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Sample records for c100-3 hcr43 ns5

  1. Brazilian Flavivirus phylogeny based on NS5.

    Science.gov (United States)

    Baleotti, Flúvia Graciela; Moreli, Marcos Lázaro; Figueiredo, Luiz Tadeu Moraes

    2003-04-01

    In this work, a comprehensive phylogenetic study based on 600 base pair nucleotide and on putative 200 amino acid sequences of NS5 was carried out in order to establish genetic relationships among 15 strains of 10 Brazilian flaviviruses: Bussuquara, Cacipacore, dengue type 1, 2 and 4, Iguape, Ilheus, Rocio, Saint Louis encephalitis (SLE), and yellow fever. Phylogenetic trees were created by neighbor-joining and maximum parsimony methods. These trees showed Brazilian flaviviruses grouped into three main branches: yellow fever branch, dengue branch subdivided in types 1, 2 and 4 branches, and Japanese encephalitis virus (JEV) complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara. Viruses transmitted by Aedes mosquitoes, such as dengue and urban yellow fever, that are also the only Flavivirus causing hemorrhagic fevers in Brazil, were grouped in the same cluster. Encephalitis associated viruses, transmitted by Culex mosquitoes such as JEV complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara were also grouped in the same clade.

  2. HCV NS5A and NS5B Enhance Expression of Human Ceramide Glucosyltransferase Gene

    Institute of Scientific and Technical Information of China (English)

    Jia Guo; Ran Yan; Guo-dong Xu; Cong-yi Zheng

    2012-01-01

    Host genes involved in lipid metabolism are differentially affected during the early stages of hepatitis C virus (HCV) infection.Here we demonstrate that artificial up-regulation of fatty acid biosynthesis has a positive effect on the replication of the HCV full-length replicon when cells were treated with nystatin.Conversely,the HCV RNA replication was decreased when fatty acid biosynthesis was inhibited with 25-hydroxycholesterol and PDMP(D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol).In agreement with these results,the expression level of GlcT-1(ceramide glucosyltransferase),a host glucosyltransferase in the first step of GSL (glycosphingolipid) biosynthesis,was found to be closely associated with the expression and replication of HCV RNA.On the other hand,the viral RNA can also activate GlcT-1 in the early stage of viral RNA transfection in vitro.To identify viral factors that are responsible for GlcT-1 activation,we constructed ten stable Vero cell lines that express individual HCV proteins.Based on the analyses of these cell lines and transient transfection assay of the GlcT-1 promoter regions,we conclude that HCV proteins,especially NS5A and NS5B,have positive effects on the expression of GlcT-1.It is possible that NS5A and NS5B stimulate transcription factor(s) to activate the expression of GlcT-1 by increasing its transcription level.

  3. Tetrahydrobenzothiophene inhibitors of hepatitis C virus NS5B polymerase.

    Science.gov (United States)

    Laporte, M G; Lessen, T A; Leister, L; Cebzanov, D; Amparo, E; Faust, C; Ortlip, D; Bailey, T R; Nitz, T J; Chunduru, S K; Young, D C; Burns, C J

    2006-01-01

    A novel series of selective HCV NS5B RNA dependent RNA polymerase inhibitors has been disclosed. These compounds contain an appropriately substituted tetrahydrobenzothiophene scaffold. This communication will detail the SAR and activities of this series.

  4. NS5A Resistance: Clinical Implications and Treatment Possibilities.

    Science.gov (United States)

    Calleja, José Luis; Llerena, Susana; Perelló, Christie; Crespo, Javier

    2016-01-01

    Treatments with interferon-free direct-acting antiviral agents have high efficacy, with sustained virological response rates of more than 90%. Nevertheless, they fail to eliminate the infection in 1-7% of patients. The majority of virological failures are due to relapse following treatment discontinuation, while virological rebound during therapy is rare. Although not the only factor, the presence of resistance-associated variants is one of the major causes for said failure. Resistance-associated variants affect the sequence involved in protein synthesis on which different direct-acting antiviral agents act (NS3/4A, NS5A, NS5B). Of all these variants, the ones with the greatest impact are resistance-associated variants that affect the NS5A region due to their long-term persistence. In this article we will describe the most significant NS5A resistance-associated variants, the clinical relevance of their detection both before and after treatment, their persistence over time, and lastly, we will devote particular attention to discussing what approach to adopt when dealing with treatment failure to an antiviral regimen that includes NS5A inhibitors.

  5. Structures of NS5 Methyltransferase from Zika Virus

    Directory of Open Access Journals (Sweden)

    Javier Coloma

    2016-09-01

    Full Text Available The Zika virus (ZIKV poses a major public health emergency. To aid in the development of antivirals, we present two high-resolution crystal structures of the ZIKV NS5 methyltransferase: one bound to S-adenosylmethionine (SAM and the other bound to SAM and 7-methyl guanosine diphosphate (7-MeGpp. We identify features of ZIKV NS5 methyltransferase that lend to structure-based antiviral drug discovery. Specifically, SAM analogs with functionalities on the Cβ atom of the methionine portion of the molecules that occupy the RNA binding tunnel may provide better specificity relative to human RNA methyltransferases.

  6. Hepatitis C virus expressing reporter tagged NS5A protein

    DEFF Research Database (Denmark)

    2014-01-01

    Hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A were developed. A deletion upstream of the inserted reporter gene sequence conferred favorable...

  7. Phylogenetic analysis of the NS5 gene of Zika virus.

    Science.gov (United States)

    Adiga, Rama

    2016-10-01

    ZIKV infection has become a global threat spreading across 31 countries in Central America, South America, and the Caribbean. However, little information is available about the molecular epidemiology of ZIKV. Shared mutation of a threonine residue to alanine at the same position in the C terminal of NS5 sequences was observed in sequences from Colombia, Mexico, Panama, and Martinique. The sequences in the phylogenetic tree fell within the same cluster. Based on shared mutation the presence of a Latin American genotype was proposed. Comparison of African and Asian lineages yielded R29N, N273S, H383Q, and P391S mutation. The study highlights that mutation of amino acids at NS5 may contribute to neutropism of ZIKV. J. Med. Virol. 88:1821-1826, 2016. © 2016 Wiley Periodicals, Inc.

  8. Discovery of an irreversible HCV NS5B polymerase inhibitor.

    Science.gov (United States)

    Zeng, Qingbei; Nair, Anilkumar G; Rosenblum, Stuart B; Huang, Hsueh-Cheng; Lesburg, Charles A; Jiang, Yueheng; Selyutin, Oleg; Chan, Tin-Yau; Bennett, Frank; Chen, Kevin X; Venkatraman, Srikanth; Sannigrahi, Mousumi; Velazquez, Francisco; Duca, Jose S; Gavalas, Stephen; Huang, Yuhua; Pu, Haiyan; Wang, Li; Pinto, Patrick; Vibulbhan, Bancha; Agrawal, Sony; Ferrari, Eric; Jiang, Chuan-kui; Li, Cheng; Hesk, David; Gesell, Jennifer; Sorota, Steve; Shih, Neng-Yang; Njoroge, F George; Kozlowski, Joseph A

    2013-12-15

    The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 μM), highly selective, and safe in in vitro and in vivo assays.

  9. Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Gottwein, Judith M; Mikkelsen, Lotte S;

    2011-01-01

    Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates...... of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-a....

  10. Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core*

    Science.gov (United States)

    Camus, Gregory; Herker, Eva; Modi, Ankit A.; Haas, Joel T.; Ramage, Holly R.; Farese, Robert V.; Ott, Melanie

    2013-01-01

    The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy. PMID:23420847

  11. Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

    Science.gov (United States)

    Kaushik-Basu, Neerja; Bopda-Waffo, Alain; Talele, Tanaji T.; Basu, Amartya; Costa, Paulo R. R.; da Silva, Alcides J. M.; Sarafianos, Stefan G.; Noël, François

    2008-01-01

    The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B–RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC50 values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors. PMID:18203743

  12. The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing

    Science.gov (United States)

    Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G.; Mammi, Pablo; Yanovsky, Marcelo J.; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V.

    2016-01-01

    Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. PMID:27575636

  13. HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding

    Institute of Scientific and Technical Information of China (English)

    Guo-Zhong Gong; Yong-Fang Jiang; Yan He; Li-Ying Lai; Ying-Hua Zhu; Xian-Shi Su

    2004-01-01

    AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function.METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter.Western blot experiment was used for detection of HCV NS5A and p53 proteins expression.RESULTS: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression.CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.

  14. Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Humes, Daryl

    2014-01-01

    BACKGROUND & AIMS: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A)...

  15. Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients

    OpenAIRE

    Yoshida,C.F.T.; Takahashi, Y.; B.O.M. Vanderborght; Rouzere,C. D.; França,M. S. de; Takahashi,C.; Takamizawa, A; Yoshida, I.; Schatzmayr, H. G.

    1993-01-01

    Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent inf...

  16. Structure and function of the Zika virus full-length NS5 protein

    Science.gov (United States)

    Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; Chuang, Yin-Chih; Vaughan, Robert C.; Sankaran, Banumathi; Kao, C. Cheng; Li, Pingwei

    2017-01-01

    The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication. PMID:28345656

  17. The structure of Zika virus NS5 reveals a conserved domain conformation

    Science.gov (United States)

    Wang, Boxiao; Tan, Xiao-Feng; Thurmond, Stephanie; Zhang, Zhi-Min; Lin, Asher; Hai, Rong; Song, Jikui

    2017-01-01

    The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of flavivirus NS5 proteins. The activity of this NS5 protein is verified through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identification of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antivirals for ZIKV. PMID:28345600

  18. Resistance Patterns Associated with HCV NS5A Inhibitors Provide Limited Insight into Drug Binding

    Directory of Open Access Journals (Sweden)

    Moheshwarnath Issur

    2014-11-01

    Full Text Available Direct-acting antivirals (DAAs have significantly improved the treatment of infection with the hepatitis C virus. A promising class of novel antiviral agents targets the HCV NS5A protein. The high potency and broad genotypic coverage are favorable properties. NS5A inhibitors are currently assessed in advanced clinical trials in combination with viral polymerase inhibitors and/or viral protease inhibitors. However, the clinical use of NS5A inhibitors is also associated with new challenges. HCV variants with decreased susceptibility to these drugs can emerge and compromise therapy. In this review, we discuss resistance patterns in NS5A with focus prevalence and implications for inhibitor binding.

  19. NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV Nonstructural Protein 5A (NS5A via MEK/Extracellular Signal Regulated Kinase (ERK Pathway

    Directory of Open Access Journals (Sweden)

    Xuesong Gao

    2013-05-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein 5A (NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.

  20. Approaches to hepatitis C treatment and cure using NS5A inhibitors

    Directory of Open Access Journals (Sweden)

    Kohler JJ

    2014-03-01

    Full Text Available James J Kohler,1,2 James H Nettles,1,2 Franck Amblard,1,2 Selwyn J Hurwitz,1,2 Leda Bassit,1,2 Richard A Stanton,1 Maryam Ehteshami,1 Raymond F Schinazi1,2 1Center for AIDS Research and Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA; 2Atlanta Veterans Affairs Medical Center, Decatur, GA, USA Abstract: Recent progress in the understanding of hepatitis C virus (HCV biology and the availability of in vitro models to study its replication have facilitated the development of direct-acting antiviral agents (DAAs that target specific steps in the viral replication cycle. Currently, there are three major classes of DAA in clinical development: NS3/4A protease inhibitors, NS5B polymerase inhibitors, and NS5A directed inhibitors. Several compounds thought to bind directly with NS5A are now in various clinical trial phases, including the most advanced, daclatasvir (BMS-790052, ledipasvir (GS-5885, and ABT-267. While many NS5A-targeted compounds demonstrate picomolar potency, the exact mechanism(s of their action is still unclear. In the clinic, NS5A HCV inhibitors show promise as important components in DAA regimens and have multifunctionality. In addition to inhibiting viral replication, they may synergize with other DAAs, possibly by modulating different viral proteins, to help suppress the emergence of resistant viruses. Structure-based models have identified target interaction domains and spatial interactions that explain drug resistance for mutations at specific positions (eg, residues 93 and 31 within NS5A and potential binding partners. This review provides, insights into the unique complexity of NS5A as a central platform for multiple viral/host protein interactions, and possible mechanism(s for the NS5A inhibitors currently undergoing clinical trials that target this nonstructural viral protein. Keywords: HCV replication complex, direct acting antivirals (DAAs, clinical trials

  1. Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients.

