficient vaccines against hepatitis B virus (HBV) infections, these still represent a serious threat to human health worldwide. Acute HBV infections often become chronic, marked by liver cirrhosis and hepatocellular carcinoma. Promising results with interferons alpha or gamma (IFN-α, γ) or nucleoside/nucleotide analogs in inhibiting HBV replication in vitro have led to therapeutic applications to chronic HBV patients, however, their results so far have not been satisfactory. The treatments were either not effective in all patients or had adverse effects. Certain progress was expected from expression of interferons targeted to liver by adenovirus vectors, however, this approach turned out to be limited by undesired expression of toxic viral genes and high production costs. Therefore, in this study, we attempted to inhibit HBV replication in HepG2.2.15 cells by human IFN-γ expressed through a non-viral vector, an eukaryotic plasmid. The results demonstrated that IFN-γ, targeted to HBV-replicating cells, significantly inhibited the virus growth without inducing apoptosis and indicated that local expression of this kind of cytokine may be a promising strategy of gene therapy. PMID:24294955

  16. The importance of alfalfa mosaic virus coat protein dimers in the initiation of replication.

    Science.gov (United States)

    Choi, Jiwon; Kim, Bong-Suk; Zhao, Xiaoxia; Loesch-Fries, Sue

    2003-01-01

    Deletion and substitution mutations affecting the oligomerization of alfalfa mosaic virus (AMV) coat protein (CP) were studied in protoplasts to determine their effect on genome activation, an early step in AMV replication. The CP mutants that formed dimers, CPDeltaC9 and CPC-A(R)F, were highly active in initiating replication with 63-84% of wild-type (wt) CP activity. However, all mutants that did not form dimers, CPDeltaC18, CPDeltaC19, CPC-WFP, and CPC-W, were much less active with 19-33% of wt CP activity. The accumulation and solubility of mutant CPs expressed from a virus-based vector in Nicotiana benthamiana were similar to that of wt CP. Analysis of CP-RNA interactions indicated that CP dimers and CP monomers interacted very differently with AMV RNA 3' ends. These results suggest that CP dimers are more efficient for replication than CP monomers because of differences in RNA binding rather than differences in expression and accumulation of the mutant CPs in infected cells. PMID:12504539

  17. 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. PMID:26968901

  18. 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.

  19. Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy.

    Directory of Open Access Journals (Sweden)

    Janet Lei

    Full Text Available The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA, human tyrosinase-related protein 2 (TRP-2, and oncoprotein E7 of human papillomavirus type 16 (HPV16E7. Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication

  20. 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.

  1. HIV-1 Nef control of cell signalling molecules: multiple strategies to promote virus replication

    Indian Academy of Sciences (India)

    Alison L Greenway; Gavan Holloway; Dale A McPhee; Phoebe Ellis; Alyssa Cornall; Michael Lidman

    2003-04-01

    HIV-1 has at its disposal numerous proteins encoded by its genome which provide the required arsenal to establish and maintain infection in its host for a considerable number of years. One of the most important and enigmatic of these proteins is Nef. The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. This small protein of approximately 27 kDa is required for maximal virus replication and disease progression. The mechanisms by which it is able to act as a positive factor during virus replication is an area of intense research and although some controversy surrounds Nef much has been gauged as to how it functions. Its ability to modulate the expression of key cellular receptors important for cell activation and control signal transduction elements and events by interacting with numerous cellular kinases and signalling molecules, including members of the Src family kinases, leading to an effect on host cell function is likely to explain at least in part its role during infection and represents a finely tuned mechanism where this protein assists HIV-1 to control its host.

  2. 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. PMID:7682254

  3. RNA interference modulates replication of dengue virus in Drosophila melanogaster cells

    Directory of Open Access Journals (Sweden)

    Hanley Kathryn A

    2010-04-01

    Full Text Available Abstract Background Mosquito-borne dengue virus (DENV, genus Flavivirus has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. RNA interference (RNAi induced by exogenous small interfering RNAs (siRNAs has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. However, the impact of RNAi triggered by authentic virus infection on replication of DENV, or any flavivirus, has received little study. The objectives of the current study were threefold: first, to assess the utility of Drosophila melanogaster S2 cells for the study of DENV, second to investigate the impact of multiple enzymes in the RNAi pathway on DENV replication; and third to test for variation in the response of the four serotypes of DENV to modulation of RNAi. Results Three strains from each of the four DENV serotypes showed replication in S2 cells following infection at multiplicity of infection (MOI 0.1 and MOI 10; each strain achieved titers > 4.0 log10pfu/ml five days after infection at MOI 10. The four serotypes did not differ in mean titer. S2 cells infected with DENV-1, 2, 3 or 4 produced siRNAs, indicating that infection triggered an RNAi response. Knockdown of one of the major enzymes in the RNAi pathway, Dicer-2 (Dcr-2, resulted in a 10 to 100-fold enhancement of replication of all twelve strains of DENV in S2 cells. While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown. Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2, Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown. Conclusions These findings indicate that DENV can

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    International Nuclear Information System (INIS)

    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)

  6. Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

    OpenAIRE

    Liang, Libin; Deng, Guohua; Shi, Jianzhong; Wang, Shuai; Zhang, Qianyi; Kong, Huihui; Gu, Chunyang; Guan, Yuntao; Suzuki, Yasuo; Li, Yanbing; Jiang, Yongping; Tian, Guobin; Liu, Liling; Li, Chengjun; Chen, Hualan

    2016-01-01

    ABSTRACT H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that...

  7. 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. PMID:21190707

  8. Oleanane triterpenes from the flowers of Camellia japonica inhibit porcine epidemic diarrhea virus (PEDV) replication.

    Science.gov (United States)

    Yang, Jun-Li; Ha, Thi-Kim-Quy; Dhodary, Basanta; Pyo, Euisun; Nguyen, Ngoc Hieu; Cho, Hyomoon; Kim, Eunhee; Oh, Won Keun

    2015-02-12

    Porcine epidemic diarrhea virus (PEDV) infections have resulted in a severe economic loss in the swine industry in many countries due to no effective treatment approach. Fifteen oleanane triterpenes (1-15), including nine new ones (1-4 and 10-14), were isolated from the flowers of Camellia japonica, and their molecular structures were determined by extensive spectroscopic methods. These compounds were evaluated for their antiviral activity against PEDV replication, and the structure-activity relationships (SARs) were discussed. Compounds 6, 9, 11, and 13 showed most potent inhibitory effects on PEDV replication. They were found to inhibit PEDV genes encoding GP6 nucleocapsid, GP2 spike, and GP5 membrane protein synthesis based on RT-PCR data. Western blot analysis also demonstrated their inhibitory effects on PEDV GP6 nucleocapsid and GP2 spike protein synthesis during viral replication. The present study suggested the potential of compounds 6, 9, 11, and 13 as promising scaffolds for treating PEDV infection via inhibiting viral replication. PMID:25568928

  9. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

    International Nuclear Information System (INIS)

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

  10. Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Min Lin; Qun Chen; Li-Ye Yang; Wen-Yu Li; Xi-Biao Cao; Jiao-Ren Wu; You-Peng Peng; Mo-Rui Chen

    2007-01-01

    AIM:To investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODS:The human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTS:A stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSION:HBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.

  11. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon; Yang, Sujeong; Choi, Seunga [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Kang, Misun [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); Rho, Jaerang, E-mail: jrrho@cnu.ac.kr [Department of Microbiology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); BK21 Bio Brain Center, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of); GRAST, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejon 305-764 (Korea, Republic of)

    2010-01-01

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain and examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.

  12. 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.

  13. Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses

    International Nuclear Information System (INIS)

    Vesicular stomatitis virus (VSV) infection of mice via intranasal administration results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes. We have recently shown that these glial cells express RIG-I and MDA5, cytosolic pattern recognition receptors for viral RNA. However, it is unclear whether VSV can replicate in glial cells or if such replication is required for their inflammatory responses. Here we demonstrate that primary microglia and astrocytes are permissive for VSV infection and limited productive replication. Importantly, we show that viral replication is required for robust inflammatory mediator production by these cells. Finally, we have confirmed that in vivo VSV administration can result in viral infection of glial cells in situ. These results suggest that viral replication within resident glial cells might play an important role in CNS inflammation following infection with VSV and possibly other neurotropic nonsegmented negative-strand RNA viruses.

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

    Science.gov (United States)

    Heilbronn, R; zur Hausen, H

    1989-01-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of six HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensible for SV40 DNA amplification. Our results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo. Images PMID:2547992

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

    Energy Technology Data Exchange (ETDEWEB)

    Heilbronn, R.; zur Hausen, H. (Deutsches Krebsforschungszentrum, Heidelberg (West Germany))

    1989-09-01

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

  16. Modeling the intracellular replication of influenza A virus in the presence of defective interfering RNAs.

    Science.gov (United States)

    Laske, Tanja; Heldt, Frank Stefan; Hoffmann, Helene; Frensing, Timo; Reichl, Udo

    2016-02-01

    Like many other viral pathogens, influenza A viruses can form defective interfering particles (DIPs). These particles carry a large internal deletion in at least one of their genome segments. Thus, their replication depends on the co-infection of cells by standard viruses (STVs), which supply the viral protein(s) encoded by the defective segment. However, DIPs also interfere with STV replication at the molecular level and, despite considerable research efforts, the mechanism of this interference remains largely elusive. Here, we present a mechanistic mathematical model for the intracellular replication of DIPs. In this model, we account for the common hypothesis that defective interfering RNAs (DI RNAs) possess a replication advantage over full-length (FL) RNAs due to their reduced length. By this means, the model captures experimental data from yield reduction assays and from studies testing different co-infection timings. In addition, our model predicts that one important aspect of interference is the competition for viral proteins, namely the heterotrimeric viral RNA-dependent RNA polymerase (RdRp) and the viral nucleoprotein (NP), which are needed for encapsidation of naked viral RNA. Moreover, we find that there may be an optimum for both the DI RNA synthesis rate and the time point of successive co-infection of a cell by DIPs and STVs. Comparing simulations for the growth of DIPs with a deletion in different genome segments suggests that DI RNAs derived from segments which encode for the polymerase subunits are more competitive than others. Overall, our model, thus, helps to elucidate the interference mechanism of DI RNAs and provides a novel hypothesis why DI RNAs derived from the polymerase-encoding segments are more abundant in DIP preparations. PMID:26592173

  17. Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis.

    OpenAIRE

    Ariga, H

    1984-01-01

    The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixtur...

  18. Gaining replicability in a nonhost compromises the silencing suppression activity of Tobacco mild green mosaic virus in a host.

    Science.gov (United States)

    Ishibashi, Kazuhiro; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-02-01

    Natural isolates of Tobacco mild green mosaic virus (TMGMV) fail to infect tomato because the tomato tm-1 protein binds to the replication proteins of TMGMV and prevents RNA replication. Previously, we isolated a TMGMV mutant that overcomes tm-1-mediated resistance and multiplies in tomato plants. Here, we show that the causal mutations in the replication protein gene that abolish the interaction with tm-1 reduce its ability to suppress RNA silencing in host plant Nicotiana benthamiana. The results suggest that the multifunctionality of the replication proteins is an evolutionary constraint of tobamoviruses that restricts their host ranges. PMID:21106731

  19. Gaining Replicability in a Nonhost Compromises the Silencing Suppression Activity of Tobacco Mild Green Mosaic Virus in a Host▿

    Science.gov (United States)

    Ishibashi, Kazuhiro; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-01-01

    Natural isolates of Tobacco mild green mosaic virus (TMGMV) fail to infect tomato because the tomato tm-1 protein binds to the replication proteins of TMGMV and prevents RNA replication. Previously, we isolated a TMGMV mutant that overcomes tm-1-mediated resistance and multiplies in tomato plants. Here, we show that the causal mutations in the replication protein gene that abolish the interaction with tm-1 reduce its ability to suppress RNA silencing in host plant Nicotiana benthamiana. The results suggest that the multifunctionality of the replication proteins is an evolutionary constraint of tobamoviruses that restricts their host ranges. PMID:21106731

  20. Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

    OpenAIRE

    Lodeiro, María F.; Filomatori, Claudia V.; Andrea V Gamarnik

    2008-01-01

    The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG region (5′UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replicatio...

  1. Viscerotropic velogenic Newcastle disease virus replication in feathers of infected chickens

    Science.gov (United States)

    Lee, Dong-Hun; Kwon, Jung-Hoon; Noh, Jin-Yong; Park, Jae-Keun; Yuk, Seong-Su; Erdene-Ochir, Tseren-Ochir; Nahm, Sang-Soep; Kwon, Yong-Kuk; Lee, Sang-Won

    2016-01-01

    Newcastle disease viruses (NDVs) cause systemic diseases in chickens with high mortality. However, little is known about persistence of NDVs in contaminated tissues from infected birds. In this study, we examined viral replication in the feather pulp of chickens inoculated with viscerotropic velogenic NDV (vvNDV) genotype VII. Reverse transcription real-time PCR and immunohistochemistry were used to investigate viral persistence in the samples. vvNDV was detected in the oropharynx and cloaca and viral antigens were detected in the feathers, suggesting that feathers act as sources of viral transmission. PMID:27051348

  2. cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

    OpenAIRE

    Hartl, M.; Willnow, T; Fanning, E

    1990-01-01

    Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of ...

  3. Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

    Directory of Open Access Journals (Sweden)

    Anna Maisa

    Full Text Available BACKGROUND: Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor. CONCLUSIONS/SIGNIFICANCE: Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  4. 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.

  5. 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....

  6. Studies on photoinactivation by various phthalocyanines of a free or replicating non-enveloped virus.

    Science.gov (United States)

    Gaspard, S; Tempête, C; Werner, G H

    1995-12-01

    The non-enveloped picornaviruses, which are particularly resistant to physicochemical inactivation, include the aetiological agents of poliomyelitis, hepatitis A and E and infectious common cold (rhinovirus). In this work we used human rhinovirus type 5 (RV-5) cultivated in VERO cells to study the photoinactivating effects of several phthalocyanines and naphthobenzoporphyrazines. Free RV-5 was photoinactivated by aluminium trisulphonated naphthobenzoporphyrazine at 5 x 10(-8) M concentration. This photosensitizer was also active on replicating virus when the infected VERO cells were treated with 5 x 10(-6) M concentration followed by a very short illumination period. On the other hand, the ZnPc(3-MeO-Py)4 phthalocyanine, which possesses four positive charges, does not photoinactivate free rhinovirus, but this molecule protects VERO cells against RV-5 infection when added to the cultures before virus inoculation, in the presence or absence of subsequent illumination, and may therefore be considered as an antiviral agent in itself. PMID:8583283

  7. Characterization of the activity of 2'-C-methylcytidine against dengue virus replication.

    Science.gov (United States)

    Lee, Jin-Ching; Tseng, Chin-Kai; Wu, Yu-Hsuan; Kaushik-Basu, Neerja; Lin, Chun-Kuang; Chen, Wei-Chun; Wu, Huey-Nan

    2015-04-01

    Dengue virus (DENV) is a severe mosquito-borne viral pathogen. Neither vaccines nor antiviral therapy is currently available to treat DENV infection. Nucleoside inhibitors targeting viral polymerase have proved promising for the development of drugs against viruses. In this study, we report a nucleoside analog, 2'-C-methylcytidine (2CMC), which exerts potent anti-DENV activity in DENV subgenomic RNA replicon and infectious systems, with an IC50 value of 11.2±0.3μM. This study utilized both cell-based and cell-free reporter assay systems to reveal the specific anti-DENV RNA polymerase activity of 2CMC. In addition, both xenograft bioluminescence-based DENV replicon and DENV-infected Institute of Cancer Research (ICR) suckling mice models evaluated the anti-DENV replication activity of 2CMC in vivo. Collectively, these findings provide a promising compound for the development of direct-acting antivirals against DENV infection. PMID:25614455

  8. Cofolding organizes alfalfa mosaic virus RNA and coat protein for replication.

    Science.gov (United States)

    Guogas, Laura M; Filman, David J; Hogle, James M; Gehrke, Lee

    2004-12-17

    Alfalfa mosaic virus genomic RNAs are infectious only when the viral coat protein binds to the RNA 3' termini. The crystal structure of an alfalfa mosaic virus RNA-peptide complex reveals that conserved AUGC repeats and Pro-Thr-x-Arg-Ser-x-x-Tyr coat protein amino acids cofold upon interacting. Alternating AUGC residues have opposite orientation, and they base pair in different adjacent duplexes. Localized RNA backbone reversals stabilized by arginine-guanine interactions place the adenosines and guanines in reverse order in the duplex. The results suggest that a uniform, organized 3' conformation, similar to that found on viral RNAs with transfer RNA-like ends, may be essential for replication. PMID:15604410

  9. Replication-deficient rabies virus-based vaccines are safe and immunogenic in mice and nonhuman primates.

    Science.gov (United States)

    Cenna, Jonathan; Hunter, Meredith; Tan, Gene S; Papaneri, Amy B; Ribka, Erin P; Schnell, Matthias J; Marx, Preston A; McGettigan, James P

    2009-10-15

    Although current postexposure prophylaxis rabies virus (RV) vaccines are effective, approximately 40,000-70,000 rabies-related deaths are reported annually worldwide. The development of effective formulations requiring only 1-2 applications would significantly reduce mortality. We assessed in mice and nonhuman primates the efficacy of replication-deficient RV vaccine vectors that lack either the matrix (M) or phosphoprotein (P) gene. A single dose of M gene-deficient RV induced a more rapid and efficient anti-RV response than did P gene-deficient RV immunization. Furthermore, the M gene-deleted RV vaccine induced 4-fold higher virus-neutralizing antibody (VNA) levels in rhesus macaques than did a commercial vaccine within 10 days after inoculation, and at 180 days after immunization rhesus macaques remained healthy and had higher-avidity antibodies, higher VNA titers, and a more potent antibody response typical of a type 1 T helper response than did animals immunized with a commercial vaccine. The data presented in this article suggest that the M gene-deleted RV vaccine is safe and effective and holds the potential of replacing current pre- and postexposure RV vaccines. PMID:19764884

  10. Soluble human CD4 elicits an antibody response in rhesus monkeys that inhibits simian immunodeficiency virus replication

    International Nuclear Information System (INIS)

    Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac) demonstrate significant virologic and clinical improvement as a result of treatment with human recombinant soluble CD4 (rsCD4). The authors show that human rsCD4 does not efficiently inhibit SIVmac replication in bone marrow macrophages of rhesus monkeys and does not significantly augment bone marrow hematopoietic colony formation in vitro. However, plasma of human rsCD4-treated rhesus monkeys does exhibit significant anti-SIVmac activity in vitro. Plasma of these animals efficiently blocks SIVmac replicaton in peripheral blood lymphocytes and bone marrow macrophages. It also increases granulocyte/macrophage colony formation in vitro by bone marrow cells of SIVmac-infected monkeys. This plasma and the IgG fraction of plasma from a rhesus monkey immunized with human rsCD4 in adjuvant demonstrate reactivity with a soluble form of the rhesus monkey CD4 molecule, exhibit binding to CD4+ but not CD8+ concanavalin A-activated rhesus monkey peripheral blood lymphocytes, and precipitate the CD4 molecule from surface-labeled activated rhesus monkey peripheral blood lymphocytes. Moreover, anti-viral activity is demonstrable in the IgG fraction of plasma from a human rsCD4-immunized monkey. These studies raise the possibility that a modified human CD4 molecule serving as an immunogen might elicit an antibody response that could potentially induce a beneficial therapeutic response in human immunodeficiency virus-infected individuals

  11. Assessment of the Role of the Central DNA Flap in Human Immunodeficiency Virus Type 1 Replication by Using a Single-Cycle Replication System

    OpenAIRE

    Ao, Zhujun; Yao, Xiaojian; Éric A Cohen

    2004-01-01

    In this study, reverse transcriptase (RT)- and integrase (IN)-defective human immunodeficiency virus type 1 (HIV-1) was transcomplemented with Vpr-RT-IN fusion proteins to delineate pol sequences important for HIV-1 replication. Our results reveal that a 194-bp sequence encompassing the 3′end of the IN gene and containing the central DNA flap is necessary and sufficient for efficient HIV-1 single-cycle replication in dividing and nondividing cells. Furthermore, we show that the central DNA fl...

  12. Proteome analysis of virus-host cell interaction: rabies virus replication in Vero cells in two different media.

    Science.gov (United States)

    Kluge, Sabine; Rourou, Samia; Vester, Diana; Majoul, Samy; Benndorf, Dirk; Genzel, Yvonne; Rapp, Erdmann; Kallel, Héla; Reichl, Udo

    2013-06-01

    The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection. PMID:23674149

  13. Construction and Use of a Replication-Competent Human Immunodeficiency Virus (HIV-1) that Expresses the Chloramphenicol Acetyltransferase Enzyme

    Science.gov (United States)

    Terwilliger, E. F.; Godin, B.; Sodroski, J. G.; Haseltine, W. A.

    1989-05-01

    The construction and properties of an infectious human immunodeficiency virus (HIV) that expresses the bacterial gene chloramphenicol acetyltransferase are described. This virus can be used in vitro to screen for drugs that inhibit HIV infection. The marked virus may also be used to trace the routes of infection from the site of inoculation in animal experiments.

  14. Inducible gene expression of the human immunodeficiency virus LTR in a replication-incompetent herpes simplex virus vector.

    Science.gov (United States)

    Warden, M P; Weir, J P

    1996-12-01

    Although replication-incompetent herpes simplex virus (HSV) vectors have the capability to express foreign genes, successful development of these vectors for gene delivery would require that expression of the foreign gene be regulated. To investigate the feasibility of obtaining inducible expression of a foreign gene in such a vector, a replication-incompetent HSV vector, vd120/LTR beta, was developed that used the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) to express the Escherichia coli lacZ gene. Examination of lacZ expression from the HIV-1 LTR in vd120/LTR beta-infected cells indicated that the LTR was active as a promoter under both replicating and nonreplicating conditions, although expression of lacZ under nonreplicating conditions was approximately 4-fold lower. In addition, the LTR expressed lacZ in a manner distinct from that of well-characterized HSV-1 promoters of each temporal class. The effect of the HIV-1 regulatory protein Tat on expression from the LTR in vd120/LTR beta was examined by infection of two different HeLa-derived cell lines that constitutively expressed Tat, HL2/3, and HLtat. Compared to infection of HeLa cells, lacZ expression from vd120/LTR beta-infected HL2/3 and HLtat cells increased from 4- to 24-fold, depending on the multiplicity of vector infection. Sustained expression of lacZ from the LTR in vd120/LTR beta-infected cells was not observed even in the continuous presence of Tat, although vector could be recovered for up to 5 days after infection. However, the amount of recoverable vector decreased during this time, suggesting that cellular cytotoxicity may account for some of the decrease in Tat-mediated expression from the LTR. PMID:8941330

  15. A Functional Role for ADAM10 in Human Immunodeficiency Virus Type-1 Replication

    Directory of Open Access Journals (Sweden)

    Rubin Donald H

    2011-05-01

    Full Text Available Abstract Background Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted ADAM10 (A Disintegrin and Metalloprotease 10 allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1. Results Silencing ADAM10 expression with small interfering RNA (siRNA 48 hours before infection significantly inhibited HIV-1 replication in primary human monocyte-derived macrophages and in CD4+ cell lines. In agreement, ADAM10 over-expression significantly increased HIV-1 replication. ADAM10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of ADAM10 expression. Integration of HIV-1 cDNA was reduced in ADAM10 down-regulated cells; however, concomitant 2-LTR circle formation was not detected, suggesting that HIV-1 does not enter the nucleus. Further, ADAM10 silencing inhibited downstream reporter gene expression and viral protein translation. Interestingly, we found that while the metalloprotease domain of ADAM10 is not required for HIV-1 replication, ADAM15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of ADAM10 from the plasma membrane, respectively do support productive infection. Conclusions We propose that ADAM10 facilitates replication at the level of nuclear trafficking. Collectively, our data support a model whereby ADAM10 is cleaved by ADAM15 and γ-secretase and that the ADAM10 intracellular domain directly facilitates HIV-1 nuclear trafficking. Thus, ADAM10 represents a novel cellular target class for development of antiretroviral drugs.

  16. Inhibition of visna virus replication and cytopathic effect in sheep choroid plexus cell cultures by selected anti-HIV agents.

    Science.gov (United States)

    Thormar, H; Balzarini, J; Debyser, Z; Witvrouw, M; Desmyter, J; De Clercq, E

    1995-05-01

    Several anti-HIV agents were tested against visna virus replication and cytopathic effect (CPE) in sheep choroid plexus cell cultures. Sulphated polysaccharides (i.e., dextran sulphate, pentosan polysulphate and heparin) and plant lectins, which inhibit viral adsorption and fusion, were found to be 10- to 40-fold less active against visna virus than against HIV. Bicyclam derivatives were at least 250-fold less active against visna virus and the highly HIV-1 specific TIBO derivatives were without a significant inhibitory effect on visna virus at subtoxic concentrations. In contrast, several 2',3'-dideoxynucleosides and acyclic nucleoside phosphonate analogues, which inhibit reverse transcription, were found to be very effective inhibitors of visna virus replication and viral CPE in cell culture. PMID:7486958

  17. Comparison of the replication and transmissibility of two infectious laryngotracheitis virus chicken embryo origin vaccines delivered via drinking water.

    Science.gov (United States)

    Coppo, Mauricio J C; Devlin, Joanne M; Noormohammadi, Amir H

    2012-01-01

    Infectious laryngotracheitis (ILT) is an acute infectious viral disease that affects chickens, causing respiratory disease, loss of production and mortality in severe cases. Biosecurity measures and administration of attenuated viral vaccine strains are commonly used to prevent ILT. It is notable that most recent ILT outbreaks affecting the intensive poultry industry have been caused by vaccine-related virus strains. The purpose of this study was to characterize and compare viral replication and transmission patterns of two attenuated chicken embryo origin ILT vaccines delivered via the drinking water. Two groups of specific pathogen free chickens were each inoculated with SA-2 ILT or Serva ILT vaccine strains. Unvaccinated birds were then placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days over a period of 60 days and examined for the presence and amount of virus using a quantitative polymerase chain reaction. A rapid increase in viral genome copy numbers was observed shortly after inoculation with SA-2 ILT virus. In contrast, a comparatively delayed virus replication was observed after vaccination with Serva ILT virus. Transmission to in-contact birds occurred soon after exposure to Serva ILT virus but only several days after exposure to SA-2 ILT virus. Results from this study demonstrate in vivo differences between ILT vaccine strains in virus replication and transmission patterns. PMID:22515537

  18. Pathogenesis mediated by proviral host factors involved in translation and replication of plant positive-strand RNA viruses.

    Science.gov (United States)

    Hyodo, Kiwamu; Okuno, Tetsuro

    2016-04-01

    Viral pathogenesis comes from complex interactions between viruses and hosts. All the processes of viral infection, including translation of viral factors and replication of viral genomes, define viral pathogenesis; therefore, molecular insights into the mechanisms underlying viral replication strategies unambiguously pave the way for our comprehensive understanding of viral pathogenesis and disease outcome, as well as for developing new antiviral strategies against plant virus disease. Recent studies of plant positive-strand RNA [(+)RNA] viruses have advanced our understanding of co-opted host factors and their roles in viral translation and replication. It is becoming clear that plant (+)RNA viruses harness host factors involved in membrane trafficking and lipid metabolism to establish the viral replication complex (VRC). In this review, we aim to discuss the contribution of co-opted host factors in translation and genome replication of plant (+)RNA viruses mainly focusing on those involved in the biogenesis of the VRC, which may act as a central hub in almost all the processes of viral infection as well as viral pathogenesis. PMID:26651023

  19. Human Immunodeficiency Virus Replication in a Primary Effusion Lymphoma Cell Line Stimulates Lytic-Phase Replication of Kaposi’s Sarcoma-Associated Herpesvirus

    OpenAIRE

    Varthakavi, Vasundhara; Browning, Philip J.; Spearman, Paul

    1999-01-01

    Human immunodeficiency virus (HIV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) coinfect many individuals in North America and in parts of Africa. Infection with HIV is a leading risk factor for the development of Kaposi’s sarcoma (KS). In this study, we tested the hypothesis that HIV infection of common or adjacent cells would stimulate replication and spread of KSHV. Infection of a primary effusion lymphoma cell line by vesicular stomatitis virus type G-pseudotyped HIV type 1 led to a...

  20. 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. PMID:26143474

  1. Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549 cells

    Directory of Open Access Journals (Sweden)

    Wang Jianwei

    2011-08-01

    Full Text Available Abstract Background Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. Results We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP family, was significantly induced in human lung epithelial (A549 cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. Conclusions LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.

  2. 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

    Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) 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 to...... findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly...

  3. PKR activation enhances replication of classical swine fever virus in PK-15 cells.

    Science.gov (United States)

    Liu, Wen-Jun; Yang, You-Tian; Zhao, Ming-Qiu; Dong, Xiao-Ying; Gou, Hong-Chao; Pei, Jing-Jing; Chen, Jin-Ding

    2015-06-01

    Classical swine fever (CSF) is a highly contagious swine disease that is responsible for economic losses worldwide. Protein kinase R (PK)R is an important protein in the host viral response; however, the role of PKR in CSFV infection remains unknown. This issue was addressed in the present study using the PK-15 swine kidney cell line. We found that CSFV infection increased the phosphorylation of eukaryotic translation initiation factor (eIF)2α and its kinase PKR. However, the expression of viral proteins continued to increase. Furthermore, PKR overexpression enhanced CSFV replication, while PKR inhibition resulted in reduced CSFV replication and an increase in interferon (IFN) induction. In addition, PKR was responsible for eIF2α phosphorylation in CSFV-infected cells. These results suggest that the activation of PKR during CSFV infection is beneficial to the virus. The virus is able to commandeer the host cell's translation machinery for viral protein synthesis while evading innate immune defenses. PMID:25899421

  4. Transient fasting enhances replication of oncolytic herpes simplex virus in glioblastoma

    Science.gov (United States)

    Esaki, Shinichi; Rabkin, Samuel D; Martuza, Robert L; Wakimoto, Hiroaki

    2016-01-01

    Short-term nutritional restriction (fasting) has been shown to enhance the efficacy of chemotherapy by sensitizing cancer cells and protecting normal cells in a variety of cancer models, including glioblastoma (GBM). Cancer cells, unlike normal cells, respond to fasting by promoting oncogenic signaling and protein synthesis. We hypothesized that fasting would increase the replication of oncolytic herpes simplex virus (oHSV) in GBM. Patient-derived GBM cell lines were fasted by growth in glucose and fetal calf serum restricted culture medium. “Transient fasting”, 24-hour fasting followed by 24-hour recovery in complete medium, increased late virus gene expression and G47Δ yields about 2-fold in GBM cells, but not in human astrocytes, and enhanced G47Δ killing of GBM cells. Mechanistically, “transient fasting” suppressed phosphorylation of the subunit of eukaryotic initiation factor 2α (eIF2α) and c-Jun N-terminal kinases (JNK) in GBM cells, but not in astrocytes. Pharmacological inhibition of JNK also increased G47Δ yield. In vivo, transient fasting (48-hour food restriction and 24-hour recovery) doubled luciferase activity after intratumoral G47Δ-US11fluc injection into orthotopic GBM xenografts. Thus, “transient fasting” increases G47Δ replication and oncolytic activity in human GBM cells. These results suggest that “transient fasting” may be effectively combined to enhance oncolytic HSV therapy of GBM. PMID:27186404

  5. Establishment of hepatitis C virus RNA-replicating cell lines possessing ribavirin-resistant phenotype.

    Directory of Open Access Journals (Sweden)

    Shinya Satoh

    Full Text Available Ribavirin (RBV is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.By repetitive RBV (100 μM treatment (10 weeks of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM. By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the

  6. Asthma and influenza virus infection:focusing on cell death and stress pathways in influenza virus replication.

    Directory of Open Access Journals (Sweden)

    Behzad Yeganeh

    2013-03-01

    Full Text Available Asthma is one of the fastest growing syndromes in many countries and is adding a huge cost to the health care system. Increasing reports have linked airway infectious diseases to asthma. Influenza is one of the most serious airway infectious diseases and in recent years there have been some serious influenza virus pandemics which caused increased fatality in numerous different populations. Diverse host response pathways during virus infection have been identified, including different cell death and survival pathways. These pathways include1 programmed cell death I (apoptosis, 2 programmed cell death II (autophagy, and 3 endoplasmic reticulum stress with subsequent unfolded protein response (UPR. There has been extensive research on the regulatory roles of these pathways during the influenza virus life cycle. These studies address the benefits of enhancing or inhibiting these pathways on viral replication. Here we review the most recent and significant knowledge in this area for possible  benefits  to  clinicians and  basic  scientist researchers  in  different  areas  of  the respiratory and virology sciences.

  7. Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication.

    Science.gov (United States)

    Wang, Yijin; Wang, Wenshi; Xu, Lei; Zhou, Xinying; Shokrollahi, Ehsan; Felczak, Krzysztof; van der Laan, Luc J W; Pankiewicz, Krzysztof W; Sprengers, Dave; Raat, Nicolaas J H; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2016-05-01

    Viruses 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 enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway

  8. The N-terminus of classical swine fever virus (CSFV) nonstructural protein 2 modulates viral genome RNA replication.

    Science.gov (United States)

    Li, Ling; Wu, Rui; Zheng, Fengwei; Zhao, Cheng; Pan, Zishu

    2015-12-01

    Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but poorly understood role in viral RNA replication and infectious virus production. In the present study, based on sequence analysis, we mutated several representative conserved residues within the N-terminus of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA replication and infectious virus production. Our results demonstrated that the mutation of two aspartic acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in modulating viral RNA replication. PMID:26232654

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

    OpenAIRE

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

    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 a...

  10. Interferon-inducible MyD88 protein inhibits hepatitis B virus replication

    International Nuclear Information System (INIS)

    Myeloid differential primary response protein (MyD88) is a critical component in the signaling cascade through Toll-like receptors (TLRs) and is induced by α interferon (IFN-α). To examine the role of MyD88 in the antiviral activity of IFN-α against hepatitis B virus (HBV), we established MyD88 stably expressing cell lines and studied HBV replication in these lines after transient transfection. The levels of HBV proteins and viral replicative intermediates were effectively reduced in MyD88-expressing cells. A significant reduction of total and cytoplasmic viral RNAs in MyD88 stably expressing cells was also observed. Using a nuclear factor-κB (NF-κB) dependent reporter assay, it was shown that activation of NF-κB was moderately increased in the presence of expression of MyD88, and further significantly increased by co-expression of HBV. These results suggest a novel mechanism for the inhibition of HBV replication by IFN-α via expression of MyD88 protein involving activation of NF-κB signaling pathway and downregulation of viral transcription

  11. 2',3'-cyclic nucleotide 3'-phosphodiesterases inhibit hepatitis B virus replication.

    Science.gov (United States)

    Ma, Hui; Zhao, Xing-Liang; Wang, Xue-Yan; Xie, Xing-Wang; Han, Jin-Chao; Guan, Wen-Li; Wang, Qin; Zhu, Lin; Pan, Xiao-Ben; Wei, Lai

    2013-01-01

    2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV. PMID:24260477

  12. 2',3'-cyclic nucleotide 3'-phosphodiesterases inhibit hepatitis B virus replication.

    Directory of Open Access Journals (Sweden)

    Hui Ma

    Full Text Available 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.

  13. 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.

  14. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Directory of Open Access Journals (Sweden)

    Nicholas D Weber

    Full Text Available Despite an existing effective vaccine, hepatitis B virus (HBV remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB, imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

  15. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    Science.gov (United States)

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  16. The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

    International Nuclear Information System (INIS)

    The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function

  17. Na~+/K~+-ATPase β1 subunit interacts with M2 proteins of influenza A and B viruses and affects the virus replication

    Institute of Scientific and Technical Information of China (English)

    GAO; George; Fu

    2010-01-01

    Interplay between the host and influenza virus has a pivotal role for the outcome of infection.The matrix proteins M2/BM2 from influenza (A and B) viruses are small type III integral membrane proteins with a single transmembrane domain,a short amino-terminal ectodomain and a long carboxy-terminal cytoplasmic domain.They function as proton channels,mainly forming a membrane-spanning pore through the transmembrane domain tetramer,and are essential for virus assembly and release of the viral genetic materials in the endosomal fusion process.However,little is known about the host factors which interact with M2/BM2 proteins and the functions of the long cytoplasmic domain are currently unknown.Starting with yeast two-hybrid screening and applying a series of experiments we identified that the β1 subunit of the host Na+/K+-ATPase β1 subunit (ATP1B1) interacts with the cytoplasmic domain of both the M2 and BM2 proteins.A stable ATP1B1 knockdown MDCK cell line was established and we showed that the ATP1B1 knockdown suppressed influenza virus A/WSN/33 replication,implying that the interaction is crucial for influenza virus replication in the host cell.We propose that influenza virus M2/BM2 cytoplasmic domain has an important role in the virus-host interplay and facilitates virus replication.

  18. The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

    2014-06-15

    The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.

  19. 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. PMID:26873065

  20. Cluster-cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

    OpenAIRE

    Alves, S. G.; M. L. Martins

    2010-01-01

    Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and \\textit{in vitro} drug testing. In the present paper, a cluster-cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simula...

  1. Foot-and-mouth disease virus replicates independently of phosphatidylinositol 4-phosphate and type III phosphatidylinositol 4-kinases.

    Science.gov (United States)

    Berryman, Stephen; Moffat, Katy; Harak, Christian; Lohmann, Volker; Jackson, Terry

    2016-08-01

    Picornaviruses form replication complexes in association with membranes in structures called replication organelles. Common themes to emerge from studies of picornavirus replication are the need for cholesterol and phosphatidylinositol 4-phosphate (PI4P). In infected cells, type III phosphatidylinositol 4-kinases (PI4KIIIs) generate elevated levels of PI4P, which is then exchanged for cholesterol at replication organelles. For the enteroviruses, replication organelles form at Golgi membranes in a process that utilizes PI4KIIIβ. Other picornaviruses, for example the cardioviruses, are believed to initiate replication at the endoplasmic reticulum and subvert PI4KIIIα to generate PI4P. Here we investigated the role of PI4KIII in foot-and-mouth disease virus (FMDV) replication. Our results showed that, in contrast to the enteroviruses and the cardioviruses, FMDV replication does not require PI4KIII (PI4KIIIα and PI4KIIIβ), and PI4P levels do not increase in FMDV-infected cells and PI4P is not seen at replication organelles. These results point to a unique requirement towards lipids at the FMDV replication membranes. PMID:27093462

  2. Partial deletion of stem-loop 2 in the 3' untranslated region of foot-and-mouth disease virus identifies a region that is dispensable for virus replication.

    Science.gov (United States)

    Biswal, Jitendra K; Subramaniam, Saravanan; Ranjan, Rajeev; Pattnaik, Bramhadev

    2016-08-01

    The 3' untranslated region (3' UTR) of the foot-and-mouth disease virus (FMDV) genome plays an essential role in virus replication, but the properties of the 3' UTR are not completely defined. In order to determine the role of different regions of the 3' UTR in FMDV replication, we conducted site-directed mutagenesis of the 3' UTR of FMDV serotype O IND R2/1975 using a cDNA clone. Through independent serial deletions in various regions of the 3' UTR, we demonstrated that deletion of nucleotides between the stem-loop (SL) structures and in the beginning and the end regions of the SL2 structure could be lethal for FMDV replication. However, a block deletion of 20 nucleotides (nt 60 to 79) in the middle of SL2 did not affect the viability of FMDV in cultured cells. Characterisation of the deletion mutant virus (O(R2/1975-Δ3'UTR 60-79)) revealed no significant difference in growth kinetics or RNA replication ability compared to the parental virus. However, the mutant virus produced slightly larger plaques when compared to the parental virus. This is the first description of a dispensable 20-nucleotide region in SL2 of the FMDV 3' UTR. PMID:27233801

  3. Inhibitory effects of Pycnogenol® on hepatitis C virus replication.

    Science.gov (United States)

    Ezzikouri, Sayeh; Nishimura, Tomohiro; Kohara, Michinori; Benjelloun, Soumaya; Kino, Yoichiro; Inoue, Kazuaki; Matsumori, Akira; Tsukiyama-Kohara, Kyoko

    2015-01-01

    Chronic hepatitis C virus (HCV) infection increases the risk of liver cirrhosis and hepatocellular carcinoma. In the last decade, the current standard HCV treatment, pegylated interferon and ribavirin, have limited efficacy and significant side effects. Novel direct acting antivirals show promise, but escape mutants are expected, along with potential side effects. Pycnogenol®, a French maritime pine extract, has been reported to have antioxidant and antiviral effects. Here, we evaluated the effect of Pycnogenol® on HCV replication. Wild-type and protease inhibitor (VX-950; telaprevir)-resistant HCV replicon cells were treated with Pycnogenol®, Pycnogenol® and interferon-alpha, and ribavirin and telaprevir. Pycnogenol® effects on replication were also evaluated in HCV-infected chimeric mice. Pycnogenol® treatment showed antiviral effects without cytotoxicity at doses up to 50 μg/mL. Pycnogenol® in combination with interferon-alpha or ribavirin showed synergistic effects. Moreover, Pycnogenol® inhibited HCV replication in telaprevir-resistant replicon cells; telaprevir and Pycnogenol® acted additively to reduce HCV RNA levels in wild-type HCV replicon cells without significantly increasing cytotoxicity. Pycnogenol® antiviral activity was higher than its components procyanidin and taxifolin. Further, treatment of infected chimeric mice with Pycnogenol® suppressed HCV replication and showed a synergistic effect with interferon-alpha. In addition, Pycnogenol® treatment resulted in dose-dependent reduction of reactive oxygen species in HCV replicon cell lines. Pycnogenol® is a natural product that may be used to improve the efficacy of the current standard antiviral agents and even to eliminate resistant HCV mutants. PMID:25446333

  4. TAGLN2, a novel regulator involved in Hepatitis B virus transcription and replication.

    Science.gov (United States)

    Yu, Youjia; He, Zhiliang; Cao, Yong; Tang, Hong; Huang, Feijun

    2016-09-01

    Hepatitis B virus (HBV) infection is one of the major health problems in the world. Transgelin-2 (TAGLN2) expression has been revealed to be significantly altered in previous studies concerning HBV-host interaction. The present study investigated TAGLN2 expression patterns in HBV related hepatocellular carcinoma (HCC) tissues and its role in HBV transcription and replication. We collected 59 HBV related HCC tissue samples, their adjacent non-tumoral tissues and 16 normal livers to make the tissue microarray. TAGLN2 protein was detected by immunohistochemistry and the transcriptional levels of TAGLN2, HBc, HBs and HBx were detected by qRT-PCR. Then we investigated the function of TAGLN2 on HBV transcription and replication in vitro by ectopic expressing or knocking down TAGLN2 in HepG2 and HepG2.2.15 cell lines. We further studied the effect of HBx on TAGLN2 expression with a Tet-on HBx expressing cell line. TAGLN2 protein expression was lower in normal livers and HBV-HCC tissues comparing to adjacent non-tumoral tissues. The transcriptional levels of TAGLN2 in HBV-HCC tissues and their adjacent tissues were positively related to that of HBc, HBs and HBx (P < 0.05). Ectopic expression of TAGLN2 in vitro could enhance HBV transcription and replication while suppressing TAGLN2 had the contrary effect. TAGLN2 could be induced by HBx in a dose-dependent manner. Our data demonstrated that TAGLN2 might be an HBx induced positive host factor involved in HBV transcription and replication and HBx related liver fibrosis and tumorigenesis. PMID:27402267

  5. A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

    Directory of Open Access Journals (Sweden)

    Hui Feng

    Full Text Available BACKGROUND: The specific interaction between hepatitis B virus (HBV polymerase (P protein and the ε RNA stem-loop on pregenomic (pg RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP, to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.