    Science.gov (United States)

    Yoshida, C F; Takahashi, Y; Vanderborght, B O; Rouzere, C D; França, M S; Takahashi, C; Takamizawa, A; Yoshida, I; Schatzmayr, H G

    1993-01-01

    Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15% and reached 86.5% for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8% of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7% (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.

  2. Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5

    Science.gov (United States)

    Upadhyay, Anup K.; Longenecker, Kenton; Tripathi, Rakesh; Sun, Chaohong; Kempf, Dale J.

    2017-01-01

    The rapid spread of the recent Zika virus (ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 of Zika virus (ZIKV-NS5) is critical for ZIKV replication through the 5′-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively. The crystal structure of the full-length ZIKV-NS5 protein has been determined at 3.05 Å resolution from a crystal belonging to space group P21212 and containing two protein molecules in the asymmetric unit. The structure is similar to that reported for the NS5 protein from Japanese encephalitis virus and suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain. PMID:28291746

  3. The Many Faces of the Flavivirus NS5 Protein in Antagonism of Type I Interferon Signaling.

    Science.gov (United States)

    Best, Sonja M

    2017-02-01

    The vector-borne flaviviruses cause severe disease in humans on every inhabited continent on earth. Their transmission by arthropods, particularly mosquitoes, facilitates large emergence events such as witnessed with Zika virus (ZIKV) or West Nile virus in the Americas. Every vector-borne flavivirus examined thus far that causes disease in humans, from dengue virus to ZIKV, antagonizes the host type I interferon (IFN-I) response by preventing JAK-STAT signaling, suggesting that suppression of this pathway is an important determinant of infection. The most direct and potent viral inhibitor of this pathway is the nonstructural protein NS5. However, the mechanisms utilized by NS5 from different flaviviruses are often quite different, sometimes despite close evolutionary relationships between viruses. The varied mechanisms of NS5 as an IFN-I antagonist are also surprising given that the evolution of NS5 is restrained by the requirement to maintain function of two enzymatic activities critical for virus replication, the methyltransferase and RNA-dependent RNA polymerase. This review discusses the different strategies used by flavivirus NS5 to evade the antiviral effects of IFN-I and how this information can be used to better model disease and develop antiviral countermeasures.

  4. Point mutations in E2, NS3 and NS5A of hepatitis G virus

    Institute of Scientific and Technical Information of China (English)

    Wei Dong Liu; Toshikazu Hada; Ji Dong Cheng; Kazuya Higashino

    2000-01-01

    AIM To compare the point mutation deviations of HGV among E2, NS3 and NSSA.METHODS Seven patients with hepatic diseases from Japan and China were selected for this study. RNAwas extracted and amplified by semi-nested RT-PCR; and the PCR products were sequenced directly.RESULTS The point mutation deviations of HGV ia E2, NS3 and NS5A were 10% - 17%, 11% -23%,and 0% - 5%, in nuclcotide sequences and 4% - 12%, 0%, and 0% - 6% in amino acid sequencesrespectively.CONCLUSION The frequency of variation at the nucleotide level was in the order NS3>E2>NS5A, whileat the amino acid level the order was E2 >NS5A>NS3. The detected sequences from the N-terminus of E2may be the poorly conserved region of HGV.

  5. Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

    Directory of Open Access Journals (Sweden)

    Hyock Joo Kwon

    Full Text Available Ledipasvir, a direct acting antiviral agent (DAA targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.

  6. Hagedorn Behavior of Little String Theories from string corrections to NS5-branes

    DEFF Research Database (Denmark)

    Harmark, Troels; Obers, N. A.

    2000-01-01

    We examine the Hagedorn behavior of little string theory using its conjectured duality with near-horizon NS5-branes. In particular, by studying the string-corrected NS5-brane supergravity solution, it is shown that tree-level corrections to the temperature vanish, while the leading one-loop string...... correction generates the correct temperature dependence of the entropy near the Hagedorn temperature. Finally, the Hagedorn behavior of ODp-brane theories, which are deformed versions of little string theory, is considered via their supergravity duals....

  7. Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Antonysamy, Stephen S.; Aubol, Brandon; Blaney, Jeff; Browner, Michelle F.; Giannetti, Anthony M.; Harris, Seth F.; Hébert, Normand; Hendle, Jörg; Hopkins, Stephanie; Jefferson, Elizabeth; Kissinger, Charles; Leveque, Vincent; Marciano, David; McGee, Ethel; Nájera, Isabel; Nolan, Brian; Tomimoto, Masaki; Torres, Eduardo; Wright, Tobi (SGX); (Roche)

    2009-07-22

    Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with {approx}1-10 mM binding affinity (K{sub D}) were iteratively optimized to give leads with {approx}200 nM biochemical activity and low {micro}M cellular activity in a Replicon assay.

  8. NS5 Brane and Little String Duality in the pp-wave Limit

    CERN Document Server

    Matlock, P; Viswanathan, K S; Yang, Y

    2003-01-01

    We study NSR strings in the Nappi-Witten background, which is the Penrose limit of a certain NS5-brane supergravity solution. We solve the theory in the light-cone gauge, obtaining the spectrum, which is space-time supersymmetric. In light of the LST/NS5-brane duality, this spectrum should be in correspondence with the states of little string theory in the appropriate limit. A semiclassical analysis verifies that the relationship between energy and angular momentum, after a field redefinition, matches that found for a flat background.

  9. HCV RNA traffic and association with NS5A in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.; Van Der Hoek, Kylie [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia); Betz-Stablein, Brigit; Luciani, Fabio [Systems Immunology, School of Medical Sciences, University of New South Wales, Sydney, NSW (Australia); Chopra, Abha [Institute for Immunology and infectious diseases (IIID), Murdoch University, Perth, WA (Australia); Beard, Michael R., E-mail: michael.beard@adelaide.edu.au [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia)

    2016-06-15

    The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.

  10. Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients Anticorpos contra os antígenos não estrutural c100/3 e estrutural core do vírus da hepatite C (HCV) em pacientes de hemodiálise

    OpenAIRE

    Yoshida,C.F.T.; Takahashi, Y.; B.O.M. Vanderborght; Rouzere,C. D.; França,M. S. de; Takahashi,C.; Takamizawa, A; Yoshida, I.; Schatzmayr, H. G.

    1993-01-01

    Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent inf...

  11. Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system.

    Science.gov (United States)

    Ivanov, Alexander V; Tunitskaya, Vera L; Ivanova, Olga N; Mitkevich, Vladimir A; Prassolov, Vladimir S; Makarov, Alexander A; Kukhanova, Marina K; Kochetkov, Sergey N

    2009-01-22

    Hepatitis C virus (HCV) NS5A phosphoprotein is a component of virus replicase. Here we demonstrate that in vitro unphosphorylated NS5A protein inhibits HCV RNA-dependent RNA polymerase (RdRp) activity in polyA-oligoU system but has little effect on synthesis of viral RNA. The phosphorylated casein kinase (CK) II NS5A protein causes the opposite effect on RdRp in each of these systems. The phosphorylation of NS5A protein with CKII does not affect its affinity to the HCV RdRp and RNA. The NS5A phosphorylation with CKI does not change the RdRp activity. Herein we report evidence that the NS5A prevents template binding to the RdRp.

  12. Screening of hepatocyte proteins binding to NS5ABP37 protein by yeast-two hybrid system

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes.Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus(HCV)by cloning the gene of NS5ABP37 protein into pGBKT7,then the recombinant plasmid DNA was transformed into yeast AH109(α type).The transformed yeast AH109 was mated with yeast Y187(α type)containing liver cDNA library plasmid in 2×YPDA medium.Diploid yeast was plated on synthetic dropout ...

  13. 丙型肝炎病毒NS5B基因在昆虫细胞中的表达%Expression of Hepatitis C Virus NS5B Gene in Insect Cells

    Institute of Scientific and Technical Information of China (English)

    李磊; 郜金荣; 徐进平; 叶林柏; 张斌; 吴正辉

    2001-01-01

    将含有丙型肝炎病毒(HCV)NS5B基因的转移载体pBlueBac5B与野生型杆状病毒(AcNPV)DNA共转染Sf9昆虫细胞,通过空斑纯化获得带有NS5B基因的重组病毒AcNS5B.提取重组病毒基因组DNA进行Southern分析,发现有一条1.8 kb的DNA片段与标记的探针发生杂交.AcNS5B感染Sf9细胞后,在细胞中表达了分子量为66×103 的蛋白,用Western blot方法检查这种蛋白质,能与兔抗HCVNS5B抗血清发生特异的强烈反应.%The plasmid pBlueBac 5B containing Hepatitis C Virus NS5B gene was cotransfected with the genome DNA of wild type AcNPV to Sf9 insect cells. The recombinant baculovirus AcNS5B was obtained by plaque selection. Southern blot indicates that the NS5B gene be integrated into the genome of AcNS5B and recombinant HCV NS5B protein be expressed by Sf9 cells infected with AcNS5B. This protein can react with the anti-NS5B sera of rabbit as evidence of Western blots.

  14. Crystal structure of Zika virus NS5 RNA-dependent RNA polymerase

    Science.gov (United States)

    Godoy, Andre S.; Lima, Gustavo M. A.; Oliveira, Ketllyn I. Z.; Torres, Naiara U.; Maluf, Fernando V.; Guido, Rafael V. C.; Oliva, Glaucius

    2017-01-01

    The current Zika virus (ZIKV) outbreak became a global health threat of complex epidemiology and devastating neurological impacts, therefore requiring urgent efforts towards the development of novel efficacious and safe antiviral drugs. Due to its central role in RNA viral replication, the non-structural protein 5 (NS5) RNA-dependent RNA-polymerase (RdRp) is a prime target for drug discovery. Here we describe the crystal structure of the recombinant ZIKV NS5 RdRp domain at 1.9 Å resolution as a platform for structure-based drug design strategy. The overall structure is similar to other flaviviral homologues. However, the priming loop target site, which is suitable for non-nucleoside polymerase inhibitor design, shows significant differences in comparison with the dengue virus structures, including a tighter pocket and a modified local charge distribution. PMID:28345596

  15. Comparative Investigation of Normal Modes and Molecular Dynamics of Hepatitis C NS5B Protein

    Science.gov (United States)

    Asafi, M. S.; Yildirim, A.; Tekpinar, M.

    2016-04-01

    Understanding dynamics of proteins has many practical implications in terms of finding a cure for many protein related diseases. Normal mode analysis and molecular dynamics methods are widely used physics-based computational methods for investigating dynamics of proteins. In this work, we studied dynamics of Hepatitis C NS5B protein with molecular dynamics and normal mode analysis. Principal components obtained from a 100 nanoseconds molecular dynamics simulation show good overlaps with normal modes calculated with a coarse-grained elastic network model. Coarse-grained normal mode analysis takes at least an order of magnitude shorter time. Encouraged by this good overlaps and short computation times, we analyzed further low frequency normal modes of Hepatitis C NS5B. Motion directions and average spatial fluctuations have been analyzed in detail. Finally, biological implications of these motions in drug design efforts against Hepatitis C infections have been elaborated.

  16. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

    Directory of Open Access Journals (Sweden)

    Hongping Dong

    Full Text Available RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7GpppAm of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4 specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of

  17. Discovery of ravidasvir (PPI-668) as a potent pan-genotypic HCV NS5A inhibitor.

    Science.gov (United States)

    Zhong, Min; Peng, Eric; Huang, Ningwu; Huang, Qi; Huq, Anja; Lau, Meiyen; Colonno, Richard; Li, Leping

    2016-09-15

    This Letter describes the synthesis, representative structure activity relationship (SAR), activity and PK profiles of a series of functionalized benzimidazole-naphthylene-imidazole derivatives as HCV NS5A inhibitors. This effort successfully led to the discovery of ravidasvir (PPI-668), which has been well tolerated and shown high sustained viral response rates as a key component in all-oral combination regimens in multiple human clinical trials.

  18. Progress on New Hepatitis C Virus Targets: NS2 and NS5A

    Science.gov (United States)

    Marcotrigiano, Joseph

    Hepatitis C virus (HCV) is a major global health problem, affecting about 170 million people worldwide. Chronic infection can lead to cirrhosis and liver cancer. The replication machine of HCV is a multi-subunit membrane associated complex, consisting of nonstructural proteins (NS2-5B), which replicate the viral RNA genome. The structures of NS5A and NS2 were recently determined. NS5A is an essential replicase component that also modulates numerous cellular processes ranging from innate immunity to cell growth and survival. The structure reveals a novel protein fold, a new zinc coordination motif, a disulfide bond and a dimer interface. Analysis of molecular surfaces suggests the location of the membrane interaction surface of NS5A, as well as hypothetical protein and RNA binding sites. NS2 is one of two virally encoded proteases that are required for processing the viral polyprotein into the mature nonstructural proteins. NS2 is a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer and the nucleophilic cysteine by the other. The C-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. The structure also reveals possible sites of membrane interaction, a rare cis-proline residue, and highly conserved dimer contacts. The novel features of both structures have changed the current view of HCV polyprotein replication and present new opportunities for antiviral drug design.

  19. Hepatitis C virus (HCV) NS5A inhibitor- Daclatasvir%丙型肝炎病毒NS5A抑制剂--达卡他韦

    Institute of Scientific and Technical Information of China (English)

    张晓; 钟武

    2016-01-01

    Daclatasvir is an oral tablet for the treatment of hepatitis C virus (HCV) NS5A inhibitor developed by Bristol-Myers Squibb. After Daclatasvir was approved to combine with other antiviral drugs by EU in Augst 2014 for treating adults with genotype 1-4 of HCV, it was also approved for combined use with sofosbuvir for the treatment of patients with chronic hepatitis C virus (HCV) genotype 3 infection by the FDA in July 2015. It is the first drug that has indicated safety and efficiency of chronic hepatitis C virus (HCV) genotype 3 infection without interferon or ribavirin. This provids a new choice for patients who are infected with HCV genotype 3, including individuals who can not tolerate ribavirin. The properties, mechanism, pharmacodynamics, pharmacokinetics, clinical trials, and development process of Daclatasvir were reviewed in this article, which could provide reference to the doctors and researchers.%达卡他韦(Daklinza)是由美国百时美-施贵宝公司(BMS)研制的口服丙型肝炎病毒(HCV)NS5A抑制剂。继2014年欧盟批准其与联合其他抗病毒药用于基因型1-4慢性丙型肝炎成人患者的治疗后,2015年7月美国FDA批准其与索非布韦[sofosbuvir]联合用于慢性HCV基因型3感染的治疗。达卡他韦是第一个无需同时给予干扰素或利巴韦林即可有效治疗基因型3 HCV感染的药物,为该类患者提供了一个新选择,包括那些不能耐受利巴韦林患者。笔者就达卡他韦的基本信息、作用机制、药代动力学、药物相互作用、临床试验及应用等研发动态作一概述,以期能为医院临床用药及药物研究开发提供参考。

  20. Using NS5B Sequencing for Hepatitis C Virus Genotyping Reveals Discordances with Commercial Platforms

    Science.gov (United States)

    Chueca, Natalia; Rivadulla, Isidro; Lovatti, Rubén; Reina, Gabriel; Blanco, Ana; Fernandez-Caballero, Jose Angel; Cardeñoso, Laura; Rodriguez-Granjer, Javier; Fernandez-Alonso, Miriam; Aguilera, Antonio; Alvarez, Marta

    2016-01-01

    We aimed to evaluate the correct assignment of HCV genotypes by three commercial methods—Trugene HCV genotyping kit (Siemens), VERSANT HCV Genotype 2.0 assay (Siemens), and Real-Time HCV genotype II (Abbott)—compared to NS5B sequencing. We studied 327 clinical samples that carried representative HCV genotypes of the most frequent geno/subtypes in Spain. After commercial genotyping, the sequencing of a 367 bp fragment in the NS5B gene was used to assign genotypes. Major discrepancies were defined, e.g. differences in the assigned genotype by one of the three methods and NS5B sequencing, including misclassification of subtypes 1a and 1b. Minor discrepancies were considered when differences at subtype levels, other than 1a and 1b, were observed. The overall discordance with the reference method was 34% for Trugene and 15% for VERSANT HCV2.0. The Abbott assay correctly identified all 1a and 1b subtypes, but did not subtype all the 2, 3, 4 and 5 (34%) genotypes. Major discordances were found in 16% of cases for Trugene HCV, and the majority were 1b- to 1a-related discordances; major discordances were found for VERSANT HCV 2.0 in 6% of cases, which were all but one 1b to 1a cases. These results indicated that the Trugene assay especially, and to a lesser extent, Versant HCV 2.0, can fail to differentiate HCV subtypes 1a and 1b, and lead to critical errors in clinical practice for correctly using directly acting antiviral agents. PMID:27097040

  1. Inflation driven by single geometric tachyon with D-brane orbiting around NS5-branes

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Pyung Seong, E-mail: bskwon@ks.ac.kr [Department of Physics, Kyungsung University, Pusan 608-736 (Korea, Republic of); Jun, Gyeong Yun, E-mail: gyjun@ks.ac.kr [Department of Physics, Kyungsung University, Pusan 608-736 (Korea, Republic of); Panigrahi, Kamal L., E-mail: panigrahi@phy.iitkgp.ernet.in [Department of Physics and Meteorology and Centre for Theoretical Studies, Indian Institute of Technology Kharagpur, 721 302 (India); Sami, M., E-mail: sami@iucaa.ernet.in [Center for Theoretical Physics, Jamia Millia Islamia, New Delhi 110092 (India)

    2012-05-30

    We investigate models in which inflation is driven by a single geometrical tachyon. We assume that the D-brane as a probe brane in the background of NS5-branes has non-zero angular momentum which is shown to play similar role as the number of the scalar fields of the assisted inflation. We demonstrate that the angular momentum corrected effective potential allows to account for the observational constraint on COBE normalization, spectral index n{sub S} and the tensor to scalar ratio of perturbations consistent with WMAP seven years data.

  2. Inflation driven by single geometric tachyon with D-brane orbiting around NS5-branes

    CERN Document Server

    Kwon, Pyung Seong; Panigrahi, Kamal L; Sami, M

    2011-01-01

    We investigate models in which inflation is driven by a single geometrical tachyon. We assume that the D-brane as a probe brane in the background of NS5-branes has non-zero angular momentum which is shown to play similar role as the number of the scalar fields of the assisted inflation. We demonstrate that the angular momentum corrected effective potential allows to account for the observational constraint on COBE normalization, spectral index $n_S$ and the tensor to scalar ratio of perturbations consistent with WMAP seven years data.

  3. Prokaryotic expression and subcellular localization of Japanese encephalitis virus NS5 protein%乙型脑炎病毒NS5蛋白原核表达与亚细胞定位研究

    Institute of Scientific and Technical Information of China (English)

    栗永茂; 曹胜波; 陈龙; 霍雨艳; 熊涛; 吴光旭

    2011-01-01

    The NS5 gene of Japanese encephalitis virus was amplified by PCR and cloned into the prokaryotic expression vector pET-28a. The recombinant NS5 protein was expressed highly under induction of IPTG in the E. Coli. Using BALB/c female mice immuned with the purified recombinant NS5 protein, NS5 protein-specific polyclonal antibody were prepared. Meanwhile, BHK-21 cells were transfected with eukaryotic expression plasmid pcDNA-NS5-EGFP of NS5 gene and green fluorescent protein (EGFP) fusion expression. Finally, the subcellular localization of NS5 protein in BHK-21 cells was observed by using laser confocal fluorescence microscope. The result indicates that the protein particles are irregularly distributed in the whole cell and particularly in the cytoplasm.%目的 对乙型脑炎病毒NS5蛋白进行原核表达和亚细胞定位研究.方法 通过PCR扩增乙型脑炎病毒NS5基因,并将之克隆至原核表达载体pET-28a(+)中,构建的重组表达载体转化大肠杆菌感受态细胞,经IPTG诱导表达目的蛋白.将表达产物纯化后,免疫BALB/c小鼠,制备NS5蛋白特异性的多克隆抗体.同时,构建NS5基因与绿色荧光蛋白(EGFP)融合表达的真核表达质粒pcDNA-NS5-EGFP,将之转染BHK-21细胞,利用激光共聚焦荧光显微镜观察NS5蛋白在BHK-21细胞中的亚细胞定位.结果 NS5蛋白在大肠杆菌中获得了高效表达,表达蛋白分子量大小约为103kD,该蛋白在真核细胞中的表达呈颗粒状不均匀分布于整个细胞,且颗粒状主要分布在细胞质中.结论 成功表达乙型脑炎病毒NS5蛋白,并对NS5蛋白的亚细胞定位进行了分析,为进一步研究NS5蛋白提供依据.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-04-15

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

  5. Crystal Structure of a Novel Dimeric Form of NS5A Domain I Protein from Hepatitis C Virus

    Energy Technology Data Exchange (ETDEWEB)

    Love, Robert A.; Brodsky, Oleg; Hickey, Michael J.; Wells, Peter A.; Cronin, Ciarán N.; Pfizer

    2009-07-10

    A new protein expression vector design utilizing an N-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis C virus NS5A sequence has resulted in a more straightforward purification method and improved yields of purified NS5A domain I protein. High-resolution diffracting crystals of NS5A domain I (amino acids 33 to 202) [NS5A(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is organized differently from that published previously (T. L. Tellinghuisen, J. Marcotrigiano, and C. M. Rice, Nature 435:374-379, 2005) yet is consistent with a membrane association model for NS5A. The monomer-monomer interface of NS5A(33-202) features an extensive buried surface area involving the most-highly conserved face of each monomer. The two alternate structural forms of domain I now available may be indicative of the multiple roles emerging for NS5A in viral RNA replication and viral particle assembly.

  6. Screening of hepatocyte proteins binding to NS5ABP37 protein by yeast-two hybrid system

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Qing-yong Ma; Xian-kui Meng; Kang Li; Jun Cheng

    2009-01-01

    Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes. Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus (HCV) by cloning the gene of NS5ABP37 protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (α type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we made a sequence analysis by bioinformatics. Results We screened twenty-five proteins binding to NS5ABP37, including Homo sapiens cyclin Ⅰ (CCNI) gene, Homo sapiens matrix metallopeptidase 25 (MMP25) and Homo sapiens talin 1. Conclusion The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with NS5ABP37 of HCV. And the biological function of NS5ABP37 may be associated with glycometabolism, lipid metabolism and apoptosis.

  7. NS5ATP6 modulates intracellular triglyceride content through FGF21 and independently of SIRT1 and SREBP1.

    Science.gov (United States)

    Li, Zhongshu; Feng, Shenghu; Zhou, Li; Liu, Shunai; Cheng, Jun

    2016-06-17

    The prevalence of nonalcoholic fatty liver disease (NAFLD) is rising strikingly in Western countries and China. The molecular biological mechanism of NAFLD remains unclear, with no effective therapies developed so far. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone, with safe lipid lowering effects. FGF21 analogs are being developed for clinical application. Here we demonstrated that a novel gene, NS5ATP6, modulated intracellular triglyceride (TG) content independently of sirtuin1 (SIRT1) and sterol regulatory element binding protein 1 (SREBP1) in HepG2 cells. Interestingly, NS5ATP6 regulated FGF21 expression both at the mRNA and protein levels. The modulatory effects of NS5ATP6 on intracellular TG content depended upon FGF21. Further studies revealed that NS5ATP6 decreased the promoter activity of FGF21. In addition, NS5ATP6 regulated the expression of miR-577, which directly targeted and regulated FGF21. Therefore, miR-577 might be involved in NS5ATP6 regulation of FGF21 at the post-transcriptional level. In conclusion, NS5ATP6 regulates the intracellular TG level via FGF21, and independently of SIRT1 and SREBP1.