  6. Animal Models for Influenza Viruses: Implications for Universal Vaccine Development

    Directory of Open Access Journals (Sweden)

    Irina Margine

    2014-10-01

    Full Text Available Influenza virus infections are a significant cause of morbidity and mortality in the human population. Depending on the virulence of the influenza virus strain, as well as the immunological status of the infected individual, the severity of the respiratory disease may range from sub-clinical or mild symptoms to severe pneumonia that can sometimes lead to death. Vaccines remain the primary public health measure in reducing the influenza burden. Though the first influenza vaccine preparation was licensed more than 60 years ago, current research efforts seek to develop novel vaccination strategies with improved immunogenicity, effectiveness, and breadth of protection. Animal models of influenza have been essential in facilitating studies aimed at understanding viral factors that affect pathogenesis and contribute to disease or transmission. Among others, mice, ferrets, pigs, and nonhuman primates have been used to study influenza virus infection in vivo, as well as to do pre-clinical testing of novel vaccine approaches. Here we discuss and compare the unique advantages and limitations of each model.

  7. 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

  8. Replication of Infectious Bronchitis Virus in the Chicken Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    M.H. Mohammed

    2012-06-01

    Full Text Available The susceptibility of the chicken mesenchymal stem cells to infectious bronchitis virus was characterized after twenty consecutive passages in chicken mesenchymal stemm cells. Virus replication was monitored by cytopathic observation, indirect immunoperoxidase, and reverse transcription polymerase chain reaction. At 72 h post-infection (p.i. in third passage, the cytopathic effect was characterized by rounding up of cell, monolayer detachment, intracytoplasmic brownish colouration was readily observed by from 24h p.i in third passage, and at all times the extracted viral RNA from IBV-infected monolayers was demonstrated by reverse transcription polymerase chain reaction. Tissue culture effective dose50 was used to measure virus titration performed on chicken mesenchymal stem cells and the titres in twenty passages was 108.6 TID50/ml. The results obtained in this study suggested that the chicken mesenhymal stem cells can be used for adaptation IBV and may be considered a step forward for the use of these cells in the future for IBV vaccine production

  9. Extra-hepatic replication and infection of hepatitis E virus in neuronal-derived cells.

    Science.gov (United States)

    Drave, S A; Debing, Y; Walter, S; Todt, D; Engelmann, M; Friesland, M; Wedemeyer, H; Neyts, J; Behrendt, P; Steinmann, E

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. Infection usually leads to acute hepatitis that can become fulminant, particularly among pregnant women and in patients with preexisting liver disease, or may evolve to a chronic state, especially in immunosuppressed individuals. HEV has been shown to produce a range of extra-hepatic manifestations including aplastic anaemia, acute thyroiditis, glomerulonephritis as well as neurological disorders such as Guillain-Barré syndrome, neuralgic amyotrophy and encephalitis. The pathogenesis of these neurological injuries remains largely unknown, and it is also uncertain whether or not HEV can directly infect neuronal cells. In this study, we investigated whether HEV is capable of completing the viral life cycle in human neuronal-derived cell lines such as neuroepithelioma (SK-N-MC), desmoplastic cerebellar medulloblastoma (DAOY), glioblastoma multiforme (DBTRG), glioblastoma astrocytoma (U-373 MG) and oligodendrocytic (M03.13) cells. Following transfection of these cells with HEV Gaussia luciferase reporter virus, all tested cell lines supported HEV RNA replication. Furthermore, extra- and intracellular viral capsid was detected by an HEV antigen ELISA as a marker for virus assembly and release. Permissiveness for HEV cell entry could be demonstrated for the oligodendrocytic cell line M03.13. In conclusion, these results indicate that HEV tropism is not restricted to the liver and HEV can potentially complete the full viral life cycle in neuronal-derived tissues explaining neurologic disorders during HEV infection. PMID:26891712

  10. Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

    Science.gov (United States)

    Herod, Morgan R.; Ferrer-Orta, Cristina; Loundras, Eleni-Anna; Ward, Joseph C.; Verdaguer, Nuria; Rowlands, David J.

    2016-01-01

    ABSTRACT The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome

  11. Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication.

    Science.gov (United States)

    Li, Yinzi; Xiong, Ruyi; Bernards, Mark; Wang, Aiming

    2016-01-01

    Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb. PMID:27456972

  12. RNASEK Is a V-ATPase-Associated Factor Required for Endocytosis and the Replication of Rhinovirus, Influenza A Virus, and Dengue Virus

    Directory of Open Access Journals (Sweden)

    Jill M. Perreira

    2015-08-01

    Full Text Available Human rhinovirus (HRV causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.

  13. Experimental infection of mice with Borna disease virus (BDV): replication and distribution of the virus after intracerebral infection.

    Science.gov (United States)

    Enbergs, H K; Vahlenkamp, T W; Kipar, A; Müller, H

    2001-06-01

    To develop an animal model resembling natural asymptomatic Borna disease virus (BDV) infections, BDV He/80 rat brain homogenate was passaged four times in adult SJL/J mice. Within 12 months of observation, mice did not develop overt signs of disease. Nucleotide sequencing of the rat isolate and the mouse isolates at the fourth passage revealed no difference in the deduced amino acids. Viral RNA was found in brain, heart, kidney, lung, liver, and urinary bladder. Infectious virus was isolated from brain, but also from heart and lung tissue. Immunohistochemically, BDV was demonstrated in nerves in the abdominal cavity, ganglion coeliacum, and adrenal glands, but not in organ parenchyma. Occasionally, viral RNA was detected in mononuclear blood cells. PMID:11517401

  14. Lamivudine Inhibits the Replication of ALV-J Associated Acutely Transforming Virus and its Helper Virus and Tumor Growth In vitro and In vivo.

    Science.gov (United States)

    Wang, Yixin; Xu, Shuzhen; Li, Sifei; Su, Hongqin; Chang, Shuang; Li, Yang; Sun, Xiaolong; Zhao, Peng; Cui, Zhizhong

    2015-01-01

    To study the antiviral effects of lamivudine on avian leukosis virus subgroup J (ALV-J) and its inhibitory effect on the growth of fibrosarcomas caused by acute transforming avian leukosis virus, a series of experiments were performed in chicken embryo fibroblast cultures and 1-day-old chickens inoculated with an acutely transforming viral stock Fu-J (SDAU1005). This stock was prepared from an acutely fibrosarcoma of field cases in chicken farms and contained both the replication-defective virus Fu-J carrying v-fps oncogene and its helper virus ALV-J strain SDAU1005. The results from three different assays in cell cultures demonstrated the significant inhibitory effect of lamivudine on the replication of both SDAU1005 and Fu-J viruses. Furthermore, the effect was dose dependent in the concentration range of 1-4 μg/ml. In chicken experiments, lamivudine could decrease the viral loads of SDAU1005 and Fu-J in the plasma of inoculated chickens, delay the appearance of acute sarcomas, and decrease chicken mortality in the early stage. This model may be used to directly evaluate the inhibitory effects of lamivudine on such tumors and to understand the relationship between the replication-defective virus and its helper virus while also assessing tumor processes. PMID:26648914

  15. Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model

    Science.gov (United States)

    Li, Zejun; Chen, Hualan; Jiao, Peirong; Deng, Guohua; Tian, Guobin; Li, Yanbing; Hoffmann, Erich; Webster, Robert G.; Matsuoka, Yumiko; Yu, Kangzhen

    2005-01-01

    We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans. PMID:16140781

  16. mRNA decay factor AUF1 binds the internal ribosomal entry site of enterovirus 71 and inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Jing-Yi Lin

    Full Text Available AU-rich element binding factor 1 (AUF1 has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES of enterovirus 71 (EV71 and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

  17. Replication characteristics of infectious laryngotracheitis virus in the respiratory and conjunctival mucosa.

    Science.gov (United States)

    Reddy, Vishwanatha R A P; Steukers, Lennert; Li, Yewei; Fuchs, Walter; Vanderplasschen, Alain; Nauwynck, Hans J

    2014-01-01

    Avian infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus of poultry that is spread worldwide. ILTV enters its host via the respiratory tract and the eyes. Although ILTV has been known for a long time, the replication characteristics of the virus in the respiratory and conjunctival mucosa are still poorly studied. To study these characteristics, two in vitro explant models were developed. Light microscopy and fluorescent terminal deoxynucleotidyl transferase dUTP nick end-labelling staining were used to evaluate the viability of mucosal explants, which were found to be viable up to the end of the experiment at 96 h of cultivation. The tracheal and conjunctival mucosal explants were inoculated with ILTV and collected at 0, 24, 48 and 72 h post inoculation (p.i.). ILTV spread in a plaque-wise manner in both mucosae. A reproducible quantitative analysis of this mucosal spread was evaluated by measuring plaque numbers, plaque latitude and invasion depth underneath the basement membrane. No major differences in plaque numbers were observed over time. Plaque latitude progressively increased to 70.4 ± 12.9 μm in the trachea and 97.8 ± 9.5 μm in the conjunctiva at 72 h p.i. The virus had difficulty crossing the basement membrane and was first observed only at 48 h p.i. The virus was observed at 72 h p.i. in 56% (trachea) and 74% (conjunctiva) of the plaques. Viability analysis of infected explants indicated that ILTV blocks apoptosis in infected cells of both mucosae but activates apoptosis in bystander cells. PMID:25144137

  18. Replication of a chimeric origin containing elements from Epstein-Barr virus ori P and bovine papillomavirus minimal origin.

    Science.gov (United States)

    Kivimäe, S; Allikas, A; Kurg, R; Ustav, M

    2001-05-01

    The bovine papillomavirus E2 protein is a multifunctional protein that activates viral transcription, co-operates in initiation of viral DNA replication, and is required for long-term episomal maintenance of viral genomes. The EBNA1 protein of Epstein-Barr virus is required for synthesis and maintenance of Epstein-Barr virus genomes. Both viral proteins act through direct interactions with their respective DNA sequences in their origins of replication. The chimeric protein E2:EBNA1, which consists of an transactivation domain of E2 and DNA binding domain of EBNA1 supported the replication of the chimeric origin that contained EBNA1 binding sites in place of the E2 binding sites principally as full-length E2 did in the case of papillomavirus minimal origin. This indicates that the chimeric protein E2:EBNA1 is competent to assemble a replication complex similar to the E2 protein. These data confirm the earlier observations that the only part of E2 specifically required for its activity in replication is the N-terminal activation domain and the function of the DNA binding domain of E2 in the initiation of replication is to tether the transactivation domain of E2 to the origin of replication. PMID:11311423

  19. Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

    International Nuclear Information System (INIS)

    The West Nile virus strain Kunjin virus (WNVKUN) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNVKUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNVKUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein

  20. Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Ambrose, R.L.; Mackenzie, J.M., E-mail: jason.mackenzie@unimelb.edu.au

    2015-07-15

    The West Nile virus strain Kunjin virus (WNV{sub KUN}) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV{sub KUN} replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV{sub KUN} replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein.

  1. The UL47 gene of avian infectious laryngotracheitis virus is not essential for in vitro replication but is relevant for virulence in chickens.

    Science.gov (United States)

    Helferich, Dorothee; Veits, Jutta; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

    2007-03-01

    The genome of infectious laryngotracheitis virus (ILTV) exhibits several differences from those of other avian and mammalian alphaherpesviruses. One of them is the translocation of the conserved UL47 gene from the unique long (UL) to the unique short (US) genome region, where UL47 is inserted upstream of the US4 gene homologue. As in other alphaherpesviruses, UL47 encodes a major tegument protein of ILTV particles, whereas the US4 gene product is a non-structural glycoprotein, gG, which is secreted from infected cells. For functional characterization, an ILTV recombinant was isolated in which US4 together with the 3'-terminal part of UL47 was replaced by a reporter gene cassette encoding green fluorescent protein. From this virus, UL47 and US4 single-gene deletion mutants without foreign sequences were derived and virus revertants were also generated. In vitro studies revealed that both genes were non-essential for ILTV replication in cultured cells. Whereas US4-negative ILTV exhibited no detectable growth defects, maximum virus titres of the double deletion mutant and of UL47-negative ILTV were reduced about 10-fold compared with those of wild-type virus and rescued virus. Experimental infection of chickens demonstrated that UL47-negative ILTV was significantly attenuated in vivo and was shed in reduced amounts, whereas wild-type and rescued viruses caused severe disease and high mortality rates. As all immunized animals were protected against subsequent challenge infection with virulent ILTV, the UL47 deletion mutant might be suitable as a live-virus vaccine. PMID:17325345

  2. Membrane-bound SIV envelope trimers are immunogenic in ferrets after intranasal vaccination with a replication-competent canine distemper virus vector.

    Science.gov (United States)

    Zhang, Xinsheng; Wallace, Olivia; Wright, Kevin J; Backer, Martin; Coleman, John W; Koehnke, Rebecca; Frenk, Esther; Domi, Arban; Chiuchiolo, Maria J; DeStefano, Joanne; Narpala, Sandeep; Powell, Rebecca; Morrow, Gavin; Boggiano, Cesar; Zamb, Timothy J; Richter King, C; Parks, Christopher L

    2013-11-01

    We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination. PMID:24074564

  3. H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets.

    Science.gov (United States)

    de Jonge, Jørgen; Isakova-Sivak, Irina; van Dijken, Harry; Spijkers, Sanne; Mouthaan, Justin; de Jong, Rineke; Smolonogina, Tatiana; Roholl, Paul; Rudenko, Larisa

    2016-05-01

    Avian influenza viruses continue to cross the species barrier, and if such viruses become transmissible among humans, it would pose a great threat to public health. Since its emergence in China in 2013, H7N9 has caused considerable morbidity and mortality. In the absence of a universal influenza vaccine, preparedness includes development of subtype-specific vaccines. In this study, we developed and evaluated in ferrets an intranasal live attenuated influenza vaccine (LAIV) against H7N9 based on the A/Leningrad/134/17/57 (H2N2) cold-adapted master donor virus. We demonstrate that the LAIV is attenuated and safe in ferrets and induces high hemagglutination- and neuraminidase-inhibiting and virus-neutralizing titers. The antibodies against hemagglutinin were also cross-reactive with divergent H7 strains. To assess efficacy, we used an intratracheal challenge ferret model in which an acute severe viral pneumonia is induced that closely resembles viral pneumonia observed in severe human cases. A single- and two-dose strategy provided complete protection against severe pneumonia and prevented virus replication. The protective effect of the two-dose strategy appeared better than the single dose only on the microscopic level in the lungs. We observed, however, an increased lymphocytic infiltration after challenge in single-vaccinated animals and hypothesize that this a side effect of the model. PMID:26796670

  4. CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

    Science.gov (United States)

    Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T

    2016-01-01

    Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications. PMID:26796669

  5. Epithelial Distribution and Replication of Foot-and-Mouth Disease Virus RNA in Infected Pigs

    DEFF Research Database (Denmark)

    Durand, S.; Murphy, C.; Zhang, Z.;

    2008-01-01

    Although the pathogenesis of foot-and-mouth disease (FMD) has been extensively investigated relatively few studies have addressed the localization of FMD virus (FMDV) and in particular its replication in relation to the typical in-vivo sites of FMD lesions. In the present study, pigs were infected...... experimentally with FMDV serotype O UKG 34/2001 and tissue samples were collected from I to 4 clays post-infection. Samples were stored at -70 degrees C and frozen sections were prepared for in-situ hybridization (ISH). A digoxigenin-labelled RNA probe complementary to a coding part of the RNA-dependent RNA...... polymerase (3D) genomic region was prepared. The FMDV positive strand RNA was prominent in the basal layers of the epithelium. A diffuse positive signal was also noted in the cytoplasm of cells of the stratum spinosum of lesional epithelium, but there was no signal in the stratum corneum. Detection of FMDV...

  6. 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. PMID:27106619

  7. 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.

  8. Large Animal Models for Foamy Virus Vector Gene Therapy

    Directory of Open Access Journals (Sweden)

    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.

  9. A unicellular algal virus, Emiliania huxleyi virus 86, exploits an animal-like infection strategy.

    Science.gov (United States)

    Mackinder, Luke C M; Worthy, Charlotte A; Biggi, Gaia; Hall, Matthew; Ryan, Keith P; Varsani, Arvind; Harper, Glenn M; Wilson, William H; Brownlee, Colin; Schroeder, Declan C

    2009-09-01

    Emiliania huxleyi virus 86 (EhV-86) belongs to the family Phycodnaviridae, a group of viruses that infect a wide range of freshwater and marine eukaryotic algae. Phycodnaviridae is one of the five families that belong to a large and phylogenetically diverse group of viruses known as nucleocytoplasmic large dsDNA viruses (NCLDVs). To date, our understanding of algal NCLDV entry is based on the entry mechanisms of members of the genera Chlorovirus and Phaeovirus, both of which consist of non-enveloped viruses that 'inject' their genome into their host via a viral inner-membrane host plasma membrane fusion mechanism, leaving an extracellular viral capsid. Using a combination of confocal and electron microscopy, this study demonstrated for the first time that EhV-86 differs from its algal virus counterparts in two fundamental areas. Firstly, its capsid is enveloped by a lipid membrane, and secondly, EhV-86 enters its host via either an endocytotic or an envelope fusion mechanism in which an intact nucleoprotein core still encapsulated by its capsid is seen in the host cytoplasm. Real-time fluorescence microscopy showed that viral internalization and virion breakdown took place within the host on a timescale of seconds. At around 4.5 h post-infection, virus progeny were released via a budding mechanism during which EhV-86 virions became enveloped with host plasma membrane. EhV-86 therefore appears to have an infection mechanism different from that employed by other algal NCLDVs, with entry and exit strategies showing a greater analogy to animal-like NCLDVs. PMID:19474246

  10. Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

    OpenAIRE

    Lodmell, D L; Esposito, J J; Ewalt, L C

    1993-01-01

    We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not ...

  11. Mutations in the M-Gene Segment Can Substantially Increase Replication Efficiency of NS1 Deletion Influenza A Virus in MDCK Cells

    NARCIS (Netherlands)

    Wielink, van R.; Harmsen, M.M.; Martens, D.E.; Peeters, B.P.H.; Wijffels, R.H.; Moormann, R.J.M.

    2012-01-01

    Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce high amounts of interferon (IFN). They are therefore considered as candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter

  12. Triterpenoid Saponins Isolated from Platycodon grandiflorum Inhibit Hepatitis C Virus Replication

    Directory of Open Access Journals (Sweden)

    Jong-Woo Kim

    2013-01-01

    Full Text Available Hepatitis C virus (HCV infection is a major cause of liver disease, including cirrhosis and hepatocellular carcinoma. Due to significant adverse effects and emergence of resistant strains of currently developed anti-HCV agents, plant extracts have been considered to be potential sources of new bioactive compounds against HCV. The aim of this study was to evaluate the functional effects of triterpenoid saponins contained in the root extract of Platycodon grandiflorum (PG on viral enzyme activities and replication in both HCV replicon cells and cell culture grown HCV- (HCVcc- infected cells. Inhibitory activities of triterpenoid saponins from PG were verified by NS5B RNA-dependent RNA polymerase assay and were further confirmed in the context of HCV replication. Six triterpenoid saponins (platycodin D, platycodin D2, platycodin D3, deapioplatycodin D, deapioplatycodin D2, and platyconic acid A, PG saponin mixture (PGSM, were identified as active components exerting anti-HCV activity. Importantly, PGSM exerted synergistic anti-HCV activity in combination with either interferon-α or NS5A inhibitors. We demonstrated that combinatorial treatment of PGSM and IFN-α efficiently suppressed colony formation with significant reduction in drug resistant variant of HCV. These data suggest that triterpenoid saponin may represent a novel anti-HCV therapeutic agent.