  8. Classification of HCV NS5B Polymerase Inhibitors Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Changyuan Yu

    2012-03-01

    Full Text Available Using a support vector machine (SVM, three classification models were built to predict whether a compound is an active or weakly active inhibitor based on a dataset of 386 hepatitis C virus (HCV NS5B polymerase NNIs (non-nucleoside analogue inhibitors fitting into the pocket of the NNI III binding site. For each molecule, global descriptors, 2D and 3D property autocorrelation descriptors were calculated from the program ADRIANA.Code. Three models were developed with the combination of different types of descriptors. Model 2 based on 16 global and 2D autocorrelation descriptors gave the highest prediction accuracy of 88.24% and MCC (Matthews correlation coefficient of 0.789 on test set. Model 1 based on 13 global descriptors showed the highest prediction accuracy of 86.25% and MCC of 0.732 on external test set (including 80 compounds. Some molecular properties such as molecular shape descriptors (InertiaZ, InertiaX and Span, number of rotatable bonds (NRotBond, water solubility (LogS, and hydrogen bonding related descriptors performed important roles in the interactions between the ligand and NS5B polymerase.

  9. The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A.

    Directory of Open Access Journals (Sweden)

    Simon Reiss

    2013-05-01

    Full Text Available The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα is an essential host factor of hepatitis C virus (HCV replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A. This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58, indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56. PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis

  10. HCV非结构蛋白NS5A基因的斑马鱼肝脏表达模型%A zebrafish model for liver-specific expression of HCV NS5A gene

    Institute of Scientific and Technical Information of China (English)

    赵晔; 胡占英; 佟军威; 赵立勋; 蒋建东; 张靖溥

    2011-01-01

    Objective To establish a zebrafish model for liver-specific expression of HCV non-structural protein NS5A, and to explore the effects of HCV NS5A on liver pathological marker genes in vivo.Methods Liver-specific gene expression regulating element zebrafish fatty acid binding protein enhancer (eFABP) sequence and HCV NS5A was cloned. By ligating the eFABP, CMV promoter, NS5A, the IRES of encephalomyocarditis virus (EMCV) and DsRed in turn, a liver-specific gene expression vector pFC-5AiR was constructed. Reporter gene DsRed was detected in Huh7 cells transfected with pFC-5AiR. The linearized vector was subsequently microinjected into zebrafish embryos. Liver-specific expression of pFC-5AiR was detected by observation under fluorescence microscopy and whole mount in situ hybridization methods. Moreover, the transcriptional and translational levels of NS5A were investigated by RT-PCR and western blot. Finally, expression level of some liver pathology associated genes was investigated by RT-PCR.Results Cell transfection results showed that red fluorescent protein gene DsRed in pFC-5AiR is able to be expressed in Huh7 cells. After microinjection of the vector into zebrafish embryos, red fluorescence was observed to be located in the zebrafish liver under fluorescent microscopy. RT-PCR and western blot results indicated that NS5A is able to be expressed in zebrafish. In situ hybridization results further confirmed that the expression of NS5A in zebrafish liver. In addition, some pathological marker genes, such as Adiponectin, Argsyn, LDLR, TGF-β and HMGR etc., were up-regulated in response to HCV NS5A expression, suggesting that NS5A may be involved in liver lipid metabolism disorder and fibrosis.Conclusion A zebrafish model for HCV NS5A expression in liver has been successfully established. Preliminary investigation shows that HCV NS5A expression in vivo is associated with aberrant genes expression involved in liver pathological process. This model can be used to study

  11. Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV in hemodialysis patients Anticorpos contra os antígenos não estrutural c100/3 e estrutural core do vírus da hepatite C (HCV em pacientes de hemodiálise

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    C.F.T. Yoshida

    1993-08-01

    Full Text Available Two groups of patients undergoing hemodialysis (HD maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV. Forty-six patients (Group 1 never presented liver abnormalities during HD treatment, while 52 patients (Group 2 had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1. The rate of anti-c100/3 in patients of Group 2 was 71.15% and reached 86.5% for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8% of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7% (Group 2 was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.Dois grupos de pacientes em tratamento hemodialítico foram avaliados quanto às respostas de anticorpos contra as proteínas não estrutural c100/3 (anti-c100/3 e estrutural core (anti-HCV core do vírus da hepatite C (HCV. Quarenta e seis pacientes (Grupo 1 nunca apresentaram alterações dos níveis de alanina

  12. I. Novel HCV NS5B polymerase inhibitors: Discovery of indole 2-carboxylic acids with C3-heterocycles

    Energy Technology Data Exchange (ETDEWEB)

    Anilkumar, Gopinadhan N.; Lesburg, Charles A.; Selyutin, Oleg; Rosenblum, Stuart B.; Zeng, Qingbei; Jiang, Yueheng; Chan, Tin-Yau; Pu, Haiyan; Vaccaro, Henry; Wang, Li; Bennett, Frank; Chen, Kevin X.; Duca, Jose; Gavalas, Stephen; Huang, Yuhua; Pinto, Patrick; Sannigrahi, Mousumi; Velazquez, Francisco; Venkatraman, Srikanth; Vibulbhan, Bancha; Agrawal, Sony; Butkiewicz, Nancy; Feld, Boris; Ferrari, Eric; He, Zhiqing; Jiang, Chuan-kui; Palermo, Robert E.; Mcmonagle, Patricia; Huang, H.-C.; Shih, Neng-Yang; Njoroge, George; Kozlowski, Joseph A. (Merck)

    2012-05-03

    SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC{sub 50} = 0.9 {micro}M, replicon EC{sub 50} > 100 {micro}M) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC{sub 50} = 0.032 {micro}M, replicon EC{sub 50} = 1.4 {micro}M) and 7r (NS5B IC{sub 50} = 0.017 {micro}M, replicon EC{sub 50} = 0.3 {micro}M) with improved enzyme and replicon activity.

  13. Genetic diversity of NS5A protein from hepatitis C virus genotype 3a and its relationship to therapy response

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    Rahal Paula

    2010-02-01

    Full Text Available Abstract Background The quasispecies nature of HCV may have important implications for viral persistence, pathogenicity and resistance to antiviral agents. The variability of one of the viral proteins, NS5A, is believed to be related to the response to IFN therapy, the standard treatment for infection. In this study we analyzed the quasispecies composition of NS5A protein in patients infected with HCV genotype 3a, before IFN therapy. Methods Viral RNA was isolated from samples of 12 patients: four sustained virological responders (SVR, four non-responders (NR, and four end-of-treatment responders (ETR. cDNA was synthesized, the NS5A region was amplified and the fragments obtained were cloned. Fifteen clones from each patient were sequenced with eight primers, generating 179 contigs. Results Higher values for substitution (either synonymous or non-synonymous and for distance were found in the SVR group. However, the NR group showed relatively more non-synonymous mutations than the other groups, owing to the higher values of dN/dS in complete NS5A and most specific regions. Overall, NS5A protein is undergoing purifying selection, since all dN/dS ratios values are below 0.5. Conclusions Our study provides an overview of the genetic variability of complete NS5A protein in HCV genotype 3a.

  14. Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A.

    Science.gov (United States)

    Toroney, Rebecca; Nallagatla, Subba Rao; Boyer, Joshua A; Cameron, Craig E; Bevilacqua, Philip C

    2010-07-16

    Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2alpha, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5'-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of approximately 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.

  15. Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

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    Brittney R Henderson

    Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

  16. Confirm the HCV NS5A protein interaction with aconitase 1%HCV NS5A结合蛋白顺乌头酸酶1的验证研究

    Institute of Scientific and Technical Information of China (English)

    王晓杰; 相久全; 韩乐强; 张锦前; 成军

    2011-01-01

    Objective To screen proteins of human liver cDNA library interacting with HCV NS5A and confirm the interaction between HCV N55A protein and aconitate 1 protein.Methods Yeast two hybrid was performed by mating AH109 ( HCV NS5A) with Y187 ( liver cDNA lihrary of human) .The interacted proteins with HCV NS5A were screened from the library.The aconitate 1 gene was amplified at first.The expression vectors of aconitate 1 were constructed.Then the interaction between HCV N55A protein and Homo sapiens aconitate 1 protein was confirmed using co-imruunoprecipitation and mammalian two hybrid experiment.Results The eukaryotic expression vectors of aconitate 1 were constructed.These results showed that Homo sapiens aconitate 1 protein interacted with HCV NS5A protein by co-immunoprecipitation and mammalian two hybrids experiment Conclusion HCV NS5A and Homo sapiens aconitate 1 protein can interact in HepCJ2 cells.%目的 筛选人肝脏cDNA文库中与HCV NS5A的结合蛋白基因,验证其中顺乌头酸酶1与HCV NS5A的相互作用.方法 应用酵母双杂交系统3筛选人肝脏cDNA文库中的HCV NS5A结合蛋白基因,应用哺乳动物双杂交及免疫共沉淀技术验证其中顺乌头酸酶1蛋白与HCV NS5A之间的相瓦作用.结果 成功筛选出人肝脏cD-NA文库中与HCV NS5A存在相瓦作用的蛋白基因,哺乳动物双杂交及免疫共沉淀实验结果 证实HCV NS5A与顺乌头酸酶1蛋白在HepG2细胞内存在相互作用.结论 本实验成功筛选人肝脏cDNA文库中的HCV NS5A结合蛋白基因,并且在体外水平即细胞内证实HCV NS5A与其中的顺乌头酸酶1蛋白之间的相互作用,为进一步细胞内及体内的糖、脂类代谢等功能研究奠定基础.

  17. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling

    Science.gov (United States)

    Lim, Siew Pheng; Noble, Christian Guy; Seh, Cheah Chen; Soh, Tingjin Sherryl; El Sahili, Abbas; Chan, Grace Kar Yarn; Lescar, Julien; Arora, Rishi; Benson, Timothy; Nilar, Shahul; Manjunatha, Ujjini; Wan, Kah Fei; Dong, Hongping; Xie, Xuping; Yokokawa, Fumiaki

    2016-01-01

    Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo” initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the “GDD” active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1–2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses. PMID:27500641

  18. Epitope mapping of the NS4 and NS5 gene products of hepatitis C virus and the use of a chimeric NS4-NS5 synthetic peptide for serodiagnosis.

    Science.gov (United States)

    Rosa, C; Osborne, S; Garetto, F; Griva, S; Rivella, A; Calabresi, G; Guaschino, R; Bonelli, F

    1995-10-01

    Specific domains of the NS4 and NS5 gene products of hepatitis C virus have been identified using hydrophilicity profiles for the prediction of potential immunogenic regions, and epitope scanning techniques. Peptides synthesised on the basis of such data show excellent reactivity in the ELISA format. Introduction of a glycine-glycine spacer between two peptides (NS4-12 and NS5-44) to give a single chimeric peptides does not appear to impair immunoreactivity. An ELISA based on the chimeric peptide and a Core-NS3 recombinant protein correctly diagnoses a cohort of haemodialysed patients, three commercial HCV panels and the sera of a negative control population.

  19. Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B.

    Science.gov (United States)

    Li, Yan; Yang, Zexiao

    2015-12-01

    Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups.

  20. Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

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    Sandra Regina Maruyama

    2014-02-01

    Full Text Available Transcripts similar to those that encode the nonstructural (NS proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG complementary DNA (cDNA library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

  1. The interaction between the hepatitis C proteins NS4B and NS5A is involved in viral replication.

    Science.gov (United States)

    David, Naama; Yaffe, Yakey; Hagoel, Lior; Elazar, Menashe; Glenn, Jeffrey S; Hirschberg, Koret; Sklan, Ella H

    2015-01-15

    Hepatitis C virus (HCV) replicates in membrane associated, highly ordered replication complexes (RCs). These complexes include viral and host proteins necessary for viral RNA genome replication. The interaction network among viral and host proteins underlying the formation of these RCs is yet to be thoroughly characterized. Here, we investigated the association between NS4B and NS5A, two critical RC components. We characterized the interaction between these proteins using fluorescence resonance energy transfer and a mammalian two-hybrid system. Specific tryptophan residues within the C-terminal domain (CTD) of NS4B were shown to mediate this interaction. Domain I of NS5A, was sufficient to mediate its interaction with NS4B. Mutations in the NS4B CTD tryptophan residues abolished viral replication. Moreover, one of these mutations also affected NS5A hyperphosphorylation. These findings provide new insights into the importance of the NS4B-NS5A interaction and serve as a starting point for studying the complex interactions between the replicase subunits.

  2. Therapeutic potential of Taraxacum officinale against HCV NS5B polymerase: In-vitro and In silico study.

    Science.gov (United States)

    Rehman, Sidra; Ijaz, Bushra; Fatima, Nighat; Muhammad, Syed Aun; Riazuddin, Sheikh

    2016-10-01

    Discovery of alternative and complementary regimens for HCV infection treatment is a need of time from clinical as well as economical point of views. Low cost of bioactive natural compounds production, high biochemical diversity and inexistent/milder side effects contribute to new therapies. Aim of this study is to clarify anti-HCV role of Taraxacum officinale, a natural habitat plant rich of flavonoids. In this study, methanol extract of T. officinale leaves was initially analyzed for its cytotoxic activity in human hepatoma (Huh-7) and CHO cell lines. Hepatoma cells were transfected with pCR3.1/Flagtag/HCV NS5B gene cloned vector (genotype 1a) along with T. officinale extract. Considering NS5B polymerase as potential therapeutic drug target, twelve phytochemicals of T. officinale were selected as ligands for molecular interaction with NS5B protein using Molecular Operating Environment (MOE) software. Sofosbuvir (Sovaldi: brand name) currently approved as new anti-HCV drug, was used as standard in current study for comparative analysis in computational docking screening. HCV NS5B polymerase as name indicates plays key role in viral genome replication. On the basis of which NS5B gene is targeted for determining antiviral role of T. officinale extract and 65% inhibition of NS5B expression was documented at nontoxic dose concentration (200μg/ml) using Real-time PCR. In addition, 57% inhibition of HCV replication was recorded when incubating Huh-7 cells with high titer serum of HCV infected patients along with leaves extract. Phytochemicals for instance d-glucopyranoside (-31.212 Kcal/mol), Quercetin (-29.222 Kcal/mol), Luteolin (-26.941 Kcal/mol) and some others displayed least binding energies as compared to standard drug Sofosbuvir (-21.0746 Kcal/mol). Results of our study strongly revealed that T. officinale leaves extract potentially blocked the viral replication and NS5B gene expression without posing any toxic effect on normal fibroblast cells of body.

  3. Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

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    Canard Bruno

    2011-10-01

    Full Text Available Abstract Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4. Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

  4. Legalon-SIL downregulates HCV core and NS5A in human hepatocytes expressing full-length HCV

    Institute of Scientific and Technical Information of China (English)

    Marjan Mehrab-Mohseni; Hossein Sendi; Nury Steuerwald; Sriparna Ghosh; Laura W Schrum; Herbert L Bonkovsky

    2011-01-01

    AIM: To determine the effect of Legalon-SIL (LS) on hepatitis C virus (HCV) core and NS5A expression and on heme oxygenase-1 (HMOX-1) and its transcriptionalregulators in human hepatoma cells expressing full length HCV genotype 1b.METHODS: CON1 cells were treated with 50 μmol/or 200 μmol/L LS. Cells were harvested after 2, 6 and 24 h. HCV RNA and protein levels were determined byquantitative real-time polymerase chain reaction and Western blotting, respectively.RESULTS: HCV RNA (core and NS5A regions) was decreased after 6 h with LS 200 μmol/L (P < 0.05).Both 50 and 200 μmol/L LS decreased HCV RNA levels[core region (by 55% and 88%, respectively) and NS5A region (by 62% and 87%, respectively) after 24 h compared with vehicle (dimethyl sulphoxide) control (P< 0.01). Similarly HCV core and NS5A protein were decreased(by 85%, P < 0.01 and by 65%, P < 0.05, respectively)by LS 200 μmol/L. Bach1 and HMOX-1 RNAwere also downregulated by LS treatment (P < 0.01),while Nrf2 protein was increased (P < 0.05).CONCLUSION: Our results demonstrate that treatment with LS downregulates HCV core and NS5A expression in CON1 cells which express full length HCVgenotype 1b, and suggests that LS may prove to be a valuable alternative or adjunctive therapy for the treatment of HCV infection.

  5. Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7

    DEFF Research Database (Denmark)

    Galli, Andrea; Scheel, Troels K H; Prentoe, Jannick C;

    2013-01-01

    recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core......Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (c......LDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain...

  6. 3D-QSAR and molecular docking studies on designing inhibitors of the hepatitis C virus NS5B polymerase

    Science.gov (United States)

    Li, Wenlian; Si, Hongzong; Li, Yang; Ge, Cuizhu; Song, Fucheng; Ma, Xiuting; Duan, Yunbo; Zhai, Honglin

    2016-08-01

    Viral hepatitis C infection is one of the main causes of the hepatitis after blood transfusion and hepatitis C virus (HCV) infection is a global health threat. The HCV NS5B polymerase, an RNA dependent RNA polymerase (RdRp) and an essential role in the replication of the virus, has no functional equivalent in mammalian cells. So the research and development of efficient NS5B polymerase inhibitors provides a great strategy for antiviral therapy against HCV. A combined three-dimensional quantitative structure-activity relationship (QSAR) modeling was accomplished to profoundly understand the structure-activity correlation of a train of indole-based inhibitors of the HCV NS5B polymerase to against HCV. A comparative molecular similarity indices analysis (COMSIA) model as the foundation of the maximum common substructure alignment was developed. The optimum model exhibited statistically significant results: the cross-validated correlation coefficient q2 was 0.627 and non-cross-validated r2 value was 0.943. In addition, the results of internal validations of bootstrapping and Y-randomization confirmed the rationality and good predictive ability of the model, as well as external validation (the external predictive correlation coefficient rext2 = 0.629). The information obtained from the COMSIA contour maps enables the interpretation of their structure-activity relationship. Furthermore, the molecular docking study of the compounds for 3TYV as the protein target revealed important interactions between active compounds and amino acids, and several new potential inhibitors with higher activity predicted were designed basis on our analyses and supported by the simulation of molecular docking. Meanwhile, the OSIRIS Property Explorer was introduced to help select more satisfactory compounds. The satisfactory results from this study may lay a reliable theoretical base for drug development of hepatitis C virus NS5B polymerase inhibitors.

  7. Cell penetrable humanized-VH/V(HH that inhibit RNA dependent RNA polymerase (NS5B of HCV.

    Directory of Open Access Journals (Sweden)

    Kanyarat Thueng-in

    Full Text Available NS5B is pivotal RNA dependent RNA polymerase (RdRp of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(HH that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(HH from a humanized-camel VH/V(HH display library. VH/V(HH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(HH hallmark and were designated V(HH6 and V(HH24; other clones were conventional VH, designated VH9 and VH13. All VH/V(HH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(HH6 and V(HH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(HH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

  8. Cell penetrable humanized-VH/V(H)H that inhibit RNA dependent RNA polymerase (NS5B) of HCV.

    Science.gov (United States)

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Bangphoomi, Kunan; Poungpair, Ornnuthchar; Maneewatch, Santi; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

    2012-01-01

    NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(H)H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(H)H from a humanized-camel VH/V(H)H display library. VH/V(H)H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(H)H hallmark and were designated V(H)H6 and V(H)H24; other clones were conventional VH, designated VH9 and VH13. All VH/V(H)H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(H)H6 and V(H)H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(H)H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

  9. Characterization of a peptide domain within the GB virus C NS5A phosphoprotein that inhibits HIV replication.

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    Jinhua Xiang

    Full Text Available BACKGROUND: GBV-C infection is associated with prolonged survival in HIV-infected people and GBV-C inhibits HIV replication in co-infection models. Expression of the GBV-C nonstructural phosphoprotein 5A (NS5A decreases surface levels of the HIV co-receptor CXCR4, induces the release of SDF-1 and inhibits HIV replication in Jurkat CD4+ T cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Jurkat cell lines stably expressing NS5A protein and peptides were generated and HIV replication in these cell lines assessed. HIV replication was significantly inhibited in all cell lines expressing NS5A amino acids 152-165. Substitution of an either alanine or glycine for the serine at position 158 (S158A or S158G resulted in a significant decrease in the HIV inhibitory effect. In contrast, substituting a phosphomimetic amino acid (glutamic acid; S158E inhibited HIV as well as the parent peptide. HIV inhibition was associated with lower levels of surface expression of the HIV co-receptor CXCR4 and increased release of the CXCR4 ligand, SDF-1 compared to control cells. Incubation of CD4+ T cell lines with synthetic peptides containing amino acids 152-167 or the S158E mutant peptide prior to HIV infection resulted in HIV replication inhibition compared to control peptides. CONCLUSIONS/SIGNIFICANCE: Expression of GBV-C NS5A amino acids 152-165 are sufficient to inhibit HIV replication in vitro, and the serine at position 158 appears important for this effect through either phosphorylation or structural changes in this peptide. The addition of synthetic peptides containing 152-167 or the S158E substitution to Jurkat cells resulted in HIV replication inhibition in vitro. These data suggest that GBV-C peptides or a peptide mimetic may offer a novel, cellular-based approach to antiretroviral therapy.

  10. Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein.

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    Dorothee A Vogt

    Full Text Available The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web and assists viral assembly in the close vicinity of lipid droplets (LDs. To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1-31, a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47 as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon, indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47--via its interaction with NS5A--serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.

  11. HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

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    Kenji Nakashima

    Full Text Available Hepatitis C virus (HCV infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV implied that NS5A was tyrosine phosphorylated by pervanadate (PV treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST-fusion proteins of various Src homology 2 (SH2 domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3 domain. Substitution of Arg(176 to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334 was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

  12. Discovery of Novel Thiophene-Based, Thumb Pocket 2 Allosteric Inhibitors of the Hepatitis C NS5B Polymerase with Improved Potency and Physicochemical Profiles.

    Science.gov (United States)

    Court, John J; Poisson, Carl; Ardzinski, Andrzej; Bilimoria, Darius; Chan, Laval; Chandupatla, Kishan; Chauret, Nathalie; Collier, Philip N; Das, Sanjoy Kumar; Denis, Francois; Dorsch, Warren; Iyer, Ganesh; Lauffer, David; L'Heureux, Lucille; Li, Pan; Luisi, Brian S; Mani, Nagraj; Nanthakumar, Suganthi; Nicolas, Olivier; Rao, B Govinda; Ronkin, Steven; Selliah, Subajini; Shawgo, Rebecca S; Tang, Qing; Waal, Nathan D; Yannopoulos, Constantin G; Green, Jeremy

    2016-07-14

    The hepatitis C viral proteins NS3/4A protease, NS5B polymerase, and NS5A are clinically validated targets for direct-acting antiviral therapies. The NS5B polymerase may be inhibited directly through the action of nucleosides or nucleotide analogues or allosterically at a number of well-defined sites. Herein we describe the further development of a series of thiophene carboxylate allosteric inhibitors of NS5B polymerase that act at the thumb pocket 2 site. Lomibuvir (1) is an allosteric HCV NS5B inhibitor that has demonstrated excellent antiviral activity and potential clinical utility in combination with other direct acting antiviral agents. Efforts to further explore and develop this series led to compound 23, a compound with comparable potency and improved physicochemical properties.

  13. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

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    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. A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A.

    Science.gov (United States)

    McCormick, Christopher J; Brown, David; Griffin, Stephen; Challinor, Lisa; Rowlands, David J; Harris, Mark

    2006-01-01

    Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

  15. Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution

    Energy Technology Data Exchange (ETDEWEB)

    Saw, Wuan Geok; Tria, Giancarlo; Grüber, Ardina; Subramanian Manimekalai, Malathy Sony; Zhao, Yongqian; Chandramohan, Arun; Srinivasan Anand, Ganesh; Matsui, Tsutomu; Weiss, Thomas M.; Vasudevan, Subhash G.; Grüber, Gerhard

    2015-10-31

    Infection by the four serotypes ofDengue virus(DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all fourDengue virusserotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.