  13. Triterpenoid Saponins Isolated from Platycodon grandiflorum Inhibit Hepatitis C Virus Replication.

    Science.gov (United States)

    Kim, Jong-Woo; Park, Sang Jin; Lim, Jong Hwan; Yang, Jae Won; Shin, Jung Cheul; Lee, Sang Wook; Suh, Joo Won; Hwang, Soon B

    2013-01-01

    Hepatitis C virus (HCV) infection is a major cause of liver disease, including cirrhosis and hepatocellular carcinoma. Due to significant adverse effects and emergence of resistant strains of currently developed anti-HCV agents, plant extracts have been considered to be potential sources of new bioactive compounds against HCV. The aim of this study was to evaluate the functional effects of triterpenoid saponins contained in the root extract of Platycodon grandiflorum (PG) on viral enzyme activities and replication in both HCV replicon cells and cell culture grown HCV- (HCVcc-) infected cells. Inhibitory activities of triterpenoid saponins from PG were verified by NS5B RNA-dependent RNA polymerase assay and were further confirmed in the context of HCV replication. Six triterpenoid saponins (platycodin D, platycodin D2, platycodin D3, deapioplatycodin D, deapioplatycodin D2, and platyconic acid A), PG saponin mixture (PGSM), were identified as active components exerting anti-HCV activity. Importantly, PGSM exerted synergistic anti-HCV activity in combination with either interferon- α or NS5A inhibitors. We demonstrated that combinatorial treatment of PGSM and IFN- α efficiently suppressed colony formation with significant reduction in drug resistant variant of HCV. These data suggest that triterpenoid saponin may represent a novel anti-HCV therapeutic agent. PMID:24489585

  14. Ginseng Protects Against Respiratory Syncytial Virus by Modulating Multiple Immune Cells and Inhibiting Viral Replication

    Directory of Open Access Journals (Sweden)

    Jong Seok Lee

    2015-02-01

    Full Text Available Ginseng has been used in humans for thousands of years but its effects on viral infection have not been well understood. We investigated the effects of red ginseng extract (RGE on respiratory syncytial virus (RSV infection using in vitro cell culture and in vivo mouse models. RGE partially protected human epithelial (HEp2 cells from RSV-induced cell death and viral replication. In addition, RGE significantly inhibited the production of RSV-induced pro-inflammatory cytokine (TNF-α in murine dendritic and macrophage-like cells. More importantly, RGE intranasal pre-treatment prevented loss of mouse body weight after RSV infection. RGE treatment improved lung viral clearance and enhanced the production of interferon (IFN-γ in bronchoalveolar lavage cells upon RSV infection of mice. Analysis of cellular phenotypes in bronchoalveolar lavage fluids showed that RGE treatment increased the populations of CD8+ T cells and CD11c+ dendritic cells upon RSV infection of mice. Taken together, these results provide evidence that ginseng has protective effects against RSV infection through multiple mechanisms, which include improving cell survival, partial inhibition of viral replication and modulation of cytokine production and types of immune cells migrating into the lung.

  15. Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans

    Directory of Open Access Journals (Sweden)

    Fan Yi-Hsin

    2011-11-01

    Full Text Available Abstract Background Sequence and structural elements in the 3'-untranslated region (UTR of Japanese encephalitis virus (JEV are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA which is collinear with the highly conserved regions of the 3'-UTR in JEV-infected cells. However, function of the sfRNA in JEV life cycle remains unknown. Results Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Trans-acting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation. Conclusions Our results indicate that sfRNA modulates viral translation and replication in trans.

  16. Mutagenesis in ORF AV2 affects viral replication in Mungbean yellow mosaic India virus

    Indian Academy of Sciences (India)

    A Rouhibakhsh; Q M I Haq; V G Malathi

    2011-06-01

    Mungbean yellow mosaic India virus (MYMIV) is a whitefly-transmitted begomovirus with a bipartite genome. We investigate the functions of the MYMIV-AV2 protein, the open reading frame present upstream of the coat protein gene in DNA A component. The ability of MYMIV-AV2 mutants to replicate, spread and cause symptoms in legume hosts, blackgram, cowpea and French bean was analysed. Plants agroinoculated with mutants K73R, C86S and the double mutant C84S, C86S showed increase in severity of symptoms compared with the wild type. However, mutants W2S and H14Q,G15E caused marked attenuation of symptoms. While the double mutants C84S,C86S caused a 50-fold increase in double-stranded supercoiled and single-stranded DNA accumulation, the mutations W2S and H14Q,G15E showed a decrease in double-stranded supercoiled and single-stranded viral DNA accumulation. Because AV2 mutants affect the ratio between open circular and supercoiled DNA forms, we hypothesize that these mutations may modulate the functions of the replication initiation protein.

  17. Inhibition of hepatitis B virus replication by quercetin in human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Zhikui; Cheng; Ge; Sun; Wei; Guo; Yayun; Huang; Weihua; Sun; Fei; Zhao; Kanghong; Hu

    2015-01-01

    Hepatitis B virus(HBV) infection is one of the most serious and prevalent viral diseases in the world. Although several anti-HBV drugs have been used clinically, their side and adverse effects limit treatment efficacy. Therefore, it is necessary to identify novel potential anti-HBV agents. The flavonol quercetin has shown activity against some retroviruses, but its effect on HBV remains unclear. In the present study, quercetin was incubated with Hep G2.2.15 cells, as well as Hu H-7 cells transfected with an HBV plasmid. Quercetin was shown to significantly reduce Hepatitis B surface antigen(HBs Ag) and Hepatitis B e antigen(HBe Ag), secretion and HBV genomic DNA levels in both cell lines. In addition, co-incubation with lamivudine(3TC), entecavir(ETV), or adefovir(Ade) further enhanced the quercetin-induced inhibition of HBV replication. This inhibition was partially associated with decreased heat shock proteins and HBV transcription levels. The results indicate that quercetin inhibited HBV antigen secretion and genome replication in human hepatoma cell lines, which suggests that quercetin may be a potentially effective anti-HBV agent.

  18. Replication and Translation of Sperm-introduced Hepatitis B Virus Genes in Embryonic Cells

    Directory of Open Access Journals (Sweden)

    Qiu-Ju Zhang

    2016-06-01

    Full Text Available Objective: Given that hepatitis B virus (HBV can infect human sperm cells and HBV genes introduced by sperm can be transcribed in developing embryos, we sought to confirm the presence of mature HBV antigens and to assess the mechanism of viral gene replication in embryonic cells. Methods: Human sperm were transfected with recombinant plasmids pIRES2-EGFP-HBs or pIRES2-EGFP-HBc and were then used to fertilize zona-free golden hamster ova. Two-celled embryos with and without green fluorescence were collected for detection of HBV DNA or protein by fluorescence in situ hybridization (FISH, immunofluorescence assay (IA, and enzyme-linked immunosorbent assay (ELISA. Results: 1. Positive FISH signals for HBs- and HBc-DNA were observed in the sperm nuclei and in the nuclei of the two-celled embryos in the test samples but were not detected in the controls. 2. Positive IA signals for HBsAg or HBcAg were detected within the cytoplasm of the two-celled embryos in the test samples but not in the controls. 3. Using ELISA, the level of HBsAg in a single two-celled embryo was 0.064 ng/ ml. Likewise, HBcAg was detected in the five-embryo experimental group. Conclusion: The sperm-introduced HBV S or C genes were able to replicate concomitantly host embryo genome via a semiconservative mode of DNA replication and were able to express viral proteins in the embryonic cells.

  19. Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Yan-Chang Lei; Dong-Liang Yang; You-Hua Hao; Zheng-Mao Zhang; Yong-Jun Tian; Bao-Ju Wang; Yan Yang; Xi-Ping Zhao; Meng-Ji Lu; Fei-Li Gong

    2006-01-01

    AIM: 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.METHODS: 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.RESULTS: 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.CONCLUSION: 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.

  20. Cellular DNA repair cofactors affecting hepatitis B virus infection and replication

    Institute of Scientific and Technical Information of China (English)

    Fan Zhao; Ning-Bo Hou; Ting Song; Xiang He; Zi-Rui Zheng; Qing-Jun Ma; Li Li; Yan-Hong Zhang; Hui Zhong

    2008-01-01

    AIM: To investigate whether hepatitis B virus (HBV)infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication.METHODS: Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated (ATM)-Rad3-related protein (ATR), p21 and the level of phosphorylation of Chk1, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATM chemical inhibitors caffeine (CF) and theophylline (TP), or Chk1 inhibitor 7-hydroxystaurosporine (UCN01) was studied to determine whether they suppress cellular DNA damage response and MG132 inhibits proteasome.RESULTS: The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after MG132 treatment also sharply decreased the HBV DNA yield.CONCLUSION: HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatrnent of HBV infection.

  1. Animal model of mucosally transmitted human immunodeficiency virus type 1 disease: intravaginal and oral deposition of simian/human immunodeficiency virus in macaques results in systemic infection, elimination of CD4+ T cells, and AIDS.

    Science.gov (United States)

    Joag, S V; Adany, I; Li, Z; Foresman, L; Pinson, D M; Wang, C; Stephens, E B; Raghavan, R; Narayan, O

    1997-05-01

    Chimeric simian/human immunodeficiency virus (SHIV) consists of the env, vpu, tat, and rev genes of human immunodeficiency virus type 1 (HIV-1) on a background of simian immunodeficiency virus (SIV). We derived a SHIV that caused CD4+ cell loss and AIDS in pig-tailed macaques (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L. J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996) and used a cell-free stock of this virus (SHIV(KU-1)) to inoculate macaques by the intravaginal route. Macaques developed high virus burdens and severe loss of CD4+ cells within 1 month, even when inoculated with only a single animal infectious dose of the virus by the intravaginal route. The infection was characterized by a burst of virus replication that peaked during the first week following intravenous inoculation and a week later in the intravaginally inoculated animals. Intravaginally inoculated animals died within 6 months, with CD4+ counts of <30/microl in peripheral blood, anemia, weight loss, and opportunistic infections (malaria, toxoplasmosis, cryptosporidiosis, and Pneumocystis carinii pneumonia). To evaluate the kinetics of virus spread, we inoculated macaques intravaginally and euthanized them after 2, 4, 7, and 15 days postinoculation. In situ hybridization and immunocytochemistry revealed cells expressing viral RNA and protein in the vagina, uterus, and pelvic and mesenteric lymph nodes in the macaque euthanized on day 2. By day 4, virus-infected cells had disseminated to the spleen and thymus, and by day 15, global elimination of CD4+ T cells was in full progress. Kinetics of viral replication and CD4+ loss were similar in an animal inoculated with pathogenic SHIV orally. This provides a sexual-transmission model of human AIDS that can be used to study the pathogenesis of mucosal infection and to evaluate the efficacy of vaccines and drugs directed against HIV-1. PMID:9094679

  2. Virus-specific antibodies allow viral replication in the marginal zone, thereby promoting CD8+ T-cell priming and viral control

    Science.gov (United States)

    Duhan, Vikas; Khairnar, Vishal; Friedrich, Sarah-Kim; Zhou, Fan; Gassa, Asmae; Honke, Nadine; Shaabani, Namir; Gailus, Nicole; Botezatu, Lacramioara; Khandanpour, Cyrus; Dittmer, Ulf; Häussinger, Dieter; Recher, Mike; Hardt, Cornelia; Lang, Philipp A.; Lang, Karl S.

    2016-01-01

    Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-specific antibodies or virus-specific CD8+ T cells. We found that after systemic recall infection with LCMV-WE the presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast, specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain LCMV-Docile, viral replication in spleen was essential for the priming of CD8+ T cells and for viral control. In contrast to specific antibodies, memory CD8+ T cells inhibited viral replication in marginal zone but failed to protect mice from persistent viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control of persistent virus. PMID:26805453

  3. Virus-specific antibodies allow viral replication in the marginal zone, thereby promoting CD8(+) T-cell priming and viral control.

    Science.gov (United States)

    Duhan, Vikas; Khairnar, Vishal; Friedrich, Sarah-Kim; Zhou, Fan; Gassa, Asmae; Honke, Nadine; Shaabani, Namir; Gailus, Nicole; Botezatu, Lacramioara; Khandanpour, Cyrus; Dittmer, Ulf; Häussinger, Dieter; Recher, Mike; Hardt, Cornelia; Lang, Philipp A; Lang, Karl S

    2016-01-01

    Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-specific antibodies or virus-specific CD8(+) T cells. We found that after systemic recall infection with LCMV-WE the presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast, specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain LCMV-Docile, viral replication in spleen was essential for the priming of CD8(+) T cells and for viral control. In contrast to specific antibodies, memory CD8(+) T cells inhibited viral replication in marginal zone but failed to protect mice from persistent viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control of persistent virus. PMID:26805453

  4. RNA Interference Targeting VP1 Inhibits Foot-and-Mouth Disease Virus Replication in BHK-21 Cells and Suckling Mice

    OpenAIRE

    Chen, Weizao; Yan, Weiyao; Du, Qingyun; Fei, Liang; Liu, Mingqiu; Ni, Zheng; Sheng, Zutian; Zheng, Zhaoxin

    2004-01-01

    RNA interference (RNAi) is a powerful tool to silence gene expression posttranscriptionally. In this study, we evaluated the antiviral potential of small interfering RNA (siRNA) targeting VP1 of foot-and-mouth disease virus (FMDV), which is essential during the life cycle of the virus and plays a key role in virus attachment to susceptible cells. We investigated in vivo the inhibitory effect of VP1-specific siRNAs on FMDV replication in BHK-21 cells and suckling mice, a commonly used small an...

  5. Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model

    OpenAIRE

    Li, Zejun; Chen, Hualan; Jiao, Peirong; Deng, Guohua; Tian, Guobin; Li, Yanbing; Hoffmann, Erich; Webster, Robert G; Matsuoka, Yumiko; Yu, Kangzhen

    2005-01-01

    We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, ...

  6. Positive correlation between replication rate and pathotype of Marek’s disease virus strains in maternal antibody negative chickens

    Science.gov (United States)

    Pathotyping of new field strains of MDV requires both a long period of time and a large number of birds. Confirming a positive correlation of virus replication and pathotype may lead to faster and cheaper alternative pathotyping methods or as a screening assay for choosing isolates to be pathotyped....

  7. Sites of replication of bovine respiratory syncytial virus in naturally infected calves as determined by in situ hybridization

    DEFF Research Database (Denmark)

    Viuff, B.; Uttenthal, Åse; Tegtmeier, C.; Alexandersen, Søren

    1996-01-01

    Replication of bovine respiratory syncytial virus (BRSV) was studied in three naturally infected calves by in situ hybridization using strand-specific RNA probes. One of the calves was a 5-month-old Friesian, the other two calves were a 3-month-old and a 2-week-old Jersey. Two Jersey calves, 3 mo...

  8. Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution

    Directory of Open Access Journals (Sweden)

    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

  9. Signs Observed Among Animal Species Infected with Raccoon Rabies Variant Virus, Massachusetts, USA, 1992–2010

    OpenAIRE

    Han, Linda L.; Vasil Pani; Sandra Smole; Werner, Barbara G.; Xingtai Wang

    2011-01-01

    Simple Summary We analyzed signs occurring among domestic and wild terrestrial animal species with raccoon rabies variant virus in Massachusetts, 1992–2010. While aggression is a useful predictor of rabies among wild animals, combinations of other signs such as ataxia, disorientation, and salivation are useful predictors of rabies among domestic animals. Abstract We analyzed signs occurring among domestic and wild terrestrial animal species infected with raccoon rabies variant virus (RRV) in ...

  10. Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract.

    OpenAIRE

    Tian, S F; Buckler-White, A J; London, W. T.; Reck, L J; Chanock, R M; Murphy, B R

    1985-01-01

    An avian influenza A virus, A/Mallard/NY/6750/78(H2N2), was restricted in in replication in the respiratory tract of squirrel monkeys. Avian-human influenza A reassortant viruses possessing the six RNA segments coding for nonsurface proteins (i.e., internal genes) of this avian virus were as restricted in replication in squirrel monkeys as their avian influenza parent. These findings indicated that restriction of replication of the avian influenza virus is a function of one or more of its int...

  11. The cis-acting replication element of the Hepatitis C virus genome recruits host factors that influence viral replication and translation.

    Science.gov (United States)

    Ríos-Marco, Pablo; Romero-López, Cristina; Berzal-Herranz, Alfredo

    2016-01-01

    The cis-acting replication element (CRE) of the hepatitis C virus (HCV) RNA genome is a region of conserved sequence and structure at the 3' end of the open reading frame. It participates in a complex and dynamic RNA-RNA interaction network involving, among others, essential functional domains of the 3' untranslated region and the internal ribosome entry site located at the 5' terminus of the viral genome. A proper balance between all these contacts is critical for the control of viral replication and translation, and is likely dependent on host factors. Proteomic analyses identified a collection of proteins from a hepatoma cell line as CRE-interacting candidates. A large fraction of these were RNA-binding proteins sharing highly conserved RNA recognition motifs. The vast majority of these proteins were validated by bioinformatics tools that consider RNA-protein secondary structure. Further characterization of representative proteins indicated that hnRNPA1 and HMGB1 exerted negative effects on viral replication in a subgenomic HCV replication system. Furthermore DDX5 and PARP1 knockdown reduced the HCV IRES activity, suggesting an involvement of these proteins in HCV translation. The identification of all these host factors provides new clues regarding the function of the CRE during viral cycle progression. PMID:27165399

  12. 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.

  13. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    International Nuclear Information System (INIS)

    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

  14. Differential replication of avian influenza H9N2 viruses in human alveolar epithelial A549 cells

    Directory of Open Access Journals (Sweden)

    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.

  15. Cyclophilin E functions as a negative regulator to influenza virus replication by impairing the formation of the viral ribonucleoprotein complex.

    Directory of Open Access Journals (Sweden)

    Zengfu Wang

    Full Text Available BACKGROUND: The nucleoprotein (NP of influenza A virus is a multifunctional protein that plays a critical role in the replication and transcription of the viral genome. Therefore, examining host factors that interact with NP may shed light on the mechanism of host restriction barriers and the tissue tropism of influenza A virus. Here, Cyclophilin E (CypE, a member of the peptidyl-propyl cis-trans isomerase (PPIase family, was found to bind to NP and inhibit viral replication and transcription. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, CypE was found to interact with NP but not with the other components of the viral ribonucleoprotein complex (VRNP: PB1, PB2, and PA. Mutagenesis data revealed that the CypE domain comprised of residues 137-186 is responsible for its binding to NP. Functional analysis results indicated that CypE is a negative regulator in the influenza virus life cycle. Furthermore, knock-down of CypE resulted in increased levels of three types of viral RNA, suggesting that CypE negatively affects viral replication and transcription. Moreover, up-regulation of CypE inhibited the activity of influenza viral polymerase. We determined that the molecular mechanism by which CypE negatively regulates influenza virus replication and transcription is by interfering with NP self-association and the NP-PB1 and NP-PB2 interactions. CONCLUSIONS/SIGNIFICANCE: CypE is a host restriction factor that inhibits the functions of NP, as well as viral replication and transcription, by impairing the formation of the vRNP. The data presented here will help us to better understand the molecular mechanisms of host restriction barriers, host adaptation, and tissue tropism of influenza A virus.

  16. Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication

    Directory of Open Access Journals (Sweden)

    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

  17. 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)

    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

  18. 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.

  19. 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

  20. Virus-specific antibodies allow viral replication in the marginal zone, thereby promoting CD8+ T-cell priming and viral control

    OpenAIRE

    Vikas Duhan; Vishal Khairnar; Sarah-Kim Friedrich; Fan Zhou; Asmae Gassa; Nadine Honke; Namir Shaabani; Nicole Gailus; Lacramioara Botezatu; Cyrus Khandanpour; Ulf Dittmer; Dieter Häussinger; Mike Recher; Cornelia Hardt; Lang, Philipp A.

    2016-01-01

    Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-spec...

  1. High beta-chemokine expression levels in lymphoid tissues of simian/human immunodeficiency virus 89.6-vaccinated rhesus macaques are associated with uncontrolled replication of simian immunodeficiency virus challenge inoculum.

    Science.gov (United States)

    LaFranco-Scheuch, Lisa; Abel, Kristina; Makori, Norbert; Rothaeusler, Kristina; Miller, Christopher J

    2004-06-01

    Viral suppression by noncytolytic CD8+ T cells, in addition to that by classic antiviral CD8+ cytotoxic T lymphocytes, has been described for human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. However, the role of soluble effector molecules, especially beta-chemokines, in antiviral immunity is still controversial. In an attenuated vaccine model, approximately 60% of animals immunized with simian/human immunodeficiency virus (SHIV) 89.6 and then challenged intravaginally with SIVmac239 controlled viral replication (viral RNA level in plasma, Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller, J. Virol. 77:3099-3118, 2003). To determine the in vivo importance of beta-chemokine secretion and CD8+-T-cell proliferation in the control of viral replication in this vaccine model, we examined the relationship between viral RNA levels in the axillary and genital lymph nodes of vaccinated, protected (n = 20) and vaccinated, unprotected (n = 11) monkeys by measuring beta-chemokine mRNA levels and protein expression, the frequency of CD8+ T cells expressing beta-chemokines, and the extent of CD8+-T-cell proliferation. Tissues from uninfected (n = 3) and unvaccinated, SIVmac239-infected (n = 9) monkeys served as controls. Axillary and genital lymph nodes from unvaccinated and vaccinated, unprotected monkeys had significantly higher beta-chemokine mRNA expression levels and increased numbers of beta-chemokine-positive cells than did vaccinated, protected animals. Furthermore, the lymph nodes of vaccinated, unprotected monkeys had significantly higher numbers of beta-chemokine(+) CD8+ T cells than did vaccinated, protected monkeys. Lymph nodes from vaccinated, unprotected animals also had significantly more CD8+-T-cell proliferation and marked lymph node hyperplasia than the lymph nodes of vaccinated, protected monkeys. Thus, higher levels of virus replication were associated with increased beta

  2. 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.

  3. 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.

  4. 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.

    2016-01-01

    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. PMID:27080193

  5. 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

    2016-01-01

    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. PMID:27080193

  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; Shin, HY; Kim, YS; Hwang, SB

    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...

  7. 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.