  16. In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir

    Science.gov (United States)

    Tian, Yang; Doehle, Brian; Peng, Betty; Corsa, Amoreena; Lee, Yu-Jen; Gong, Ruoyu; Yu, Mei; Han, Bin; Xu, Simin; Dvory-Sobol, Hadas; Perron, Michel; Xu, Yili; Mo, Hongmei; Pagratis, Nikos; Link, John O.; Delaney, William

    2016-01-01

    Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type. PMID:26824950

  17. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

    Science.gov (United States)

    Ng, Ivan H. W.; Chan, Kitti W. K.; Zhao, Yongqian; Ooi, Eng Eong; Lescar, Julien; Jans, David A.; Forwood, Jade K.

    2016-01-01

    Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. PMID:27622521

  18. eEF1A Interacts with the NS5A Protein and Inhibits the Growth of Classical Swine Fever Virus

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    Su Li

    2015-08-01

    Full Text Available The NS5A protein of classical swine fever virus (CSFV is involved in the RNA synthesis and viral replication. However, the NS5A-interacting cellular proteins engaged in the CSFV replication are poorly defined. Using yeast two-hybrid screen, the eukaryotic elongation factor 1A (eEF1A was identified to be an NS5A-binding partner. The NS5A–eEF1A interaction was confirmed by coimmunoprecipitation, glutathione S-transferase (GST pulldown and laser confocal microscopy assays. The domain I of eEF1A was shown to be critical for the NS5A–eEF1A interaction. Overexpression of eEF1A suppressed the CSFV growth markedly, and conversely, knockdown of eEF1A enhanced the CSFV replication significantly. Furthermore, eEF1A, as well as NS5A, was found to reduce the translation efficiency of the internal ribosome entry site (IRES of CSFV in a dose-dependent manner, as demonstrated by luciferase reporter assay. Streptavidin pulldown assay revealed that eEF1A could bind to the CSFV IRES. Collectively, our results suggest that eEF1A interacts with NS5A and negatively regulates the growth of CSFV.

  19. ADP-ribosylation Factor-related Protein 1 Interacts with NS5A and Regulates Hepatitis C Virus Propagation

    Science.gov (United States)

    Lim, Yun-Sook; Ngo, Huong T. T.; Lee, Jihye; Son, Kidong; Park, Eun-Mee; Hwang, Soon B.

    2016-01-01

    The life cycle of hepatitis C virus (HCV) is tightly coupled to the lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have previously screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet (LD) formation using cell culture-grown HCV (HCVcc)-infected cells. In this study, we selected and characterized the gene encoding ADP-ribosylation factor-related protein 1 (ARFRP1). ARFRP1 is essential for LD growth and is involved in the regulation of lipolysis. siRNA-mediated knockdown of ARFRP1 significantly inhibited HCV replication in both subgenomic replicon cells and HCVcc-infected cells. ARFRP1 interacted with NS5A and NS5A partially colocalized with LD. Silencing of ARFRP1 abrogated HCV-induced LD growth and viral protein expressions. Moreover, ARFRP1 recruited synaptosomal-associated protein 23 (SNAP23) to sites in close proximity to LDs in HCV-infected cells. Silencing of ARFRP1 ablated relocalization of SNAP23 to LD. These data indicate that HCV regulates ARFRP1 for LD growth to facilitate viral propagation and thus ARFRP1 may be a potential target for antiviral therapy. PMID:27550144

  20. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    Science.gov (United States)

    Galiano, Vicente; Garcia-Valtanen, Pablo; Micol, Vicente; Encinar, José Antonio

    2016-01-01

    The dengue virus (DENV) nonstructural protein 5 (NS5) contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds) to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <−10.5 kcal/mol) were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo.

  1. Cloning and identification of NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A

    Institute of Scientific and Technical Information of China (English)

    Qian Yang; Jun Cheng; Yan Liu; Yuan Hong; Jian-Jun Wang; Shu-Lin Zhang

    2004-01-01

    AIM: To clone, identify and study new NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus nonstructural protein 5A.METHODS: On the basis of subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus, the coding sequence of new gene and its spliced variant were obtained by bioinformatics method. Polymerase chain reaction (PCR)was conducted to amplify NS5ATP2 gene.RESUJLTS: The coding sequence of a new gene and its spliced variant were cloned and identified successfully.CONCLUSION: A new gene has been recognized as the new target transactivated by HCV NS5A protein. These results brought some new clues for studying the biological functions of new genes and pathogenesis of the viral proteins.

  2. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    Directory of Open Access Journals (Sweden)

    Galiano V

    2016-10-01

    Full Text Available Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH, Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH, Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV nonstructural protein 5 (NS5 contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular

  3. Association of Hepatitis C Virus Replication Complexes with Microtubules and Actin Filaments Is Dependent on the Interaction of NS3 and NS5A▿

    OpenAIRE

    Lai, Chao-Kuen; Jeng, King-Song; Machida, Keigo; Lai, Michael M. C.

    2008-01-01

    The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron...

  4. Optimization of thienopyrrole-based finger-loop inhibitors of the hepatitis C virus NS5B polymerase.

    Science.gov (United States)

    Martin Hernando, Jose Ignacio; Ontoria, Jesus Maria; Malancona, Savina; Attenni, Barbara; Fiore, Fabrizio; Bonelli, Fabio; Koch, Uwe; Di Marco, Stefania; Colarusso, Stefania; Ponzi, Simona; Gennari, Nadia; Vignetti, Sue Ellen; Del Rosario Rico Ferreira, Maria; Habermann, Jörg; Rowley, Michael; Narjes, Frank

    2009-10-01

    Infections caused by the hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The NS5B polymerase of HCV is responsible for the replication of viral RNA and has been a prime target in the search for novel treatment options. We had discovered allosteric finger-loop inhibitors based on a thieno[3,2-b]pyrrole scaffold as an alternative to the related indole inhibitors. Optimization of the thienopyrrole series led to several N-acetamides with submicromolar potency in the cell-based replicon assay, but they lacked oral bioavailability in rats. By linking the N4-position to the ortho-position of the C5-aryl group, we were able to identify the tetracyclic thienopyrrole 40, which displayed a favorable pharmacokinetic profile in rats and dogs and is equipotent with recently disclosed finger-loop inhibitors based on an indole scaffold.

  5. Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

    Science.gov (United States)

    Nakatani, Sueli M.; Santos, Carlos A.; Riediger, Irina N.; Krieger, Marco A.; Duarte, Cesar A. B.; Lacerda, Marco A.; Biondo, Alexander W.; Carilho, Flair J.; Ono-Nita, Suzane K.

    2010-01-01

    Background Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. Methodology/Principal Findings Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. Conclusions/Significance The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification. PMID:20405017

  6. Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.

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    Sueli M Nakatani

    Full Text Available BACKGROUND: Hepatitis C virus (HCV genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. METHODOLOGY/PRINCIPAL FINDINGS: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay. Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0% and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%, NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. CONCLUSIONS/SIGNIFICANCE: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.

  7. Transient replication of a hepatitis C virus genotype 1b replicon chimera encoding NS5A-5B from genotype 3a.

    Science.gov (United States)

    Kylefjord, Helen; Danielsson, Axel; Sedig, Susanne; Belda, Oscar; Wiktelius, Daniel; Vrang, Lotta; Targett-Adams, Paul

    2014-01-01

    Although hepatitis C virus (HCV) is a pathogen of global significance, experimental therapies in current clinical development include highly efficacious all-oral combinations of HCV direct-acting antivirals (DAAs). If approved for use, these new treatment regimens will impact dramatically upon our capacity to eradicate HCV in the majority of virus-infected patients. However, recent data from late-stage clinical evaluations demonstrated that individuals infected with HCV genotype (GT) 3 responded less well to all-oral DAA combinations than patients infected with other HCV GTs. In light of these observations, the present study sought to expand the number of molecular tools available to investigate small molecule-mediated inhibition of HCV GT3 NS5A and NS5B proteins in preclinical tissue-culture systems. Accordingly, a novel subgenomic HCV replicon chimera was created by utilizing a GT1b backbone modified to produce NS5A and NS5B proteins from a consensus sequence generated from HCV GT3a genomic sequences deposited online at the European Hepatitis C Virus database. This approach avoided the need to isolate and amplify HCV genomes from sera derived from HCV-infected patients. The replicon chimera, together with a version engineered to express NS5A encoding a Y93H mutation, demonstrated levels of replication in transient assays robust enough to assess accurate antiviral activities of inhibitors representing different HCV DAA classes. Thus, the replicon chimera represents a new simple molecular tool suitable for drug discovery programmes aimed at investigating, understanding, and improving GT3a activities of HCV DAAs targeting NS5A or NS5B.

  8. Quasispecies evolution in NS5A region of hepatitis C virus genotype 1b during interferon or combined interferon-ribavirin therapy

    Institute of Scientific and Technical Information of China (English)

    Pascal Veillon; Christopher Payan; Hélène Le Guillou-Guillemette; Catherine Gaudy; Fran(c)oise Lunel

    2007-01-01

    AIM: To evaluate the implication of substitutions in the hepatitis C virus (HCV) non-structural 5A (NS5A) protein in the resistance of HCV during mono-interferon (IFN)or combined IFN-ribavirin (IFN-R) therapy. Although NS5A has been reported to interact with the HCV RNA-dependent RNA polymerase, NS5B, as well as with many cellular proteins, the function of NS5A in the life cycle of HCV remains unclear.METHODS: HCV quasispecies were studied by cloning and sequencing of sequential isolates from patients infected by HCV genotype 1b. Patients were treated by IFN-α2b for 3 mo followed by IFN-α2b alone or combined IFN-R therapy for 9 additional months. Patients were categorized intro two groups based on their response to the treatments: 7 with sustained virological response (SVR) (quasispecies = 150) and 3 non-responders (NR) to IFN-R (quasispecies = 106).RESULTS: Prior to treatment, SVR patients displayed a lower complexity of quasispecies than NR patients. Most patients had a decrease in the complexity of quasispecies during therapy. Analysis of amino acids substitutions showed that the degree of the complexity of the interferon sensitivity-determining region (ISDR) and the V3 domain of NS5A protein was able to discriminate the two groups of patients. Moreover, SVR patients displayed more variability in the NS5A region than NR patients.CONCLUSION: These results suggest that detailed molecular analysis of the NS5A region may be important for understanding its function in IFN response during HCV 1b infection.

  9. Resistance Analyses of HCV NS3/4A Protease and NS5B Polymerase from Clinical Studies of Deleobuvir and Faldaprevir

    Science.gov (United States)

    Berger, Kristi L.; Sarrazin, Christoph; Nelson, David R.; Scherer, Joseph; Sha, Nanshi; Marquis, Martin; Côté-Martin, Alexandra; Vinisko, Richard; Stern, Jerry O.; Mensa, Federico J.; Kukolj, George

    2016-01-01

    Background & Aim The resistance profile of anti-hepatitis C virus (HCV) agents used in combination is important to guide optimal treatment regimens. We evaluated baseline and treatment-emergent NS3/4A and NS5B amino-acid variants among HCV genotype (GT)-1a and -1b-infected patients treated with faldaprevir (HCV protease inhibitor), deleobuvir (HCV polymerase non-nucleoside inhibitor), and ribavirin in multiple clinical studies. Methods HCV NS3/4A and NS5B population sequencing (Sanger method) was performed on all baseline plasma samples (n = 1425 NS3; n = 1556 NS5B) and on post-baseline plasma samples from patients with virologic failure (n = 113 GT-1a; n = 221 GT-1b). Persistence and time to loss of resistance-associated variants (RAVs) was estimated using Kaplan–Meier analysis. Results Faldaprevir RAVs (NS3 R155 and D168) and deleobuvir RAVs (NS5B 495 and 496) were rare (90%). Virologic relapse was associated with RAVs in both NS3 and NS5B (53% GT-1b; 52% GT-1b); some virologic relapses had NS3 RAVs only (47% GT-1a; 17% GT-1b). Median time to loss of GT-1b NS5B P495 RAVs post-treatment (5 months) was less than that of GT-1b NS3 D168 (8.5 months) and GT-1a R155 RAVs (11.5 months). Conclusion Faldaprevir and deleobuvir RAVs are more prevalent among virologic failures than at baseline. Treatment response was not compromised by common NS3 polymorphisms; however, alanine at NS5B amino acid 499 at baseline (wild-type in GT-1a, polymorphism in GT-1b) may reduce response to this deleobuvir-based regimen. PMID:27494410

  10. Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323.