  8. Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

    Science.gov (United States)

    Suzuki, Youichi; Chin, Wei-Xin; Han, Qi'En; Ichiyama, Koji; Lee, Ching Hua; Eyo, Zhi Wen; Ebina, Hirotaka; Takahashi, Hirotaka; Takahashi, Chikako; Tan, Beng Hui; Hishiki, Takayuki; Ohba, Kenji; Matsuyama, Toshifumi; Koyanagi, Yoshio; Tan, Yee-Joo; Sawasaki, Tatsuya; Chu, Justin Jang Hann; Vasudevan, Subhash G; Sano, Kouichi; Yamamoto, Naoki

    2016-01-01

    Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells. PMID:26735137

  9. Effective inhibition of foot-and-mouth disease virus (FMDV replication in vitro by vector-delivered microRNAs targeting the 3D gene

    Directory of Open Access Journals (Sweden)

    Cai Xuepeng

    2011-06-01

    Full Text Available Abstract Background Foot-and-mouth disease virus (FMDV causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined. Results Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV. Conclusion Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

  10. 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

  11. 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.

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

    Science.gov (United States)

    Niewiesk, Stefan

    2016-03-31

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

  13. Replication Inhibition of Hepatitis B Virus and Hepatitis C Virus in Co-Infected Patients in Chinese Population.

    Directory of Open Access Journals (Sweden)

    Ge Yu

    Full Text Available Hepatitis B virus (HBV and hepatitis C virus (HCV co-infections contributes to a substantial proportion of liver disease worldwide. The aim of this study was to assess the clinical and virological features of HBV-HCV co-infection.Demographic data were collected for 3238 high-risk people from an HCV-endemic region in China. Laboratory tests included HCV antibody and HBV serological markers, liver function tests, and routine blood analysis. Anti-HCV positive samples were analyzed for HCV RNA levels and subgenotypes. HBsAg-positive samples were tested for HBV DNA.A total of 1468 patients had chronic HCV and/or HBV infections. Among them, 1200 individuals were classified as HCV mono-infected, 161 were classified as HBV mono-infected, and 107 were classified as co-infected. The HBV-HCV co-infected patients not only had a lower HBV DNA positive rate compared to HBV mono-infected patients (84.1% versus 94.4%, respectively; P < 0.001. The median HCV RNA levels in HBV-HCV co-infected patients were significantly lower than those in the HCV mono-infected patients (1.18[Interquartile range (IQR 0-5.57] versus 5.87[IQR, 3.54-6.71] Log10 IU/mL, respectively; P < 0.001. Furthermore, co-infected patients were less likely to have detectable HCV RNA levels than HCV mono-infected patients (23.4% versus 56.5%, respectively; P < 0.001. Those HBV-HCV co-infected patients had significantly lower median HBV DNA levels than those mono-infected with HBV (1.97[IQR, 1.3-3.43] versus 3.06[IQR, 2-4.28] Log10 IU/mL, respectively; P < 0.001. The HBV-HCV co-infection group had higher ALT, AST, ALP, GGT, APRI and FIB-4 levels, but lower ALB and total platelet compared to the HBV mono-infection group, and similar to that of the HCV mono-infected group.These results suggest that co-infection with HCV and HBV inhibits the replication of both viruses. The serologic results of HBV-HCV co-infection in patients suggests more liver injury compared to HBV mono-infected patients, but is

  14. Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein.

    Science.gov (United States)

    Liu, Xia; Xu, Zhijian; Hou, Chuanwei; Wang, Meng; Chen, Xinhuan; Lin, Qinghui; Song, Rui; Lou, Meng; Zhu, Lijun; Qiu, Yunqing; Chen, Zhi; Yang, Chunhao; Zhu, Weiliang; Shao, Jimin

    2016-03-01

    Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment. PMID:26774458

  15. Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

    Science.gov (United States)

    Suzuki, Saori; Mori, Ken-Ichi; Higashino, Atsunori; Iwasaki, Yuki; Yasutomi, Yasuhiro; Maki, Noboru; Akari, Hirofumi

    2016-01-01

    The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome. PMID:26634303

  16. 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.

  17. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  18. Esterase D enhances type I interferon signal transduction to suppress foot-and-mouth disease virus replication.

    Science.gov (United States)

    Li, Weiwei; Zhu, Zixiang; Cao, Weijun; Yang, Fan; Zhang, Xiangle; Li, Dan; Zhang, Keshan; Li, Pengfei; Mao, Ruoqing; Liu, Xiangtao; Zheng, Haixue

    2016-07-01

    The enzymatic activities of esterase D (ESD) are involved in many human diseases. However, no antiviral property of ESD has been described to date. Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease. In this study, we showed that FMDV infection triggered ESD expression. Overexpression of ESD significantly suppressed FMDV replication and knockdown of ESD expression enhanced virus replication, showing an essential antiviral role of ESD. Furthermore, we found that Sendai-virus-induced interferon (IFN) signaling was enhanced by upregulation of ESD, and ESD promoted activation of the IFN-β promoter simulated by IFN regulatory factor (IRF)3 or its upstream molecules (retinoic acid-inducible gene-I, melanoma differentiation-associated protein 5, virus-induced signaling adaptor and TANK binding kinase 1). Detailed analysis revealed that ESD protein enhanced IRF3 phosphorylation during FMDV infection. Overexpression of ESD also promoted the expression of various antiviral interferon-stimulated genes (ISGs) and knockdown of ESD impaired the expression of these antiviral genes during FMDV infection. Our findings demonstrate a new mechanism evolved by ESD to enhance type I IFN signal transduction and suppress viral replication during FMDV infection. PMID:27267271

  19. Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

    Institute of Scientific and Technical Information of China (English)

    Yong-Wen He; Chun-Xia Guo; Yan-Feng Pan; Cheng Peng; Zhi-Hong Weng

    2008-01-01

    AIM:To explore the inhibitory effects of pokeweed antiviral protein seed(PAP-S)and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus(HBV)replication in vitro.METHODS:HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S.HBsAg,HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively.MTT method was used to assay for cytotoxicity.HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold overlength plasmid.On d 3 after transfection,HBsAg and HBeAg were determined by using ELISA.Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot,respectively.RESULTS:The inhibitory effects of PAP-S on HBsAg,HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration.When the concentration of PAP-S was 10 μg/mL,the inhibition rates of HBsAg,HBeAg and HBV DNA were 20.9%,30.2% and 50%,respectively.After transfection of 1.0μg and 2.0μg plasmid pXF3H-PAP,the levels of HBV nucleocapsideassociated DNA were reduced by 38.0% and 74.0% respectively,the levels of HBsAg in the media by 76.8% and 99.7% respectively,and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls.Transfection with 2μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION:Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro,and the inhibitory effects were dose-dependent.

  20. Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

    Science.gov (United States)

    van Wielink, R; Harmsen, M M; Martens, D E; de Leeuw, O S; Peeters, B P H; Wijffels, R H; Moormann, R J M

    2012-12-31

    Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production. PMID:23079079

  1. A Scorpion Defensin BmKDfsin4 Inhibits Hepatitis B Virus Replication in Vitro.

    Science.gov (United States)

    Zeng, Zhengyang; Zhang, Qian; Hong, Wei; Xie, Yingqiu; Liu, Yun; Li, Wenxin; Wu, Yingliang; Cao, Zhijian

    2016-01-01

    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. PMID:27128943

  2. In Vitro Exposure of Harbor Seal Immune Cells to Aroclor 1260 Alters Phocine Distemper Virus Replication.

    Science.gov (United States)

    Bogomolni, Andrea; Frasca, Salvatore; Levin, Milton; Matassa, Keith; Nielsen, Ole; Waring, Gordon; De Guise, Sylvain

    2016-01-01

    In the last 30 years, several large-scale marine mammal mortality events have occurred, often in close association with highly polluted regions, leading to suspicions that contaminant-induced immunosuppression contributed to these epizootics. Some of these recent events also identified morbillivirus as a cause of or contributor to death. The role of contaminant exposures regarding morbillivirus mortality is still unclear. The results of this study aimed to address the potential for a mixture of polychlorinated biphenyls (PCBs), specifically Aroclor 1260, to alter harbor seal T-lymphocyte proliferation and to assess if exposure resulted in increased likelihood of phocine distemper virus (PDV USA 2006) to infect susceptible seals in an in vitro system. Exposure of peripheral blood mononuclear cells to Aroclor 1260 did not significantly alter lymphocyte proliferation (1, 5, 10, and 20 ppm). However, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), lymphocytes exposed to 20 ppm Aroclor 1260 exhibited a significant decrease in PDV replication at day 7 and a significant increase at day 11 compared with unexposed control cells. Similar and significant differences were apparent on exposure to Aroclor 1260 in monocytes and supernatant. The results here indicate that in harbor seals, Aroclor 1260 exposure results in a decrease in virus early during infection and an increase during late infection. The consequences of this contaminant-induced infection pattern in a highly susceptible host could result in a greater potential for systemic infection with greater viral load, which could explain the correlative findings seen in wild populations exposed to a range of persistent contaminants that suffer from morbillivirus epizootics. PMID:26142119

  3. Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil

    OpenAIRE

    Cláudia L Vitral; Marcelo A Pinto; Lewis-Ximenez, Lia L.; Yuri E Khudyakov; Débora R dos Santos; Ana Maria C. Gaspar

    2005-01-01

    Serological evidence of hepatitis E virus infection (HEV) has been observed in both humans and different animal species living in non-endemic areas, suggesting that animals could be important reservoir for virus transmission to man. Antibodies to HEV have been detected in some Brazilian population groups. Nevertheless, sporadic cases of acute HEV infection have never been reported. We collected 271 serum samples from several domestic animals and also from pig handlers from Southeast of Brazil...

  4. Only minimal regions of tomato yellow leaf curl virus (TYLCV) are required for replication, expression and movement

    OpenAIRE

    Gover, Ofer; Peretz, Yuval; Mozes-Koch, Rita; Maori, Eyal; Haim D. Rabinowitch; Sela, Ilan

    2014-01-01

    The IL-60 platform, consisting of a disarmed form of tomato yellow leaf curl virus (TYLCV) and auxiliary components, was previously developed as a nontransgenic universal vector system for gene expression and silencing that can express an entire operon in plants. IL-60 does not allow rolling-circle replication; hence, production of viral single-stranded (ss) DNA progeny is prevented. We used this double-stranded (ds) DNA-restricted platform (uncoupled from the dsDNA→ssDNA replication phase of...

  5. Enhancement of internal ribosome entry site-mediated translation and replication of hepatitis C virus by PD98059

    International Nuclear Information System (INIS)

    Translation initiation of hepatitis C virus (HCV) occurs in an internal ribosome entry site (IRES)-dependent manner. We found that HCV IRES-dependent protein synthesis is enhanced by PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) signaling pathway, while cellular cap-dependent translation was relatively unaffected by the compound. Treatment of cells with PD98059 allowed for robust HCV replication following cellular incubation with HCV-positive serum. Though the molecular mechanism underlying IRES enhancement remains elusive, PD98059 is a potent accelerator of HCV RNA replication

  6. Genetic Strategy to Prevent Influenza Virus Infections in Animals

    OpenAIRE

    Chen, Jianzhu; Chen, Steve C.-Y.; Stern, Patrick; Scott, Benjamin B; Lois, Carlos

    2008-01-01

    The natural reservoirs of influenza viruses are aquatic birds. After adaptation, avian viruses can acquire the ability to infect humans and cause severe disease. Because domestic poultry serves as a key link between the natural reservoir of influenza viruses and epidemics and pandemics in human populations, an effective measure to control influenza would be to eliminate or reduce influenza virus infection in domestic poultry. The development and distribution of influenza-resistant poultry rep...

  7. The conserved structures of the 5' nontranslated region of Citrus tristeza virus are involved in replication and virion assembly

    International Nuclear Information System (INIS)

    The genomic RNA of different isolates of Citrus tristeza virus (CTV) reveals an unusual pattern of sequence diversity: the 3' halves are highly conserved (homology >90%), while the 5' halves show much more dissimilarity, with the 5' nontranslated region (NTR) containing the highest diversity (homology as low as 42%). Yet, positive-sense sequences of the 5' NTR were predicted to fold into nearly identical structures consisting of two stem-loops (SL1 and SL2) separated by a short spacer region. The predicted most stable secondary structures of the negative-sense sequences were more variable. We introduced mutations into the 5' NTR of a CTV replicon to alter the sequence and/or the predicted secondary structures with or without additional compensatory changes designed to restore predicted secondary structures, and examined their effect on replication in transfected protoplasts. The results suggested that the predicted secondary structures of the 5' NTR were more important for replication than the primary structure. Most mutations that were predicted to disrupt the secondary structures fail to replicate, while compensatory mutations were allowed replication to resume. The 5' NTR mutations that were tolerated by the CTV replicon were examined in the full-length virus for effects on replication and production of the multiple subgenomic RNAs. Additionally, the ability of these mutants to produce virions was monitored by electron microscopy and by passaging the progeny nucleocapsids to another batch of protoplasts. Some of the mutants with compensatory sequence alterations predicted to rebuild similar secondary structures allowed replication at near wild-type levels but failed to passage, suggesting that the 5' NTR contains sequences required for both replication and virion assembly

  8. Cluster–cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

    International Nuclear Information System (INIS)

    Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and in vitro drug testing. In the present paper, a cluster–cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simulations reveal that in the absence of chemotaxy the mean cluster size and the total number of clusters scale in time as stretched exponentials dependent on the particle replication rate. Also, the dynamical cluster size distribution functions are represented by a scaling relation in which the scaling function involves a stretched exponential of the time. The introduction of chemoattraction among the particles leads to distribution functions decaying as power laws with exponents that decrease in time. The fractal dimensions and size distributions of the simulated clusters are qualitatively discussed in terms of those determined experimentally for several normal and tumoral cell lines growing in culture. It is shown that particle replication and chemotaxy account for the simplest cluster size distributions of cellular aggregates observed in culture

  9. RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA.

    Science.gov (United States)

    Ferriol, I; Turina, M; Zamora-Macorra, E J; Falk, B W

    2016-05-01

    Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA. PMID:26756828

  10. A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. Porcine 2', 5'-oligoadenylate synthetases inhibit Japanese encephalitis virus replication in vitro.

    Science.gov (United States)

    Zheng, Sheng; Zhu, Dan; Lian, Xue; Liu, Weiting; Cao, Ruibing; Chen, Puyan

    2016-05-01

    The 2', 5'-oligoadenylate synthetases (OAS) are antiviral proteins and several isoforms have been identified as flavivirus-resistance biomarkers in human and mouse. The expression kinetics and antiviral functions of porcine OAS family (OAS1, OAS2, and OASL) in PK-15 cells following infection by Japanese encephalitis virus (JEV) were evaluated in the present study. The endogenous expression of the three OAS genes was efficiently induced by IFN-α treatment in PK-15 cells. However, expression of pOAS1 and pOAS2 responded more quickly than pOASL. Infection by JEV also induced the expression of the pOAS isoforms, but at a significantly lower level than that observed following IFN-α stimulation. Transient overexpression of pOASL and pOAS1 inhibited JEV replication more efficiently than OAS2 overexpression. Interestingly, knockdown of pOAS2 expression by siRNA treatment led to the highest increase in JEV multiplication. Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent, while the antiviral activity of pOASL was not. In conclusion, all pOAS isoforms play a significant role in the response to JEV infection, and are differentially induced by different stimuli. The alternative pathways of antiviral activity stimulated by OASL require further study. PMID:26437676

  13. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    International Nuclear Information System (INIS)

    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.

  14. Loss of immune escape mutations during persistent HCV infection in pregnancy enhances replication of vertically transmitted viruses

    OpenAIRE

    Honegger, Jonathan R; Kim, Seungtaek; Price, Aryn A.; Kohout, Jennifer A.; McKnight, Kevin L.; Prasad, Mona R.; Lemon, Stanley M.; Grakoui, Arash; Walker, Christopher M

    2013-01-01

    Globally, about 1% of pregnant women are persistently infected with the hepatitis C virus (HCV) 1 . Vertical transmission occurs in 3–5% of cases 2 and accounts for most new childhood HCV infections 1,3 . HCV-specific CD8+ cytotoxic T-lymphocytes (CTLs) play a vital role in the clearance of acute infections 4–6 , but in the 60–80% of infections that persist these cells become functionally exhausted or select mutant viruses that escape T-cell recognition 7–9 . Increased HCV replication during ...

  15. Generation of a human hepatoma cell line supporting efficient replication of a lamivudine resistant hepatitis B virus.

    Science.gov (United States)

    Zhang, Yijun; Zhang, Yongmei; Kang, Yaoyue; Wang, Jinyu; Liu, Hongyan; Zhu, Haoxiang; Qin, Yanli; Mao, Richeng; Lin, Xu; Lu, Mengji; Zhang, Jiming

    2014-06-01

    Emergence of lamivudine (LAM) resistance causes treatment failure in patients with chronic hepatitis B and compromise the efficacy of subsequent salvage therapies with other nucleot(s)ide analogs (NAs). Establishment of cell-based assays supporting LAM-resistant hepatitis B virus (HBV) replication will not only provide tools for investigating the replication property, but also screening for antiviral agents efficiently inhibiting the replication of LAM-resistant HBV variants. Accordingly, a human hepatoma (HepG2)-derived cell line was established by stable transfection of a plasmid containing a 1.2 unit length of HBV genome harboring rtL180M and rtM204V mutations that confer LAM resistance. In addition to support efficient viral genome replication, the cell line also produces high levels of HBV virions and subviral particles. As expected, HBV DNA replication in this cell line is completely resistant to lamivudine, but sensitive to adefovir (ADV), entecavir (ETV) and tenofovir (TDF). The cell line is suitable for screening for antiviral agents that inhibit LAM-resistant HBV replication and inhibitors of HBsAg biosynthesis and secretion, which may reduce HBsAg antigenemia and ultimately help to restore host antiviral immune response against HBV and cure chronic HBV infection. PMID:24583110

  16. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul;

    2005-01-01

    defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBL(neck+CRD)) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP...... and airway fluids, as well as in blood and various mucosal locations, and could, like MBL, play a role in restricting HIV transmission or replication in vivo.......The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL...

  17. Plant subviral RNAs as a long noncoding RNA (lncRNA): Analogy with animal lncRNAs in host-virus interactions.

    Science.gov (United States)

    Shimura, Hanako; Masuta, Chikara

    2016-01-01

    Satellite RNAs (satRNAs) and viroids belong to the group called subviral agents and are the smallest pathogens of plants. In general, small satRNAs and viroids are 300-400 nt in size and do not encode any functional proteins; they are thus regarded as so-called long noncoding RNAs (lncRNAs). These lncRNAs are receiving great attention as a new RNA class involved in gene regulation to control important biological processes such as gene transcription and epigenetic regulation. A substantial number of lncRNAs in animal cells have been found to play important roles in the interactions between a virus and its host. We here discuss the pathogenicity of subviral RNAs (especially satRNAs) in plant cells and their functions as lncRNAs associated with viral diseases, using animal lncRNAs as an analogy. Because, unlike animal lncRNAs, plant subviral RNAs can replicate and accumulate at very high levels in infected cells, we here considered the unique possibility that the RNA silencing machinery of plants, an important defense mechanism against virus infection, may have brought about the replication ability of subviral molecules. In addition, we also discuss the possibility that satRNAs may have arisen from plant-virus interactions in virus-infected cells. Understanding the molecular functions of these unique lncRNAs in plants will enable us to reveal the most plausible origins of these subviral RNAs. PMID:26116900

  18. The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication.

    Science.gov (United States)

    Graham, Rachel L; Sims, Amy C; Brockway, Sarah M; Baric, Ralph S; Denison, Mark R

    2005-11-01

    The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function. PMID:16227261

  19. PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice

    International Nuclear Information System (INIS)

    A single amino acid substitution, from glutamic acid to lysine at position 627 of the PB2 protein, converts a nonlethal H5N1 influenza A virus isolated from a human to a lethal virus in mice. In contrast to the nonlethal virus, which replicates only in respiratory organs, the lethal isolate replicates in a variety of organs, producing systemic infection. Despite a clear difference in virulence and organ tropism between the two viruses, it remains unknown whether the dissimilarity is a result of differences in cell tropism or the reduced replicative ability of the nonlethal virus in mouse cells in general. To determine how this single amino acid change affects virulence and organ tropism in mice, we investigated the growth kinetics of the two H5N1 viruses both in vitro and in vivo. The identity of the PB2 amino acid at position 627 did not appreciably affect viral replicative efficiency in chicken embryo fibroblasts and a quail cell line; however, viruses with lysine at this position instead of glutamic acid grew better in the different mouse cells tested. When the effect of this substitution was investigated in mice, all of the test viruses showed the same cell tropism, but infection by viruses containing lysine at position 627 spread more rapidly than those viruses containing glutamic acid at this position. Further analysis showed a difference in local immune responses: neutrophil infiltration in lungs infected with viruses containing lysine at position 627 persisted longer than that associated with viruses lacking a glutamic acid substitution. Our data indicate that the amino acid at position 627 of the PB2 protein determines the efficiency of viral replication in mouse (not avian) cells, but not tropism among cells in different mouse organs. The presence of lysine leads to more aggressive viral replication, overwhelming the host's defense mechanisms and resulting in high mortality rates in mice

  20. Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates▿

    OpenAIRE

    Qin, Yanli; Zhang, Jiming; Garcia, Tamako; Ito, Kiyoaki; Gutelius, Danielle; Li, Jisu; Wands, Jack; Tong, Shuping

    2011-01-01

    Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomes and/or response to antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelityplus DNA polymerase and primers targeting the conserved precore region, the HBV clones thus genera...

  1. Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

    OpenAIRE

    Narita, Yohei; Murata, Takayuki; Ryo, Akihide; Kawashima, Daisuke; Sugimoto, Atsuko; Kanda, Teru; Kimura, Hiroshi; Tsurumi, Tatsuya

    2013-01-01

    Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5,...

  2. Effects of Limiting Homology at the Site of Intermolecular Recombinogenic Template Switching during Moloney Murine Leukemia Virus Replication

    OpenAIRE

    Pfeiffer, Julie K.; Telesnitsky, Alice

    2001-01-01

    A Moloney murine leukemia virus-based single-replication-cycle assay was developed to study the effects of limiting the extent of template and primer strand complementarity on recombinogenic template switching. This system mimicked forced copy choice recombination in which nascent DNA transfers from the end of a donor template to an acceptor position on the other copackaged RNA. When acceptor target regions with different extents of complementarity to the transferring DNA were tested, efficie...

  3. Low levels of human immunodeficiency virus replication in the brain tissue of children with severe acquired immunodeficiency syndrome encephalopathy.

    OpenAIRE

    Vazeux, R.; Lacroix-Ciaudo, C.; Blanche, S; Cumont, M. C.; Henin, D; Gray, F.; Boccon-Gibod, L.; Tardieu, M.

    1992-01-01

    The authors examined the autopsy brain samples of nine children infected with human immunodeficiency virus (HIV) at birth by histology, immunologic staining, and in situ hybridization. Surprisingly, although seven of these children presented with typical AIDS encephalopathy, the authors could detect a multifocal HIV infection in the brains of only three of these patients. The authors could not detect any significant HIV replication in the brain of four other children despite severe neurologic...

  4. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

    Directory of Open Access Journals (Sweden)

    Ying Wen Huang

    Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

  5. The replication of human immunodeficiency virus type 1 in macrophages is enhanced after phagocytosis of apoptotic cells

    OpenAIRE

    Lima, Rosangela G; Van Weyenbergh, Johan; Saraiva, Elvira M. B.; Barral-Netto, Manoel; Galvão-Castro, Bernardo; Bou-Habib, Dumith Chequer

    2002-01-01

    Clearance of apoptotic cells increases macrophage secretion of antiinflammatory mediators and might modulate viral replication in human immunodeficiency virus (HIV) type 1-infected macrophages. To study this, primary macrophages were infected with HIV-1 and exposed to apoptotic cells. It was found that phagocytosis of apoptotic cells potently enhanced HIV-1 growth. The peptide Arg-Gly-Asp-Ser, which binds to integrin receptors, inhibited the uptake of apoptotic cells and the subsequent enhanc...

  6. Inhibition of influenza A virus replication by influenza B virus nucleoprotein: An insight into interference between influenza A and B viruses

    Energy Technology Data Exchange (ETDEWEB)

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jaru-ampornpan, Peera; Jengarn, Juggagarn [Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120 (Thailand); Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th [Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120 (Thailand)

    2012-10-10

    Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP{sub FluB}) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP{sub FluB} suggest that functional NP{sub FluB} is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP{sub FluB}. Further analysis of NP{sub FluB} indicated that it does not affect nuclear import of NP{sub FluA}. Taken together, these findings suggest a novel role of NP{sub FluB} in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.

  7. Inhibition of influenza A virus replication by influenza B virus nucleoprotein: An insight into interference between influenza A and B viruses

    International Nuclear Information System (INIS)

    Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NPFluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NPFluB suggest that functional NPFluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NPFluB. Further analysis of NPFluB indicated that it does not affect nuclear import of NPFluA. Taken together, these findings suggest a novel role of NPFluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.

  8. Activation of nucleotide oligomerization domain 2 (NOD2 by human cytomegalovirus initiates innate immune responses and restricts virus replication.

    Directory of Open Access Journals (Sweden)

    Arun Kapoor

    Full Text Available Nucleotide-binding oligomerization domain 2 (NOD2 is an important innate immune sensor of bacterial pathogens. Its induction results in activation of the classic NF-κB pathway and alternative pathways including type I IFN and autophagy. Although the importance of NOD2 in recognizing RNA viruses has recently been identified, its role in sensing DNA viruses has not been studied. We report that infection with human cytomegalovirus (HCMV results in significant induction of NOD2 expression, beginning as early as 2 hours post infection and increasing steadily 24 hours post infection and afterwards. Infection with human herpesvirus 1 and 2 does not induce NOD2 expression. While the HCMV-encoded glycoprotein B is not required for NOD2 induction, a replication competent virion is necessary. Lentivirus-based NOD2 knockdown in human foreskin fibroblasts (HFFs and U373 glioma cells leads to enhanced HCMV replication along with decreased levels of interferon beta (IFN-β and the pro-inflammatory cytokine, IL8. NOD2 induction in HCMV-infected cells activates downstream NF-κB and interferon pathways supported by reduced nuclear localization of NF-κB and pIRF3 in NOD2 knockdown HFFs. Stable overexpression of NOD2 in HFFs restricts HCMV replication in association with increased levels of IFN-β and IL8. Similarly, transient overexpression of NOD2 in U373 cells or its downstream kinase, RIPK2, results in decreased HCMV replication and enhanced cytokine responses. However, overexpression of a mutant NOD2, 3020insC, associated with severe Crohn's disease, results in enhanced HCMV replication and decreased levels of IFN-β in U373 cells. These results show for the first time that NOD2 plays a significant role in HCMV replication and may provide a model for studies of HCMV recognition by the host cell and HCMV colitis in Crohn's disease.

  9. Passive Transfer of Antibodies Protects Immunocompetent and Immunodeficient Mice against Lethal Ebola Virus Infection without Complete Inhibition of Viral Replication

    OpenAIRE

    Gupta, Manisha; Mahanty, Siddhartha; Bray, Mike; Ahmed, Rafi; Pierre E Rollin

    2001-01-01

    Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that m...

  10. Hamster-Adapted Sin Nombre Virus Causes Disseminated Infection and Efficiently Replicates in Pulmonary Endothelial Cells without Signs of Disease

    OpenAIRE

    Safronetz, David; Prescott, Joseph; Haddock, Elaine; Scott, Dana P.; Feldmann, Heinz; Ebihara, Hideki

    2013-01-01

    To date, a laboratory animal model for the study of Sin Nombre virus (SNV) infection or associated disease has not been described. Unlike infection with Andes virus, which causes lethal hantavirus pulmonary syndrome (HPS)-like disease in hamsters, SNV infection is short-lived, with no viremia and little dissemination. Here we investigated the effect of passaging SNV in hamsters. We found that a host-adapted SNV achieves prolonged and disseminated infection in hamsters, including efficient rep...

  11. Animal Models for Influenza Virus Pathogenesis and Transmission

    OpenAIRE

    Lowen, Anice C.; Bouvier, Nicole M.

    2010-01-01

    Influenza virus infection of humans results in a respiratory disease that ranges in severity from sub-clinical infection to primary viral pneumonia that can result in death. The clinical effects of infection vary with the exposure history, age and immune status of the host, and also the virulence of the influenza strain. In humans, the virus is transmitted through either aerosol or contact-based transfer of infectious respiratory secretions. As is evidenced by most zoonotic influenza virus in...

  12. Variability of tropism and replicative capacity of two naturally occurring influenza A H9N2 viruses in cell cultures from different tissues.

    Science.gov (United States)

    Tombari, Wafa; ElBehi, Imen; Amouna, Faten; Ghram, Abdeljelil

    2016-04-01

    Studies carried out on cell permissivity are of great interest to understand virus replication and pathogenicity. We described the results of a comparative analysis of replication efficiency of two naturally occurring influenza A H9N2 variants isolated from poultry and wild birds, differing by only two substitutions Q226L and T384N, in the receptor-binding site of haemagglutinin and the 380 loop region of NA proteins, respectively. Considering the overall growth of both viruses, lung cultures ensured the most efficient growth of TUN12L226N384 strain with titres up to 10(9) TCID50/ml whereas small intestine culture was highly susceptible to the TUN51Q226T384 virus reaching a titre of 10(6) TCID50/ml. The lowest replication was shown in liver cells. The addition of trypsin was essential for the replication of either virus in primary fibroblasts, but it had a marginal positive effect on virus replication in the four other culture types with maximum titres of 10(8) TCID50/ml. This means that in chicken, the proteolytic activation of the H9N2 viruses with the cleavage motif RSSR may be mediated by other endoproteases than trypsin. Further investigations should concentrate on the production of the appropriate set of viruses by a reverse genetics approach and the examination of cellular protease expression in chicken tissues. This would lead to a more complete understanding of the tropism of low-pathogenic Influenza A viruses. PMID:26813086

  13. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  14. Ethics and Animal Numbers: Informal Analyses, Uncertain Sample Sizes, Inefficient Replications, and Type I Errors

    OpenAIRE

    Fitts, Douglas A

    2011-01-01

    To obtain approval for the use vertebrate animals in research, an investigator must assure an ethics committee that the proposed number of animals is the minimum necessary to achieve a scientific goal. How does an investigator make that assurance? A power analysis is most accurate when the outcome is known before the study, which it rarely is. A ‘pilot study’ is appropriate only when the number of animals used is a tiny fraction of the numbers that will be invested in the main study because t...

  15. The role of dipterous insects in the mechanical transmission of animal viruses.

    Science.gov (United States)

    Carn, V M

    1996-07-01

    Animals viruses may be transmitted by arthropods in two ways, either biologically or mechanically. Many different species of Diptera are implicated in mechanical transmission, but haematophagous species are the most important. The insects become contaminated with virus during normal feeding behaviour, and virus persists on their mouthparts or body until the next feed. Some viruses are inactivated rapidly on mouthparts, whereas others survive for many days or weeks, prolonging the potential period of transmission. Some viruses produce high titres in the skin of the infected vertebrate host, which facilitates transmission, whereas other viruses are transmitted even during relatively low levels of viraemia. Mechanical transmission by arthropods is important in the epidemiology of many animal diseases, and may be the major mode of horizontal transmission. In other instances vector spread is merely incidental. PMID:8791847

  16. Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

    OpenAIRE

    Scull, Margaret A.; Gillim-Ross, Laura; Santos, Celia; Roberts, Kim L.; Bordonali, Elena; Subbarao, Kanta; Barclay, Wendy S.; Pickles, Raymond J.

    2009-01-01

    Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal ...

  17. Glycyrrhizin exerts antioxidative effects in H5N1 influenza A virus-infected cells and inhibits virus replication and pro-inflammatory gene expression.

    Directory of Open Access Journals (Sweden)

    Martin Michaelis

    Full Text Available Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549 cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher. Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

  18. Inhibitory effect of small interfering RNA on dengue virus replication in mosquito cells

    Directory of Open Access Journals (Sweden)

    Li Lei

    2010-10-01

    Full Text Available Abstract Background Dengue viruses (DENs are the wildest transmitted mosquito-borne pathogens throughout tropical and sub-tropical regions worldwide. Infection with DENs can cause severe flu-like illness and potentially fatal hemorrhagic fever. Although RNA interference triggered by long-length dsRNA was considered a potent antiviral pathway in the mosquito, only limited studies of the value of small interfering RNA (siRNA have been conducted. Results A 21 nt siRNA targeting the membrane glycoprotein precursor gene of DEN-1 was synthesized and transfected into mosquito C6/36 cells followed by challenge with DEN. The stability of the siRNA in cells was monitored by flow cytometry. The antiviral effect of siRNA was evaluated by measurement of cell survival rate using the MTT method and viral RNA was quantitated with real-time RT-PCR. The presence of cells containing siRNA at 0.25, 1, 3, 5, 7 days after transfection were 66.0%, 52.1%, 32.0%, 13.5% and 8.9%, respectively. After 7 days incubation with DEN, there was reduced cytopathic effect, increased cell survival rate (76.9 ± 4.5% vs 23.6 ± 14.6% and reduced viral RNA copies (Ct value 19.91 ± 0.63 vs 14.56 ± 0.39 detected in transfected C6/36 cells. Conclusions Our data showed that synthetic siRNA against the DEN-1 membrane glycoprotein precursor gene effectively inhibited DEN-1 viral RNA replication and increased C6/36 cell survival rate. siRNA may offer a potential new strategy for prevention and treatment of DEN infection.

  19. The Foot-and-Mouth Disease Virus cis-Acting Replication Element (cre) Can Be Complemented in trans within Infected Cells

    OpenAIRE

    Tiley, Laurence; King, Andrew M. Q.; Belsham, Graham J.

    2003-01-01

    A temperature-sensitive (ts) mutation was identified within the 5′-untranslated region of foot-and-mouth disease virus (FMDV) RNA. The mutation destabilizes a stem-loop structure recently identified as a cis-acting replication element (cre). Genetic analyses indicated that the ts defect in virus replication could be complemented. Thus, the FMDV cre can function in trans. It is suggested that the cre be renamed a 3B-uridylylation site (bus).

  20. Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain

    International Nuclear Information System (INIS)

    The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

  1. Elements in the canine distemper virus M 3' UTR contribute to control of replication efficiency and virulence.

    Directory of Open Access Journals (Sweden)

    Danielle E Anderson

    Full Text Available Canine distemper virus (CDV is a negative-sense, single-stranded RNA virus within the genus Morbillivirus and the family Paramyxoviridae. The Morbillivirus genome is composed of six transcriptional units that are separated by untranslated regions (UTRs, which are relatively uniform in length, with the exception of the UTR between the matrix (M and fusion (F genes. This UTR is at least three times longer and in the case of CDV also highly variable. Exchange of the M-F region between different CDV strains did not affect virulence or disease phenotype, demonstrating that this region is functionally interchangeable. Viruses carrying the deletions in the M 3' UTR replicated more efficiently, which correlated with a reduction of virulence, suggesting that overall length as well as specific sequence motifs distributed throughout the region contribute to virulence.

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

    Directory of Open Access Journals (Sweden)

    Maohua Cao

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

  3. Unique conditionally replication competent bipartite adenoviruses-cancer terminator viruses (CTV): efficacious reagents for cancer gene therapy.

    Science.gov (United States)

    Sarkar, Devanand; Su, Zao-Zhong; Fisher, Paul B

    2006-07-01

    The frequent resistance of aggressive cancers to currently available therapies, such as radiotherapy and chemotherapy, mandates development of targeted, nontoxic and more efficacious treatment protocols. Conditionally replication competent adenoviruses (CRCAs) that induce oncolysis by cancer-specific replication are currently being evaluated in clinical trials. However, a single modality approach may not be sufficient to completely eradicate cancer in a patient, because most cancers arise from abnormalities in multiple genetic and signal transduction pathways. The promoter region of rodent progression elevated gene-3 (PEG-3), cloned and characterized in our laboratory, embodies the unique property of increased activity in a broad range of tumor cells, both rodent and human, when compared to normal counterparts. Bipartite adenoviruses were engineered to express the E1A gene, necessary for viral replication, under control of the PEG-3 promoter (PEG-Prom) and simultaneously express a second transgene in the E3 region that encodes an apoptosis-inducing and immunomodulatory cytokine, either immune interferon (IFN-gamma) or melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). These conditionally replication competent bipartite adenoviruses, referred to as cancer terminator viruses (CTVs), facilitated cancer-selective adenovirus replication, robust transgene expression and apoptosis induction with complete eradication of both primary and distant (metastatic) human cancers xenotransplanted in athymic nude mice. These findings suggest that CTVs might prove efficacious for the therapy of primary and advanced neoplastic diseases. PMID:16861924

  4. Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status.

    Science.gov (United States)

    Koumbi, Lemonica; Pollicino, Teresa; Raimondo, Giovanni; Stampoulis, Dimitrios; Khakoo, Salim; Karayiannis, Peter

    2016-07-15

    In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication. PMID:27132039

  5. Hepatitis C Virus (HCV) NS5B Nonnucleoside Inhibitors Specifically Block Single-Stranded Viral RNA Synthesis Catalyzed by HCV Replication Complexes In Vitro▿

    OpenAIRE

    Yang, Wengang; Sun, Yongnian; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2006-01-01

    Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

  6. Productive replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens.

    Science.gov (United States)

    Reddy, Vishwanatha R A P; Trus, Ivan; Desmarets, Lowiese M B; Li, Yewei; Theuns, Sebastiaan; Nauwynck, Hans J

    2016-01-01

    In the present study, the replication kinetics of nephropathogenic (B1648) and respiratory (Massachusetts-M41) IBV strains were compared in vitro in respiratory mucosa explants and blood monocytes (KUL01(+) cells), and in vivo in chickens to understand why some IBV strains have a kidney tropism. B1648 was replicating somewhat better than M41 in the epithelium of the respiratory mucosa explants and used more KUL01(+) cells to penetrate the deeper layers of the respiratory tract. B1648 was productively replicating in KUL01(+) monocytic cells in contrast with M41. In B1648 inoculated animals, 10(2.7-6.8) viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8, 10 and 12 days post inoculation (dpi), 10(2.4-4.5) viral RNA copies/mL in plasma at 2, 4, 6, 8, 10 and 12 dpi and 10(1.8-4.4) viral RNA copies/10(6) mononuclear cells in blood at 2, 4, 6 and 8 dpi. In M41 inoculated animals, 10(2.6-7.0) viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8 and 10 dpi, but viral RNA was not demonstrated in plasma and mononuclear cells (except in one chicken at 6 dpi). Infectious virus was detected only in plasma and mononuclear cells of the B1648 group. At euthanasia (12 dpi), viral RNA and antigen positive cells were detected in lungs, liver, spleen and kidneys of only the B1648 group and in tracheas of both the B1648 and M41 group. In conclusion, only B1648 can easily disseminate to internal organs via a cell-free and -associated viremia with KUL01(+) cells as important carrier cells. PMID:27412035

  7. The photosystem II oxygen-evolving complex protein PsbP interacts with the coat protein of Alfalfa mosaic virus and inhibits virus replication.

    Science.gov (United States)

    Balasubramaniam, Muthukumar; Kim, Bong-Suk; Hutchens-Williams, Heather M; Loesch-Fries, L Sue

    2014-10-01

    Alfalfa mosaic virus (AMV) coat protein (CP) is essential for many steps in virus replication from early infection to encapsidation. However, the identity and functional relevance of cellular factors that interact with CP remain unknown. In an unbiased yeast two-hybrid screen for CP-interacting Arabidopsis proteins, we identified several novel protein interactions that could potentially modulate AMV replication. In this report, we focus on one of the novel CP-binding partners, the Arabidopsis PsbP protein, which is a nuclear-encoded component of the oxygen-evolving complex of photosystem II. We validated the protein interaction in vitro with pull-down assays, in planta with bimolecular fluorescence complementation assays, and during virus infection by co-immunoprecipitations. CP interacted with the chloroplast-targeted PsbP in the cytosol and mutations that prevented the dimerization of CP abolished this interaction. Importantly, PsbP overexpression markedly reduced virus accumulation in infected leaves. Taken together, our findings demonstrate that AMV CP dimers interact with the chloroplast protein PsbP, suggesting a potential sequestration strategy that may preempt the generation of any PsbP-mediated antiviral state. PMID:24940990

  8. Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo.

    Science.gov (United States)

    Fox, James M; Hilburn, Silva; Demontis, Maria-Antonietta; Brighty, David W; Rios Grassi, Maria Fernanda; Galvão-Castro, Bernardo; Taylor, Graham P; Martin, Fabiola

    2016-03-01

    Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the "mitotic" spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset. PMID:26985903

  9. A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

    International Nuclear Information System (INIS)

    An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Δ30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host

  10. Coordinate induction of IFN-α and -γ by SARS-CoV also in the absence of virus replication

    International Nuclear Information System (INIS)

    Background:: Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV, characterized by an over-exuberant immune response with lung lymphomononuclear cells infiltration and proliferation that may account for tissue damage more than the direct effect of viral replication. This study is aimed at investigating the capability of SARS-CoV to activate IFN-α and -γ expression in lymphomonocytes (PBMC) from healthy donors, evaluating whether viral replication is necessary for this activation. Results:: SARS-CoV virus is able to induce both IFN-α and -γ mRNA accumulation and protein release in a dose-dependent manner, MOI 10 being the most effective. The time course curve indicated that IFN-α mRNA induction peaked at 24 h.p.i,. whereas IFN-γ mRNA was still increasing at 48 h.p.i. Released IFN (both types) reached a plateau after 24-48 h.p.i. and remained rather stable over a 5-day period. A transient peak of negative strand viral RNA was detected after 1-2 days of infection, but neither infectious virus progeny yield nor newly produced viral genomic RNA could be evidenced in infected cultures, even after prolonged observation time (up to 13 days). Cocultivation of PBMC with fixed SARS-CoV-infected Vero cells was even more efficient than exposure to live virus in eliciting IFN-α and -γ induction. A combination of IFN-α and -γ strongly inhibited SARS-CoV replication in Vero cells, while the single cytokines were much less effective. Conclusions:: This study provides evidence that SARS-CoV is able to induce in normal PBMC a coordinate induction of IFN-α and -γ gene expression. Virus replication is not necessary for IFN induction since efficient IFN expression could be obtained also by the cocultivation of normal PBMC with fixed SARS-CoV-infected cells. Concomitant activation of IFN-α and -γ gene expression by SARS-CoV in vivo may be relevant for the pathogenesis of the disease, both for the possible

  11. Animal Models for Influenza Viruses: Implications for Universal Vaccine Development

    OpenAIRE

    Irina Margine; Florian Krammer

    2014-01-01

    Influenza virus infections are a significant cause of morbidity and mortality in the human population. Depending on the virulence of the influenza virus strain, as well as the immunological status of the infected individual, the severity of the respiratory disease may range from sub-clinical or mild symptoms to severe pneumonia that can sometimes lead to death. Vaccines remain the primary public health measure in reducing the influenza burden. Though the first influenza vaccine preparation wa...