    Directory of Open Access Journals (Sweden)

    Kristen M Bullard

    Full Text Available Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323's antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99 and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450 isoforms thus suggesting this molecule

  11. DEB025 (Alisporivir inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A.

    Directory of Open Access Journals (Sweden)

    Lotte Coelmont

    Full Text Available DEB025/Debio 025 (Alisporivir is a cyclophilin (Cyp-binding molecule with potent anti-hepatitis C virus (HCV activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b to DEB025, requiring an average of 20 weeks (four independent experiments, compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold resistance. Replacing the NS5A gene (but not the NS5B gene from the wild type (WT genome with the corresponding sequence from the DEB025(res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res replicons. Unlike WT, DEB025(res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

  12. How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A.

    Science.gov (United States)

    Tan, S L; Katze, M G

    2001-05-25

    Interferons (IFNs) induce an antiviral state in the cell through complex and indirect mechanisms, which culminate in a direct inhibition of viral replication and stimulation of the host adaptive responses. Viruses often counteract with elaborate strategies to interfere with the induction as well as action of IFN effector molecules. This evolutionary battle between viruses and IFN components is a subject of intense research aimed at understanding the immunopathogenesis of viruses and the molecular basis of IFN signaling and action. In the case with hepatitis C virus (HCV), this may have profound implications for the therapeutic use of recombinant IFN in treating chronic hepatitis C. Depending on the subtype of HCV, current IFN-based treatment regimens are effective for only a small subset of chronic hepatitis C patients. Thus, one of the Holy Grails in HCV research is to understand the mechanisms by which the virus may evade IFN antiviral surveillance and establish persistent infection, which may eventually provide insights into new avenues for better antiviral therapy. Despite the lack of an efficient tissue culture system and an appropriate animal model for HCV infection, several mechanisms have been proposed based on clinical studies and in vitro experiments. This minireview focuses on the HCV NS5A nonstructural protein, which is implicated in playing a role in HCV tolerance to IFN treatment, possibly in part through its ability to inhibit the cellular IFN-induced PKR protein kinase.

  13. Dp-branes, NS5-branes and U-duality from nonabelian (2,0) theory with Lie 3-algebra

    CERN Document Server

    Honma, Yoshinori; Shiba, Shotaro

    2011-01-01

    We derive the super Yang-Mills action of Dp-branes on a torus T^{p-4} from the nonabelian (2,0) theory with Lie 3-algebra. Our realization is based on Lie 3-algebra with pairs of Lorentzian metric generators. The resultant theory then has negative norm modes, but it results in a unitary theory by setting VEV's of these modes. This procedure corresponds to the torus compactification, therefore by taking a transformation which is equivalent to T-duality, the Dp-brane action is obtained. We also study type IIA/IIB NS5-brane and Kaluza-Klein monopole systems by taking other VEV assignments. Such various compactifications can be realized in the nonabelian (2,0) theory, since both longitudinal and transverse directions can be compactified, which is different from the BLG theory. We finally discuss U-duality among these branes, and show that most of the moduli parameters in U-duality group are recovered. Especially in D5-brane case, the whole U-duality relation is properly reproduced.

  14. Microwave assisted synthesis of some novel Flurbiprofen hydrazide- hydrazones as anti-HCV NS5B and anticancer agents

    Directory of Open Access Journals (Sweden)

    Sevil Aydın

    2013-01-01

    Full Text Available The synthesis of a new series of flurbiprofen hydrazide-hydrazones using microwave assisted reactions is described. Substituted aldehydes were condensed with flurbiprofen hydrazide by microwave irradiation to corresponding hydrazones. Synthesis of N’-[(4-bromothiophen-2-ylmethylidene]-2-(2-fluorobiphenyl-4-yl propanehydrazide (3o employing microwave assisted process resulted in higher yields, in faster time and with less chemical waste compared to traditional techniques. (2-fluorobiphenyl-4-yl-N’-(phenylmethylidenepropanehydrazide (3p andN’-[(2-chloro-6-fluorophenyl methylidene]-2-(2-fluorobiphenyl-4-ylpropanehydrazide (3s inhibited the growth of a leukemia cancercell line HL-60 (TB by 66.37% and an ovarian cancer cell line OVCAR-4 by 77.34% (singledose, 10μM, respectively at the National Cancer Institute (NCI, but had no significant ef-fect on a panel of sixty human tumor cell lines. Flurbiprofen hydrazide-hydrazones were weak inhibitors of hepatitis C virus NS5B polymerase activity with N’-[(5-ethylfuran-2-ylmethylidene]-2-(2-fluorobiphenyl-4-ylpropanehydrazide (3m being the most active of this series. Binding mode investigations of compound 3m suggested that allosteric pocket (AP-B may be the potential binding site for flurbiprofen hydrazones and these results will alsoassist in further derivatization of 3m using the green chemistry approach and improve the potency of S-flurbiprofen hydrazide hydrazones

  15. 丙型肝炎病毒NS5A基因在昆虫细胞中的表达及其分布研究%Study on Expression and Location of Hepatitis C Virus NS5A Protein in Insect Cells

    Institute of Scientific and Technical Information of China (English)

    冯磊; 叶林柏; 阮华; 郜金荣; 徐进平; 刘源洁; 吴正辉

    2002-01-01

    应用PCR方法从含有丙肝病毒全部非结构蛋白基因的质粒pBAC25中扩增出全长的NS5A基因DNA片段(约1.34kb),PCR扩增NS5A基因片段克隆到转移载体pBlueBacHisA中.重组转移质粒pBlueBacHis5A DNA与野生型杆状病毒(AcNPV)DNA共转染SF-9昆虫细胞,通过空斑纯化获得带有NS5A基因的重组病AcNS5A.对重组病毒基因组DNA进行酶切和PCR鉴定,证实HCV NS5A基因已插入重组病毒基因组中.AcNS5A感染SF-9细胞后,在细胞中表达出一条64kD的蛋白,用Western-blot分析,结果表明这种蛋白与抗HCV HS5A特异性抗体发生强烈反应,说明NS5A基因已在细胞中得到表达,应用免疫荧光技术与免疫组化技术进一步研究NS5A蛋白在昆虫细胞中不同时间的表达情况及其分布,结果表明,NS5A蛋白在AcNS5A重组病毒感染细胞24h后主要分布在细胞质膜上,而在48h后则同时分布于细胞质膜和细胞核内,在72h则完全布满整个细胞,我们认为NS5A蛋白定位于质膜和细胞核中,暗示着在病毒复制过程中NS5A蛋白可能参与病毒RNA在质膜上复制和细胞基因表达的调控.

  16. Screening for proteins interacting with NS5 0f Japanese encephalitis virus by yeast two-hybrid technique%酵母双杂交技术筛选与乙型脑炎病毒NS5相互作用的蛋白

    Institute of Scientific and Technical Information of China (English)

    周希珍; 赵慧; 李晓峰; 邓永强; 秦成峰; 秦鄂德; 高岚

    2011-01-01

    Objective To screen possible human proteins that interact with the non-structural protein 5 (NS5) of Japanese encephalitis virus (JEV) by yeast two-hybrid system. Methods The bait plasmid pSos-NS5 carrying JEV NS5 gene was first constructed and transformed to veast cells, and then validated by the self-activation and toxicity assay. Then, a human umbilical vein endothelial cells cDNA library was used for screening with the bait plasmid pSos-NS5, and positive clones were selected based on nutritional requirements (Leucine and Uracil) and temperature change, and verified by one to one back-hybridization. Finally, the plasmids from the positive clones were sequenced and analyzed by Blast with sequences from GenBank. Results The bait plasmid pSos-NS5 was successfully constructed and confirmed to be no self-activation actiity and toxicity to the host veast cells. Three positive clones (10, 11, and 4'6) were then selected from the human umbilical vein endothelial cells cDNA library with the bait plasmid pSos-NS5. Then, the plasmid from clone 4'6 was sequenced and BLAST analysis within GenBank showed that the sequences were highly homologous to the TC21 gene. Finally, these data indicated TC21 protein might be the putative candidate protein that interacted with the NS5 protein of JEV. Conclusion A candidate host protein has been identified by the veast two-hybrid system, which will be helpful for understanding the biological function of NS5 of JEV,and further research should be warranted in the future.%目的 应用酵母双杂交技术筛选与乙型脑炎病毒NS5蛋白相互作用的宿主蛋白.方法 构建表达乙型脑炎病毒NS5蛋白的诱饵质粒pSos-NS5,经自激活和毒性实验后对人脐静脉内皮细胞cDNA文库进行筛选,挑取37℃条件下在缺失亮氨酸和尿嘧啶培养基上生长的克隆,利用一对一酵母回复性杂交实验进行验证,然后测定阳性克隆插入片段序列,通过Blast分析确定可能与NS5

  17. Resistance-Associated NS5A Variants of Hepatitis C Virus Are Susceptible to Interferon-Based Therapy.

    Directory of Open Access Journals (Sweden)

    Jun Itakura

    Full Text Available The presence of resistance-associated variants (RAVs of hepatitis C virus (HCV attenuates the efficacy of direct acting antivirals (DAAs. The objective of this study was to characterize the susceptibility of RAVs to interferon-based therapy.Direct and deep sequencing were performed to detect Y93H RAV in the NS5A region. Twenty nine genotype 1b patients with detectable RAV at baseline were treated by a combination of simeprevir, pegylated interferon and ribavirin. The longitudinal changes in the proportion of Y93H RAV during therapy and at breakthrough or relapse were determined.By direct sequencing, Y93H RAV became undetectable or decreased in proportion at an early time point during therapy (within 7 days in 57% of patients with both the Y93H variant and wild type virus at baseline when HCV RNA was still detectable. By deep sequencing, the proportion of Y93H RAV against Y93 wild type was 52.7% (5.8%- 97.4% at baseline which significantly decreased to 29.7% (0.16%- 98.3% within 7 days of initiation of treatment (p = 0.023. The proportion of Y93H RAV was reduced in 21 of 29 cases (72.4% and a marked reduction of more than 10% was observed in 14 cases (48.7%. HCV RNA reduction was significantly greater for Y93H RAV (-3.65±1.3 logIU/mL/day than the Y93 wild type (-3.35±1.0 logIU/mL/day (p<0.001.Y93H RAV is more susceptible to interferon-based therapy than the Y93 wild type.

  18. Pharmacokinetic, Pharmacodynamic, and Drug-Interaction Profile of the Hepatitis C Virus NS5B Polymerase Inhibitor Sofosbuvir.

    Science.gov (United States)

    Kirby, Brian J; Symonds, William T; Kearney, Brian P; Mathias, Anita A

    2015-07-01

    Sofosbuvir (SOVALDI(®)), a potent, once-daily, orally administered nucleotide analog prodrug inhibitor of the hepatitis C virus (HCV) NS5B polymerase is approved in the USA, EU, Canada, and other regions for the treatment of HCV infection as a component of an antiviral treatment regimen. Sofosbuvir undergoes intracellular activation to form GS-461203 (active triphosphate, not detected in plasma), and ultimately the inactive, renally eliminated metabolite GS-331007. GS-331007 was identified as the primary analyte of interest for clinical pharmacology studies as it accounted for >90 % of systemic drug-related material exposure, and provided comparable exposure-response relationships for viral kinetics as observed for sofosbuvir. GS-331007 and sofosbuvir exhibit linear pharmacokinetics with minimal accumulation upon multiple dosing. Compared to healthy subjects, HCV-infected patients had modestly lower (39 %) GS-331007 area under the plasma concentration-time curve (AUC) and higher sofosbuvir AUC (60 %). Sofosbuvir can be administered without dose modification in HCV-infected patients with any degree of hepatic impairment or mild to moderate renal impairment. Sofosbuvir has a low propensity for clinically significant drug interactions with common concomitant medications used by HCV-infected patients. Clinically significant alterations in GS-331007 or sofosbuvir exposures are limited to potent inducers of intestinal P-glycoprotein that may lower exposure. In HCV-infected patients, demographic variables do not significantly influence GS-331007 and sofosbuvir exposures and no consistent exposure-response relationships were observed for efficacy or safety. This review focuses on the clinical pharmacokinetics, pharmacodynamics, and pharmacokinetic-pharmacodynamic relationships of sofosbuvir, and summarizes a number of drug interaction studies with important concomitant medications commonly used by HCV-infected patients.