  12. Animal models for Ebola and Marburg virus infections

    OpenAIRE

    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...

  13. Hendra and Nipah viruses: pathogenesis, animal models and recent breakthroughs in vaccination

    Directory of Open Access Journals (Sweden)

    Weingartl HM

    2015-09-01

    Full Text Available Hana M Weingartl National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB, Canada Abstract: Hendra and Nipah viruses are two highly pathogenic zoonotic members of the genus Henipavirus, family Paramyxoviridae, requiring work under biosafety level 4 conditions due to a lack of effective therapy and human vaccines. Several vaccine candidates were protective in animal models: recombinant vaccinia virus expressing Nipah virus (NiV F and G proteins in hamsters against NiV; recombinant ALVAC–NiV F and G in swine against NiV; recombinant Hendra virus (HeV soluble G protein (sGHeV against HeV and NiV in cats, ferrets, horses, and African green monkeys (AGM; recombinant vesicular stomatitis virus-based vectors expressing NiV F or G against NiV in hamsters and ferrets; measles virus-based NiV G vaccine candidate in hamsters and AGMs against NiV; and adenoassociated virus expressing NiG protein, which protected hamsters against NiV. The sGHeV was licensed for use in horses (Equivac HeV® in 2012. It is the first vaccine candidate licensed against a biosafety level 4 agent. With the development of suitable animal models (ferret, hamster and, importantly, AGM, progress can be made toward development of a human vaccine.Keywords: henipavirus, equine, swine, human infection, animal models, vaccine candidates

  14. Cyclin A degradation by primate cytomegalovirus protein pUL21a counters its innate restriction of virus replication.

    Directory of Open Access Journals (Sweden)

    Nicolas Caffarelli

    Full Text Available Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC, but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.

  15. Inhibition of hepatitis B virus replication by a dNTPase-dependent function of the host restriction factor SAMHD1.

    Science.gov (United States)

    Jeong, Gi Uk; Park, Il-Hyun; Ahn, Kwangseog; Ahn, Byung-Yoon

    2016-08-01

    SAMHD1 is a cellular protein that possesses dNTPase activity and inhibits retroviruses and DNA viruses through the depletion of cellular dNTPs. However, recent evidence suggests the existence of alternative or additional mechanisms that involve novel nuclease activities. Hepatitis B virus is a DNA virus but resembles retroviruses in that its DNA genome is synthesized via reverse transcription of an RNA transcript. SAMHD1 was shown to inhibit the expression and replication of a transfected HBV DNA. We further investigated the antiviral mechanisms in a newly developed infection assay. Our data indicated that SAMHD1 exerts a profound antiviral effect. In addition, unlike previous findings, our results demonstrate the essential role of SAMHD1 dNTPase. SAMHD1 did not affect virion-derived cccDNA and gene expression but specifically inhibited viral DNA synthesis. These results indicate that SAMHD1 inhibits HBV replication at the reverse transcription step, most likely through the depletion of cellular dNTPs. PMID:27179347

  16. Analysis of in vitro replicated human hepatitis C virus (HCV for the determination of genotypes and quasispecies

    Directory of Open Access Journals (Sweden)

    Chelyapov Nickolas

    2006-09-01

    Full Text Available Abstract Isolation and self-replication of infectious HCV has been a difficult task. However, this is needed for the purposes of developing rational drugs and for the analysis of the natural virus. Our recent report of an in vitro system for the isolation of human HCV from infected patients and their replication in tissue culture addresses this challenge. At California Institute of Molecular Medicine several isolates of HCV, called CIMM-HCV, were grown for over three years in cell culture. This is a report of the analysis of CIMM-HCV isolates for subtypes and quasispecies using a 269 bp segment of the 5'UTR. HCV RNA from three patients and eleven CIMM-HCV were analyzed for this purpose. All isolates were essentially identical. Isolates of HCV from one patient were serially transmitted into fresh cells up to eight times and the progeny viruses from each transmission were compared to each other and also to the primary isolates from the patient's serum. Some isolates were also transmitted to different cell types, while others were cultured continuously without retransmission for over three years. We noted minor sequence changes when HCV was cultured for extended periods of time. HCV in T-cells and non-committed lymphoid cells showed a few differences when compared to isolates obtained from immortalized B-cells. These viruses maintained close similarity despite repeated transmissions and passage of time. There were no subtypes or quasispecies noted in CIMM-HCV.

  17. Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

    DEFF Research Database (Denmark)

    Jacobsen, Kari Stougaard; Nielsen, Kirstine Overgaard; Winther, Thilde Nordmann; Glebe, Dieter; Pociot, Flemming; Hogh, Birthe

    2016-01-01

    BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially ....... CONCLUSION: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.......BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially...... expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection...

  18. Evidence that a polyhexameric genome length is preferred, but not strictly required, for efficient mumps virus replication.

    Science.gov (United States)

    Sauder, Christian J; Simonyan, Vahan; Ngo, Laurie; Karagiannis, Konstantinos; Cong, Yu; Zhang, Cheryl; Wang, Rong; Wu, Wells W; Malik, Tahir; Rubin, Steven A

    2016-06-01

    Mumps virus (MuV) is postulated to adhere to the "rule of six" for efficient replication. To examine the requirement for MuV, minigenomes of nonpolyhexameric length (6n-1 and 6n+1) were analyzed. Expression of the reporter gene CAT was significantly reduced with minigenomes of nonpolyhexameric length compared to the wild type 6n genome, and reduction was more pronounced for the 6n-1 than for the 6n+1 minigenome. That 6n-1 genomes are impacted by nonconformance with the rule of six to a greater degree as compared to 6n+1 genomes was also suggested with MuV derived from cDNA coding for 6n+1 or 6n-1 genomes. While viruses recovered from 6n+1 cDNAs maintained a nonpolyhexameric genome length over multiple replication cycles, viruses rescued from the 6n-1 cDNAs acquired length correcting mutations rapidly following rescue. Our data indicate that polyhexameric genomes are the preferred template for the MuV RNA polymerase, but that this requirement is not absolute. PMID:27058764

  19. Genetic Adaptation of Influenza A Viruses in Domestic Animals and Their Potential Role in Interspecies Transmission: A Literature Review.

    Science.gov (United States)

    Munoz, Olga; De Nardi, Marco; van der Meulen, Karen; van Reeth, Kristien; Koopmans, Marion; Harris, Kate; von Dobschuetz, Sophie; Freidl, Gudrun; Meijer, Adam; Breed, Andrew; Hill, Andrew; Kosmider, Rowena; Banks, Jill; Stärk, Katharina D C; Wieland, Barbara; Stevens, Kim; van der Werf, Sylvie; Enouf, Vincent; Dauphin, Gwenaelle; Dundon, William; Cattoli, Giovanni; Capua, Ilaria

    2016-03-01

    In December 2011, the European Food Safety Authority awarded a Grant for the implementation of the FLURISK project. The main objective of FLURISK was the development of an epidemiological and virological evidence-based influenza risk assessment framework (IRAF) to assess influenza A virus strains circulating in the animal population according to their potential to cross the species barrier and cause infections in humans. With the purpose of gathering virological data to include in the IRAF, a literature review was conducted and key findings are presented here. Several adaptive traits have been identified in influenza viruses infecting domestic animals and a significance of these adaptations for the emergence of zoonotic influenza, such as shift in receptor preference and mutations in the replication proteins, has been hypothesized. Nonetheless, and despite several decades of research, a comprehensive understanding of the conditions that facilitate interspecies transmission is still lacking. This has been hampered by the intrinsic difficulties of the subject and the complexity of correlating environmental, viral and host factors. Finding the most suitable and feasible way of investigating these factors in laboratory settings represents another challenge. The majority of the studies identified through this review focus on only a subset of species, subtypes and genes, such as influenza in avian species and avian influenza viruses adapting to humans, especially in the context of highly pathogenic avian influenza H5N1. Further research applying a holistic approach and investigating the broader influenza genetic spectrum is urgently needed in the field of genetic adaptation of influenza A viruses. PMID:25630935

  20. No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus

    International Nuclear Information System (INIS)

    BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were stimated by the method of fiber-autoradiography and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcome virus-transformed BALB/c 3T3 cells

  1. The carboxy-terminal half of nonstructural protein 3A is not essential for foot-and-mouth disease virus replication in cultured cell lines.

    Science.gov (United States)

    Behura, Mrutyunjay; Mohapatra, Jajati K; Pandey, Laxmi K; Das, Biswajit; Bhatt, Mukesh; Subramaniam, Saravanan; Pattnaik, Bramhadev

    2016-05-01

    In foot-and-mouth disease (FMD)-endemic parts of the globe, control is mainly implemented by preventive vaccination with an inactivated purified vaccine. ELISAs detecting antibodies to the viral nonstructural proteins (NSP) distinguish FMD virus (FMDV)-infected animals in the vaccinated population (DIVA). However, residual NSPs present in the vaccines are suspected to be a cause of occasional false positive results, and therefore, an epitope-deleted negative marker vaccine strategy is considered a more logical option. In this study, employing a serotype Asia 1 FMDV infectious cDNA clone, it is demonstrated that while large deletions differing in size and location in the carboxy-terminal half of 3A downstream of the putative hydrophobic membrane-binding domain (deletion of residues 86-110, 101-149, 81-149 and 81-153) are tolerated by the virus without affecting its infectivity in cultured cell lines, deletions in the amino-terminal half (residues 5-54, 21-50, 21-80, 55-80 and 5-149) containing the dimerization and the transmembrane domains are deleterious to its multiplication. Most importantly, the virus could dispense with the entire carboxy-terminal half of 3A (residues 81-153) including the residues involved in the formation of the 3A-3B1 cleavage junction. The rescue of a replication-competent FMDV variant carrying the largest deletion ever in 3A (residues 81-153) and the fact that the deleted region contains a series of linear B-cell epitopes inspired us to devise an indirect ELISA based on a recombinant 3A carboxy-terminal fragment and to evaluate its potential to serve as a companion diagnostic assay for differential serosurveillance if the 3A-truncated virus is used as a marker vaccine. PMID:26935917

  2. Scope for using plant viruses to present epitopes from animal pathogens.

    Science.gov (United States)

    Porta; Lomonossoff

    1998-01-01

    Epitope presentation to the immune system for vaccination purposes can be achieved either via an inactivated or attenuated form of a pathogen or via its isolated antigenic sequences. When free, these peptides can adopt a variety of conformations, most of which will not exist in their native environment. Conjugation to carrier proteins restricts mobility of the peptides and increases their immunogenicity. A high local concentration of epitopes boosts the immune response further and can be generated by the use of self-aggregating carriers, such as the capsid proteins of viruses. In this regard plant viruses have in recent years started to make an impact as safer alternatives to the use of bacterial and attenuated animal viruses: the latter both require propagation in costly cell-culture systems where they can undergo reversion towards a virulent form and/or become contaminated by other pathogens. Plant virus-based vectors can be multiplied cheaply and to high yields (exceeding 1 mg/g plant tissue) in host plants. Both helical (tobacco mosaic virus, potato virus X, alfalfa mosaic virus) and icosahedral (cowpea mosaic virus, tomato bushy stunt virus) particles have been used to express a number of animal B-cell epitopes, whose immunogenic properties have been explored to varying degrees. Copyright 1998 John Wiley & Sons, Ltd. PMID:10398492

  3. Efficient Replication of Adeno-Associated Virus Type 2 Vectors: a cis-Acting Element outside of the Terminal Repeats and a Minimal Size

    OpenAIRE

    Tullis, Gregory E.; Shenk, Thomas

    2000-01-01

    Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containin...

  4. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo

    OpenAIRE

    Fenaux, M.; Meng, X.J.; F Elvinger; Opriessnig, T.; Halbur, P. G.

    2004-01-01

    Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived ...

  5. Two Amino Acid Mutations in the Capsid Protein of Type 2 Porcine Circovirus (PCV2) Enhanced PCV2 Replication In Vitro and Attenuated the Virus In Vivo

    OpenAIRE

    Fenaux, M.; Opriessnig, T.; Halbur, P. G.; F Elvinger; Meng, X.J.

    2004-01-01

    Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived ...

  6. Cytokine mRNA expression in foot-and-mouth disease virus (FMDV) persistently infected bovine pharynx cultures: effect of IFNgamma on replication of persistent virus

    Science.gov (United States)

    Foot-and-mouth disease virus (FMDV), a member of the family Picornaviridae, genus Aphtovirus, causes a highly contagious disease in livestock. Following acute infection in ruminants, up to 50% of both vaccinated and non-vaccinated animals become persistently infected asymptomatic carriers with low-l...

  7. Gamma Radiation for Sterilizing the Carcasses of Foot-and-Mouth Disease Virus Infected Animals

    International Nuclear Information System (INIS)

    Experiments on sterilization by means of gamma rays of the carcasses of animals experimentally infected with foot-and-mouth disease virus (FMDV) have been carried out. In the first part the author studied the presence and survival of FMDV in the carcasses and in the organs of infected slaughtered animals. The results obtained are sufficient to underline the problem of the sterilization of carcasses of animals infected by FMDV. The experiments on the inactivation of the FMDV by gamma irradiation in vitro showed the same radiation sensitivity of the three types, O, A and C, of FMDV inaqueous solutions and showed that the fraction of surviving virus is an exponential function of the gamma-ray dose. The results obtained confirm the remarkable resistance of viruses to the effect of radiation. As far as the dry virus is concerned special tests indicated the necessity of greater doses for inactivating the same virus in the dry as opposed to the liquid state. In the third part the author studied the possibility of utilizing gamma rays for the sterilization of carcasses of infected (or suspected of being infected) FMDV animals using some tissues of infected animals (pigs) (blood, bone marrow, vertebrae, lymph nodes). The results obtained show that the inactivation of FMDV types O, A and C in the carcasses of infected animals can be made by treatment with gamma rays. (author)

  8. Feasibility of Using the Mosquito Blood Meal for Rapid and Efficient Human and Animal Virus Surveillance and Discovery.

    Science.gov (United States)

    Yang, Yu; Garver, Lindsey S; Bingham, Karen M; Hang, Jun; Jochim, Ryan C; Davidson, Silas A; Richardson, Jason H; Jarman, Richard G

    2015-12-01

    Mosquito blood meals taken from humans and animals potentially represent a useful source of blood for the detection of blood-borne pathogens. In this feasibility study, Anopheles stephensi mosquitoes were fed with blood meals spiked with dengue virus type 2 (DENV-2) and harvested at serial time points. These mosquitoes are not competent vectors, and the virus is not expected to replicate. Ingested blood was spotted on Whatman FTA cards and stored at room temperature. Mosquito abdomens were removed and stored at -80°C. Control blood meal aliquots were stored in vials or applied onto FTA cards. After 4 weeks of storage, the samples were extracted using beadbeating and QIAamp Viral RNA kit (Qiagen Sciences, Germantown, MD). Recovered viral RNA was analyzed by DENV-2 TaqMan RT-PCR assay and next-generation sequencing (NGS). Overall viral RNA recovery efficiency was 15% from the directly applied dried blood spots and approximately 20% or higher for dried blood spots made by blotting mosquito midgut on FTA cards. Viral RNA in mosquito-ingested blood decreases over time, but remains detectable 24 hours after blood feeding. The viral sequences in FTA-stored specimens can be maintained at room temperature. The strategy has the potential utility in expedited zoonotic virus discovery and blood-borne pathogen surveillance. PMID:26416112

  9. Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

    Directory of Open Access Journals (Sweden)

    Yan Mylene L

    2011-08-01

    Full Text Available Abstract Background Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1 gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Results Five commercially available animal-component free, serum-free media (SFM were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. Conclusions We describe for the

  10. The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

    OpenAIRE

    Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Choudhury, Nirupam Roy; Karjee, Sumona; Mukherjee, Sunil Kumar

    2006-01-01

    Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem–loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA...

  11. Reprint of "Modeling the intracellular replication of influenza A virus in the presence of defective interfering RNAs.

    Science.gov (United States)

    Laske, Tanja; Heldt, Frank Stefan; Hoffmann, Helene; Frensing, Timo; Reichl, Udo

    2016-06-15

    Like many other viral pathogens, influenza A viruses can form defective interfering particles (DIPs). These particles carry a large internal deletion in at least one of their genome segments. Thus, their replication depends on the co-infection of cells by standard viruses (STVs), which supply the viral protein(s) encoded by the defective segment. However, DIPs also interfere with STV replication at the molecular level and, despite considerable research efforts, the mechanism of this interference remains largely elusive. Here, we present a mechanistic mathematical model for the intracellular replication of DIPs. In this model, we account for the common hypothesis that defective interfering RNAs (DI RNAs) possess a replication advantage over full-length (FL) RNAs due to their reduced length. By this means, the model captures experimental data from yield reduction assays and from studies testing different co-infection timings. In addition, our model predicts that one important aspect of interference is the competition for viral proteins, namely the heterotrimeric viral RNA-dependent RNA polymerase (RdRp) and the viral nucleoprotein (NP), which are needed for encapsidation of naked viral RNA. Moreover, we find that there may be an optimum for both the DI RNA synthesis rate and the time point of successive co-infection of a cell by DIPs and STVs. Comparing simulations for the growth of DIPs with a deletion in different genome segments suggests that DI RNAs derived from segments which encode for the polymerase subunits are more competitive than others. Overall, our model, thus, helps to elucidate the interference mechanism of DI RNAs and provides a novel hypothesis why DI RNAs derived from the polymerase-encoding segments are more abundant in DIP preparations. PMID:27208847

  12. Tomato bushy stunt virus and DI RNAs as a model for studying mechanisms of RNA virus replication, pathogenicity and recombination. Final technical report for 1994--1997

    Energy Technology Data Exchange (ETDEWEB)

    Morris, T.J. [Univ. of Nebraska, Lincoln, NE (United States). School of Biological Sciences; Jackson, A.O. [Univ. of California, Berkeley, CA (United States). Dept. of Plant Biology

    1997-12-31

    Tomato bushy stunt virus (TBSV) is a small icosahedral virus with a very broad host-range. The symptoms of systemic infection range from mild mosaic to severe necrosis that often results in death. The genome of TBSV is composed of a single plus stranded RNA molecule with five genes. Two 5 inch genes are translated from the viral RNA, and the remaining three are translated from two subgenomic RNAs. Prior to the DOE supported studies, TBSV gene function had been assigned solely on the basis of sequence similarity with other virus genes of known function. The two 5 inch proximal genes (p33 and p92) were thought to be involved in viral replication, the middle gene encoded the capsid protein (p41), but no clear function was assigned to two nested 3 inch genes (p19 and p22), although it was suggested that at least one could be involved in movement. This research has determined the roles of each of the viral genes in the infection process, and the authors have obtained considerable genetic information pertinent to the contributions of the coat protein and the nested genes to the disease phenotypes observed in several host plants. They have also identified another genetic element with a short open reading frame in the 3 inch-noncoding region of the genome that provides a host-dependent replication function.

  13. Interferon-alpha mediates restriction of human immunodeficiency virus type-1 replication in primary human macrophages at an early stage of replication.

    Directory of Open Access Journals (Sweden)

    Kelly M Cheney

    Full Text Available Type I interferons (IFNα and β are induced directly in response to viral infection, resulting in an antiviral state for the cell. In vitro studies have shown that IFNα is a potent inhibitor of viral replication; however, its role in HIV-1 infection is incompletely understood. In this study we describe the ability of IFNα to restrict HIV-1 infection in primary human macrophages in contrast to peripheral blood mononuclear cells and monocyte-derived dendritic cells. Inhibition to HIV-1 replication in cells pretreated with IFNα occurred at an early stage in the virus life cycle. Late viral events such as budding and subsequent rounds of infection were not affected by IFNα treatment. Analysis of early and late HIV-1 reverse transcripts and integrated proviral DNA confirmed an early post entry role for IFNα. First strand cDNA synthesis was slightly reduced but late and integrated products were severely depleted, suggesting that initiation or the nucleic acid intermediates of reverse transcription are targeted. The depletion of integrated provirus is disproportionally greater than that of viral cDNA synthesis suggesting the possibility of a least an additional later target. A role for either cellular protein APOBEC3G or tetherin in this IFNα mediated restriction has been excluded. Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect. Determining both the viral determinants and cellular proteins involved may lead to novel therapeutic approaches. Our results add to the understanding of HIV-1 restriction by IFNα.

  14. Protein-protein and protein-small-molecule inhibitor interactions in the measles virus replication complex

    OpenAIRE

    Stefanie A Krumm

    2013-01-01

    The disease measles is caused by the highly contagious measles virus (MeV). MeV belongs to the paramyxovirus family together with respiratory syncytial virus, human parainfluenza viruses and metapneumovirus. Paramyxoviruses are responsible for major pediatric morbidity and mortality. Despite the availability of an effective MeV vaccine, measles case numbers increased alarmingly in the past few years especially in Europe. The return of endemic measles in the European population can directly be...

  15. Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues.

    OpenAIRE

    Cannon, P M; Wilson, W; Byles, E; Kingsman, S M; Kingsman, A J

    1994-01-01

    Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysi...

  16. Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

    OpenAIRE

    Flamand, L.; Stefanescu, I.(Argonne National Laboratory, Argonne, IL, 60439, USA); Ablashi, D V; Menezes, J.

    1993-01-01

    One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the...

  17. 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

    of neo or the enhanced green fluorescence protein gene (EGFP). Akv-MLV's with IRES-neo and IRES-EGFP cassettes replicated with titers of about 10(6) infectious units/ml while SL3-3-MLV with IRES-neo gave about 10(3)-fold lower titers. Interestingly, RNA analysis showed a drastic reduction in the amount...... of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  18. Mutational Analysis of the Influenza Virus cRNA Promoter and Identification of Nucleotides Critical for Replication

    OpenAIRE

    Crow, Mandy; Deng, Tao; Addley, Mark; Brownlee, George G.

    2004-01-01

    Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A “corkscrew” secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authe...

  19. Inhibition of DNA virus: Herpes-1 (HSV-1) in cellular culture replication, through an antioxidant treatment extracted from rosemary spice

    OpenAIRE

    Dalva Assunção Portari Mancini; Rosângela Pavan Torres; José Ricardo Pinto; Jorge Mancini-Filho

    2009-01-01

    This work aimed to evaluate antiviral properties in antioxidants from spices. Phenolic compounds extracted from rosemary (Rosmarinus officinallis, L) by hot water, had their antioxidant activity determined by spectrophotometry using β carotene/linoleic acid system. The rosemary extract was evaluated by antiviral assay of Herpes Virus type-1 (HSV-1) replication in VERO cells, in the presence or absence of the spice. 10,000 TCID50/mL of the HSV-1 was kept for 3 h at 4º C, with 300 ppm of r...

  20. Direct entry of rabies virus into the central nervous system without prior local replication.

    OpenAIRE

    Shankar, V.; Dietzschold, B.; Koprowski, H

    1991-01-01

    Rabies virus pathogenesis was studied in a mouse model by inoculation of the masseter muscle. At different intervals, the masseter muscle, trigeminal ganglia, and brain were analyzed for virus-specific RNA with a polymerase chain reaction assay, which revealed that as early as 18 h postinfection (p.i.), virus-specific RNA was present in the trigeminal ganglia, and at 24 h p.i., viral RNA was identified in the brain stem. Analysis of the masseter muscle demonstrated virus at 1 h p.i. but no vi...

  1. An Association between BK Virus Replication in Bone Marrow and Cytopenia in Kidney-Transplant Recipients

    OpenAIRE

    Emilie Pambrun; Catherine Mengelle; Geneviève Fillola; Patrick Laharrague; Laure Esposito; Isabelle Cardeau-Desangles; Arnaud Del Bello; Jacques Izopet; Lionel Rostaing; Nassim Kamar

    2014-01-01

    The human polyomavirus BK (BKV) is associated with severe complications, such as ureteric stenosis and polyomavirus-associated nephropathy (PVAN), which often occur in kidney-transplant patients. However, it is unknown if BKV can replicate within bone marrow. The aim of this study was to search for BKV replication within the bone marrow of kidney-transplant patients presenting with a hematological disorder. Seventy-two kidney-transplant patients underwent bone-marrow aspiration for cytopenia....

  2. Rupestonic acid derivative YZH-106 suppresses influenza virus replication by activation of heme oxygenase-1-mediated interferon response.

    Science.gov (United States)

    Ma, Lin-Lin; Wang, Hui-Qiang; Wu, Ping; Hu, Jin; Yin, Jin-Qiu; Wu, Shuo; Ge, Miao; Sun, Wen-Fang; Zhao, Jiang-Yu; Aisa, Haji Akber; Li, Yu-Huan; Jiang, Jian-Dong

    2016-07-01

    Given the limitation of available antiviral drugs and vaccines, there remains to be a pressing need for novel anti-influenza drugs. Rupestonic acid derivatives were reported to have an anti-influenza virus activity, but their mechanism remains to be elucidated. Herein, we aim to evaluate the antiviral activity of YZH-106, a rupestonic acid derivative, against a broad-spectrum of influenza viruses and to dissect its antiviral mechanisms. Our results demonstrated that YZH-106 exhibited a broad-spectrum antiviral activity against influenza viruses, including drug-resistant strains in vitro. Furthermore, YZH-106 provided partial protection of the mice to Influenza A virus (IAV) infection, as judged by decreased viral load in lungs, improved lung pathology, reduced body weight loss and partial survival benefits. Mechanistically, YZH-106 induced p38 MAPK and ERK1/2 phosphorylation, which led to the activation of erythroid 2-related factor 2 (Nrf2) that up-regulated heme oxygenase-1 (HO-1) expression in addition to other genes. HO-1 inhibited IAV replication by activation of type I IFN expression and subsequent induction of IFN-stimulated genes (ISGs), possibly in a HO-1 enzymatic activity-independent manner. These results suggest that YZH-106 inhibits IAV by up-regulating HO-1-mediated IFN response. HO-1 is thus a promising host target for antiviral therapeutics against influenza and other viral infectious diseases. PMID:27107768

  3. VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    Jun Yi; Wei-Dong Gong; Ling Wang; Rui Ling; Jiang-Hao Chen; Jun Yun

    2005-01-01

    AIM: To investigate the inhibitory effect of VP22 fusion protein-based dominant negative (DN) mutant on Hepatitis Bvrus (HBV) replication.METHODS: Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein (HBc), and subcloned into pcDNA3.1 (-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR (FQ-PCR). Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.RESULTS: VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutantbearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups.CONCLUSION: VP22-based DN mutant can inhibit HBV replication effectively.

  4. Improved method for rapid and efficient determination of genome replication and protein expression of clinical hepatitis B virus isolates.

    Science.gov (United States)

    Qin, Yanli; Zhang, Jiming; Garcia, Tamako; Ito, Kiyoaki; Gutelius, Danielle; Li, Jisu; Wands, Jack; Tong, Shuping

    2011-04-01

    Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomes and/or response to antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelity(plus) DNA polymerase and primers targeting the conserved precore region, the HBV clones thus generated are replication deficient due to the inability to generate the terminally redundant pregenomic RNA essential for genome replication. The transfection experiment is further complicated by PCR errors and the presence of viral quasispecies. A previous study found that the precise removal of non-HBV sequence by SapI digestion led to HBV replication in transfected cells, possibly due to low-level genome circularization by a cellular enzyme. We released HBV genome from the cloning vector using BspQI, an inexpensive isoschizomer of SapI, and increased the efficiency of genome replication by an extra step of in vitro DNA ligation. The uncut plasmid DNA can be used for transfection if the sole purpose is to study envelope protein expression. We found significant PCR errors associated with the High Fidelity(plus) DNA polymerase, which could be greatly diminished using Phusion DNA polymerase or masked by the use of a clone pool. The reduced PCR error and modified enzymatic steps prior to transfection should facilitate a more widespread functional characterization of clinical HBV isolates, while the clone pool approach is useful for samples with significant sequence heterogeneity. PMID:21289153

  5. Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhe; XU Ze-feng; YE Jing-jia; YAO Hang-ping; ZHENG Shu; DING Jia-yi

    2005-01-01

    Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region ofHBV genome were designed and chemically synthesized.(2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR(Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs(siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs.Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

  6. The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication.

    Science.gov (United States)

    La Ferla, F M; Peluso, R W

    1989-01-01

    We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo. Images PMID:2548001

  7. MicroRNA-23b Promotes Avian Leukosis Virus Subgroup J (ALV-J) Replication by Targeting IRF1.

    Science.gov (United States)

    Li, Zhenhui; Chen, Biao; Feng, Min; Ouyang, Hongjia; Zheng, Ming; Ye, Qiao; Nie, Qinghua; Zhang, Xiquan

    2015-01-01

    Avian leukosis virus subgroup J (ALV-J) can cause several different leukemia-like proliferative diseases in the hemopoietic system of chickens. Here, we investigated the transcriptome profiles and miRNA expression profiles of ALV-J-infected and uninfected chicken spleens to identify the genes and miRNAs related to ALV-J invasion. In total, 252 genes and 167 miRNAs were differentially expressed in ALV-J-infected spleens compared to control uninfected spleens. miR-23b expression was up-regulated in ALV-J-infected spleens compared with the control spleens, and transcriptome analysis revealed that the expression of interferon regulatory factor 1 (IRF1) was down-regulated in ALV-J-infected spleens compared to uninfected spleens. A dual-luciferase reporter assay showed that IRF1 was a direct target of miR-23b. miR-23b overexpression significantly (P = 0.0022) decreased IRF1 mRNA levels and repressed IRF1-3'-UTR reporter activity. In vitro experiments revealed that miR-23b overexpression strengthened ALV-J replication, whereas miR-23b loss of function inhibited ALV-J replication. IRF1 overexpression inhibited ALV-J replication, and IRF1 knockdown enhanced ALV-J replication. Moreover, IRF1 overexpression significantly (P = 0.0014) increased IFN-β expression. In conclusion, these results suggested that miR-23b may play an important role in ALV-J replication by targeting IRF1. PMID:25980475

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

    NARCIS (Netherlands)

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

    2016-01-01

    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

  9. Metagenomic analysis of viruses associated with field-grown and retail lettuce identifies human and animal viruses.

    Science.gov (United States)

    Aw, Tiong Gim; Wengert, Samantha; Rose, Joan B

    2016-04-16

    The emergence of culture- and sequence-independent metagenomic methods has not only provided great insight into the microbial community structure in a wide range of clinical and environmental samples but has also proven to be powerful tools for pathogen detection. Recent studies of the food microbiome have revealed the vast genetic diversity of bacteria associated with fresh produce. However, no work has been done to apply metagenomic methods to tackle viruses associated with fresh produce for addressing food safety. Thus, there is a little knowledge about the presence and diversity of viruses associated with fresh produce from farm-to-fork. To address this knowledge gap, we assessed viruses on commercial romaine and iceberg lettuces in fields and a produce distribution center using a shotgun metagenomic sequencing targeting both RNA and DNA viruses. Commercial lettuce harbors an immense assemblage of viruses that infect a wide range of hosts. As expected, plant pathogenic viruses dominated these communities. Sequences of rotaviruses and picobirnaviruses were also identified in both field-harvest and retail lettuce samples, suggesting an emerging foodborne transmission threat that has yet to be fully recognized. The identification of human and animal viruses in lettuce samples in the field emphasizes the importance of preventing viral contamination on leafy greens starting at the field. Although there are still some inherent experimental and bioinformatics challenges in applying viral metagenomic approaches for food safety testing, this work will facilitate further application of this unprecedented deep sequencing method to food samples. PMID:26894328

  10. No evidence of African swine fever virus replication in hard ticks

    NARCIS (Netherlands)

    Carvalho Ferreira, de H.C.; Zúquete, S.T.; Wijnveld, M.; Weesendorp, E.; Jongejan, F.; Stegeman, J.A.; Loeffen, W.L.A.

    2014-01-01

    African swine fever (ASF) is caused by African swine fever virus (ASFV), a tick-borne DNA virus. Soft ticks of the genus Ornithodoros are the only biological vectors of ASFV recognized so far. Although other hard ticks have been tested for vector competence, two commonly found tick species in Europe

  11. Genome sequence of foot-and-mouth disease virus outside the 3A region is also responsible for virus replication in bovine cells.

    Science.gov (United States)

    Ma, Xueqing; Li, Pinghua; Sun, Pu; Lu, Zengjun; Bao, Huifang; Bai, Xingwen; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

    2016-07-15

    The deletion of residues 93-102 in non-structure protein 3A of foot-and-mouth disease virus (FMDV) is associated with the inability of FMDV to grow in bovine cells and attenuated virulence in cattle.Whereas, a previously reported FMDV strain O/HKN/21/70 harboring 93-102 deletion in 3A protein grew equally well in bovine and swine cells. This suggests that changes inFMDV genome sequence, in addition to 93-102 deletion in 3A, may also affectthe viral growth phenotype in bovine cellsduring infection and replication.However, it is nuclear that changes in which region (inside or outside of 3A region) influences FMDV growth phenotype in bovine cells.In this study, to determine the region in FMDV genomeaffecting viral growth phenotype in bovine cells, we constructed chimeric FMDVs, rvGZSB-HKN3A and rvHN-HKN3A, by introducing the 3A coding region of O/HKN/21/70 into the context of O/SEA/Mya-98 strain O/GZSB/2011 and O Cathay topotype strain O/HN/CHA/93, respectively, since O/GZSB/2011 containing full-length 3A protein replicated well in bovine and swine cells, and O/HN/CHA/93 harboring 93-102 deletion in 3A protein grew poorly in bovine cells.The chimeric virusesrvGZSB-HKN3A and rvHN-HKN3A displayed growth properties and plaque phenotypes similar to those of the parental virus rvGZSB and rv-HN in BHK-21 and primary fetal porcine kidney (FPK) cells. However, rvHN-HKN3A and rv-HN replicated poorly in primary fetal bovine kidney (FBK) cells with no visible plaques, and rvGZSB-HKN3A exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the difference present in FMDV genome sequence outside the 3A coding region also have influence on FMDV replication ability in bovine cells. PMID:27094491

  12. Limited replication of yellow fever 17DD and 17D-Dengue recombinant viruses in rhesus monkeys

    Directory of Open Access Journals (Sweden)

    Gisela F. Trindade

    2008-06-01

    Full Text Available For the development of safe live attenuated flavivirus vaccines one of the main properties to be established is viral replication. We have used real-time reverse transcriptase-polymerase chain reaction and virus titration by plaque assay to determine the replication of yellow fever 17DD virus (YFV 17DD and recombinant yellow fever 17D viruses expressing envelope proteins of dengue virus serotypes 2 and 4 (17D-DENV-2 and 17D-DENV-4. Serum samples from rhesus monkeys inoculated with YFV 17DD and 17D-DENV chimeras by intracerebral or subcutaneous route were used to determine and compare the viremia induced by these viruses. Viral load quantification in samples from monkeys inoculated by either route with YFV 17DD virus suggested a restricted capability of the virus to replicate reaching not more than 2.0 log10 PFU mL-1 or 3.29 log10 copies mL-1. Recombinant 17D-dengue viruses were shown by plaquing and real-time PCR to be as attenuated as YF 17DD virus with the highest mean peak titer of 1.97 log10 PFU mL-1 or 3.53 log10 copies mL-1. These data serve as a comparative basis for the characterization of other 17D-based live attenuated candidate vaccines against other diseases.Uma das principais propriedades a serem estabelecidas para o desenvolvimento de vacinas seguras e atenuadas de flavivirus,é a taxa de replicação viral. Neste trabalho, aplicamos a metodologia de amplificação pela reação em cadeia da polimerase em tempo real e titulação viral por plaqueamento para determinação da replicação do vírus 17DD (FA 17DD e recombinantes, expressando proteínas do envelope de dengue sorotipos 2 e 4 (17D-DENV-2 e 17D-DENV-4. As amostras de soros de macacos inoculados por via intracerebral ou subcutânea com FA 17DD ou 17D-DENV foram usadas para determinar e comparar a viremia induzida por estes vírus. A quantificação da carga viral em amostras de macacos inoculados por ambas as vias com FA 17DD sugere restrita capacidade de replicação com

  13. Vaccination of Non-Domestic Animals against Emerging Virus Infections

    OpenAIRE

    Philippa, Joost

    2007-01-01

    markdownabstract__Abstract__ Since the 1980's, emerging and re-emerging infectious diseases have made an enormous impact on public and animal health, food supply, economies, and the environment. An estimated 75% of emerging infectious diseases in humans are zoonotic (pathogens of non-human vertebrate animals that may be transmitted to humans under natural conditions), mainly of viral origin and often vector-borne. Since 1980, more than 35 new infectious agents have emerged in humans , includi...

  14. CD4+ T Cells Are Not Required for Suppression of Hepatitis B Virus Replication in the Liver of Vaccinated Chimpanzees.

    Science.gov (United States)

    Rybczynska, Jolanta; Campbell, Katherine; Kamili, Saleem; Locarnini, Stephen; Krawczynski, Krzysztof; Walker, Christopher M

    2016-01-01

    Humans vaccinated with hepatitis B virus (HBV) surface antigen (HBsAg) sometimes develop humoral and cellular immunity to HBV proteins such as core and polymerase that are not vaccine components, providing indirect evidence that vaccine-induced immunity is not sterilizing. We previously described CD4(+) T-cell immunity against HBsAg and polymerase in chimpanzees after vaccination and HBV challenge. Here, vaccinated chimpanzees with protective levels of anti-HBsAg antibodies were rechallenged with HBV after antibody-mediated CD4(+) T-cell depletion. HBV DNA was detected in liver for at least 3 months after rechallenge, but virus replication was suppressed, as revealed by the absence of HBV DNA and HBsAg in serum. These observations provide direct virological evidence for nonsterilizing immunity in individuals with anti-HBsAg antibodies and are consistent with translation of HBV proteins to prime immune responses. They also indicate that CD4(+) T cells were not required for suppression of HBV replication in previously vaccinated individuals. PMID:26324781

  15. Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages.

    Science.gov (United States)

    Ryzhova, Elena V; Vos, Robin M; Albright, Andrew V; Harrist, Alexia V; Harvey, Thomas; González-Scarano, Francisco

    2006-03-01

    Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM. PMID:16501079

  16. HSV-1-induced activation of NF-κB protects U937 monocytic cells against both virus replication and apoptosis.

    Science.gov (United States)

    Marino-Merlo, Francesca; Papaianni, Emanuela; Medici, Maria Antonietta; Macchi, Beatrice; Grelli, Sandro; Mosca, Claudia; Borner, Christoph; Mastino, Antonio

    2016-01-01

    The transcription factor nuclear factor-kappa B (NF-κB) is a crucial player of the antiviral innate response. Intriguingly, however, NF-κB activation is assumed to favour herpes simplex virus (HSV) infection rather than restrict it. Apoptosis, a form of innate response to viruses, is completely inhibited by HSV in fully permissive cells, but not in cells incapable to fully sustain HSV replication, such as immunocompetent cells. To resolve the intricate interplay among NF-κB signalling, apoptosis and permissiveness to HSV-1 in monocytic cells, we utilized U937 monocytic cells in which NF-κB activation was inhibited by expressing a dominant-negative IκBα. Surprisingly, viral production was increased in monocytic cells in which NF-κB was inhibited. Moreover, inhibition of NF-κB led to increased apoptosis following HSV-1 infection, associated with lysosomal membrane permeabilization. High expression of late viral proteins and induction of apoptosis occurred in distinct cells. Transcriptional analysis of known innate response genes by real-time quantitative reverse transcription-PCR excluded a contribution of the assayed genes to the observed phenomena. Thus, in monocytic cells NF-κB activation simultaneously serves as an innate process to restrict viral replication as well as a mechanism to limit the damage of an excessive apoptotic response to HSV-1 infection. This finding may clarify mechanisms controlling HSV-1 infection in monocytic cells. PMID:27584793

  17. Animal Models of Virus-Induced Neurobehavioral Sequelae: Recent Advances, Methodological Issues, and Future Prospects

    OpenAIRE

    Marco Bortolato; Sean C Godar

    2010-01-01

    Converging lines of clinical and epidemiological evidence suggest that viral infections in early developmental stages may be a causal factor in neuropsychiatric disorders such as schizophrenia, bipolar disorder, and autism-spectrum disorders. This etiological link, however, remains controversial in view of the lack of consistent and reproducible associations between viruses and mental illness. Animal models of virus-induced neurobehavioral disturbances afford powerful tools to test etiologica...

  18. Animal models for human T-lymphotropic virus type 1 (HTLV-1) infection and transformation

    OpenAIRE

    Lairmore, Michael D.; Silverman, Lee; Ratner, Lee

    2005-01-01

    Over the past 25 years, animal models of human T-lymphotropic virus type 1 (HTLV-1) infection and transformation have provided critical knowledge about viral and host factors in adult T-cell leukemia/lymphoma (ATL). The virus consistently infects rabbits, some non-human primates, and to a lesser extent rats. In addition to providing fundamental concepts in viral transmission and immune responses against HTLV-1 infection, these models have provided new information about the role of viral prote...

  19. Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

    OpenAIRE

    FAVORETTO Silvana Regina; Carrieri, Maria Luiza; CUNHA Elenice Maria S.; Elizabeth A.C. Aguiar; SILVA Luzia Helena Q.; Miriam M. SODRÉ; SOUZA Maria Conceição A.M.; Kotait, Ivanete

    2002-01-01

    Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cel...

  20. Exogenous avian leukosis virus-induced activation of the ERK/AP1 pathway is required for virus replication and correlates with virus-induced tumorigenesis.

    Science.gov (United States)

    Dai, Manman; Feng, Min; Ye, Yu; Wu, Xiaochan; Liu, Di; Liao, Ming; Cao, Weisheng

    2016-01-01

    A proteomics approach was used to reveal the up-regulated proteins involved in the targeted mitogen-activated protein kinase (MAPK) signal transduction pathway in DF-1 cells after ALV subgroup J (ALV-J) infection. Next, we found that ALV-J CHN06 strain infection of DF-1 cells correlated with extracellular signal-regulated kinase 2 (ERK2) activation, which was mainly induced within 15 min, a very early stage of infection, and at a late infection stage, from 108 h to 132 h post-infection. Infection with other ALV subgroup (A/B) strains also triggered ERK/MAPK activation. Moreover, when activating ERK2, ALV subgroups A, B and J simultaneously induced the phosphorylation of c-Jun, an AP1 family member and p38 activation but had no obvious effect on JNK activation at either 15 min or 120 h. Interestingly, only PD98059 inhibited the ALV-induced c-Jun phosphorylation while SP600125 or SB203580 had no influence on c-Jun activation. Furthermore, the viral gp85 and gag proteins were found to contribute to ERK2/AP1 activation. Additionally, the specific ERK inhibitor, PD980509, significantly suppressed ALV replication, as evidenced by extremely low levels of ALV promoter activity and ALV-J protein expression. In vivo analysis of ERK2 activation in tumor cells derived from ALV-J-infected chicken demonstrated a strong correlation between ERK/MAPK activation and virus-associated tumorigenesis. PMID:26754177