  19. 应用表达谱芯片筛选HCV NS5B基因转染Hun 7.5细胞所致表达差异的糖、脂类代谢基因%Screening of genes differentially expressed in Hun 7.5 cells transfected with HCV NS5B gene

    Institute of Scientific and Technical Information of China (English)

    刘文祥; 汪涛; 侯鹏

    2013-01-01

    目的 构建HCV NS5B的真核表达载体pCDNA3.1(-)HCV NS5B,研究HCV NS5B在Hun 7.5细胞中表达导致的糖、脂类代谢相关基因的差异表达.方法 真核表达载体pCDNA3.1(-)HCV NS5B转染Hun 7.5细胞,培养48h后提取细胞蛋白进行Western blot检测;将pcDNA3.1(-)HCV NS5B和pcDNA3.1(-)载体分别转染Hun 7.5细胞后,提取mRNA并逆转录为cDNA,运用基因表达谱芯片技术分析糖、脂类代谢相关的差异表达基因.结果 成功构建了HCV NS5B的真核表达载体pCDNA3.1(-) HCV NS5B;转染Hun 7.5细胞后HCV NSSB在细胞内可以表达;基因表达谱芯片技术筛选,分别发现了其中5个显著上调和3个显著下调的糖、脂类代谢相关基因.结论 筛选HCVNS5B转染Hun 7.5细胞后的糖、脂类物质代谢相关的差异表达基因,从而为丙型肝炎病毒感染合并糖尿病、脂肪肝等代谢性疾病分子生物学机制的研究提供重要依据.%Objective To detect HCV NS5B expression and screen the differential expression gene in Hun 7.5 cell transfected with HCV NS5B using gene chip technology.Methods The expression vector of pcDNA3.1(-) HCV NS5B was transfected into Hun 7.5 cell line.The expression of HCV NS5B protein was detected by western-blotting method.The differential expression of genes in the Hun 7.5 transfected with pcDNA3.1 (-)HCV NS5B and pcDNA3.1 (-) was detected respectively using cDNA microarray technique.Results The expression vector has been confirmed by restriction enzyme digestion.The expression of HCV NS5B protein has been confirmed by western blotting.High quality mRNA and cDNA had been prepared and successful microarray screening had been conducted.The results showed that 5 genes were up-regulated and 3 genes were down-regulated in Hun 7.5 cell line transfected with HCV NS5B,which was associated with metabolism of glucose and lipid.Conclusions cDNA microarray technology was successfully used to screen the genes differentially expressed in Hun 7.5 cell line

  20. New Inhibitors of the DENV-NS5 RdRp from Carpolepis laurifolia as Potential Antiviral Drugs for Dengue Treatment

    Directory of Open Access Journals (Sweden)

    Paul Coulerie

    2014-05-01

    Full Text Available Since a few decades the dengue virus became a major public health concern and no treatment is available yet. In order to propose potential antidengue compounds for chemotherapy we focused on DENV RNA polymerase (DENV-NS5 RdRp which is specific and essential for the virus replication. Carpolepis laurifolia belongs to the Myrtaceae and is used as febrifuge in traditional kanak medicine. Leaf extract of this plant has been identified as a hit against the DENV-NS5 RdRp. Here we present a bioguided fractionation of the leaf extract of C. laurifolia which is also the first phytochemical evaluation of this plant. Five flavonoids, namely quercetin (1, 6-methyl-7-methoxyapigenin (2, avicularin (3, quercitrin (4 and hyperoside (5, together with betulinic acid (6, were isolated from the leaf extract of C. laurifolia. All isolated compounds were tested individually against the DENV-NS5 RdRp and compared with four other commercial flavonoids: isoquercitrin (7, spiraeoside (8, quercetin-3,4’-di-O-glucoside (9 and rutine (10. Compounds 3, 4, 6, 8 and 10 displayed IC 50 ranging from 1.7 to 2.1 µM, and were the most active against the DENV-NS5 RdRp.

  1. The discovery and structure-activity relationships of pyrano[3,4-b]indole based inhibitors of hepatitis C virus NS5B polymerase.

    Science.gov (United States)

    LaPorte, Matthew G; Draper, Tandy L; Miller, Lori E; Blackledge, Charles W; Leister, Lara K; Amparo, Eugene; Hussey, Alison R; Young, Dorothy C; Chunduru, Srinivas K; Benetatos, Christopher A; Rhodes, Gerry; Gopalsamy, Ariamala; Herbertz, Torsten; Burns, Christopher J; Condon, Stephen M

    2010-05-01

    We describe the structure-activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.

  2. Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Prentoe, Jannick; Carlsen, Thomas H R

    2012-01-01

    directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1-7 cell culture systems, and through reverse genetics demonstrated a universal importance...... of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had...... greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases...

  3. Kushenin induces the apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A.

    Science.gov (United States)

    Zhou, Yi; Chen, Na; Liu, Xiaojing; Lin, Shumei; Luo, Wenjuan; Liu, Min

    2016-07-01

    With the increased burden induced by HCV, there is an urgent need to develop better-tolerated agents with good safety. In this study, we evaluated the anti-HCV capability of kushenin, as well as the possible mechanism to Huh7.5-HCV cells. The results demonstrated that kushenin significantly inhibited the HCV-RNA level. Similarly, the expression of HCV-specific protein NS5A was also decreased. Molecular docking results displayed that kushenin bonded well to the active pockets of HCV NS5A, further confirming the effects of kushenin on HCV replication. Coimmunoprecipitation assay determined that kushenin suppressed the interaction between PI3K and NS5A in HCV-replicon cells. Furthermore, kushenin exerted an obviously induced function on HCV-replicon cells apoptosis by inhibiting PI3K-Akt-mTOR pathway, which could be ameliorated by the specific activator IGF-1 addition. Taken together, kushenin possesses the ability to inhibit HCV replication, and contributes to the increased apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A. Our results provide important evidence for a better understanding of the pathogenesis of HCV infection, and suggest that kushenin has the potential to treat HCV disease.

  4. Effects of Resistance-Associated NS5A Mutations in Hepatitis C Virus on Viral Production and Susceptibility to Antiviral Reagents

    Science.gov (United States)

    Nitta, Sayuri; Asahina, Yasuhiro; Matsuda, Mami; Yamada, Norie; Sugiyama, Ryuichi; Masaki, Takahiro; Suzuki, Ryosuke; Kato, Nobuyuki; Watanabe, Mamoru; Wakita, Takaji; Kato, Takanobu

    2016-01-01

    Direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have potent anti-HCV effects but may provoke resistance-associated variants (RAVs). In this study, we assessed the characteristics of these RAVs and explored efficacious anti-HCV reagents using recombinant HCV with NS5A from a genotype 1b strain. We replaced the NS5A of JFH1 with that of Con1 (JFH1/5ACon1) and introduced known NS5A inhibitor resistance mutations (L31M, L31V, L31I and Y93H) individually or in combination. Susceptibilities against anti-HCV reagents were also investigated. RAVs with Y93H exhibited high extracellular core antigen levels and infectivity titers. Variants with any single mutation showed mild to moderate resistance against NS5A inhibitors, whereas variants with double mutations at both L31 and Y93 showed severe resistance. The variants with mutations exhibited similar levels of susceptibility to interferon (IFN)-α, IFN-λ1, IFN-λ3 and Ribavirin. Variants with the Y93H mutation were more sensitive to protease inhibitors compared with JFH1/5ACon1. In conclusion, the in vitro analysis indicated that the Y93H mutation enhanced infectious virus production, suggesting advantages in the propagation of RAVs with this mutation. However, these RAVs were susceptible to protease inhibitors. Thus, a therapeutic regimen that includes these reagents is a promising means to eradicate these RAVs. PMID:27703205

  5. Accounting for target flexibility and water molecules by docking to ensembles of target structures: the HCV NS5B palm site I inhibitors case study.

    Science.gov (United States)

    Barreca, Maria Letizia; Iraci, Nunzio; Manfroni, Giuseppe; Gaetani, Rosy; Guercini, Chiara; Sabatini, Stefano; Tabarrini, Oriana; Cecchetti, Violetta

    2014-02-24

    The introduction of new anti-HCV drugs in therapy is an imperative need and is necessary with a view to develop an interferon-free therapy. Thus, the discovery and development of novel small molecule inhibitors of the viral NS5B polymerase represent an exciting area of research for many pharmaceutical companies and academic groups. This study represents a contribution to this field and relies on the identification of the best NS5B model(s) to be used in structure-based computational approaches aimed at identifying novel non-nucleoside inhibitors of one of the protein allosteric sites, namely, palm site I. First, the NS5B inhibitors at palm site I were classified as water-mediated or nonwater-mediated ligands depending on their ability to interact with or displace a specific water molecule. Then, we took advantage of the available X-ray structures of the NS5B/ligand complexes to build different models of protein/water combinations, which were used to investigate the influence on docking studies of solvent sites as well as of the influence of the protein conformations. As the overall trend, we observed improved performance in the docking results of the water-mediated inhibitors by inclusion of explicit water molecules, with an opposite behavior generally happening for the nonwater-mediated inhibitors. The best performing target structures for the two ligand sets were then used for virtual screening simulations of a library containing the known NS5B inhibitors along with related decoys to assess the best performing targets ensembles on the basis of their ability to discriminate active and inactive compounds as well as to generate the correct binding modes. The parallel use of different protein structures/water sets outperformed the use of a single target structure, with the two-protein 3H98/2W-2FVC/7W and 3HKY/NoW-3SKE/NoW models resulting in the best performing ensembles for water-mediated inhibitors and nonwater-mediated inhibitors, respectively. The information

  6. Highly efficient infectious cell culture of three HCV genotype 2b strains and sensitivity to lead protease, NS5A, and polymerase inhibitors

    DEFF Research Database (Denmark)

    Ramirez, Santseharay; Li, Yi-Ping; Brun Jensen, Sanne;

    2014-01-01

    , we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct acting antivirals. NS5B inhibitors Sofosbuvir, Mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222...... systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors Sofosbuvir, Mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist......Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist...

  7. Resistance studies of a dithiazol analogue, DBPR110, as a potential hepatitis C virus NS5A inhibitor in replicon systems.

    Science.gov (United States)

    Lin, Hui-Mei; Wang, Jing-Chyi; Hu, Han-Shu; Wu, Pei-Shan; Wang, Wen-Hung; Wu, Su-Ying; Yang, Chi-Chen; Yeh, Teng-Kuang; Hsu, Tsu-An; Jiaang, Weir-Torn; Chao, Yu-Sheng; Chern, Jyh-Haur; Yueh, Andrew

    2013-02-01

    Hepatitis C virus (HCV), a member of the Flaviviridae family, affects approximately 3% of the world's population and is becoming the leading cause of liver disease in the world. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In our previous study, we identified a potential HCV NS5A inhibitor, BP008. After further systemic optimization, we discovered a more potent HCV inhibitor, DBPR110. DBPR110 reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC(50)) and a selective index value of 3.9 ± 0.9 pM and >12,800,000, respectively. DBPR110 reduced HCV2a replicon activity with an EC(50) and a selective index value of 228.8 ± 98.4 pM and >173,130, respectively. Sequencing analyses of several individual clones derived from the DBPR110-resistant RNAs purified from cells harboring genotype 1b and 2a HCV replicons revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. P58L/T and Y93H/N in genotype 1b and T24A, P58L, and Y93H in the genotype 2a replicon were the key substitutions for resistance selection. In the 1b replicon, V153M, M202L, and M265V play a compensatory role in replication and drug resistance. Moreover, DBPR110 displayed synergistic effects with alpha interferon (IFN-α), an NS3 protease inhibitor, and an NS5B polymerase inhibitor. In summary, our results present an effective small-molecule inhibitor, DBPR110, that potentially targets HCV NS5A. DBPR110 could be part of a more effective therapeutic strategy for HCV in the future.

  8. Inhibition on IFN-βExpression by hCV ns3 and ns5A in HepG2 Cells

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Objective To observe the effects of HCV protein, NS3 and NS5A on IFN-βin HepG2 cells and its regulation mechanism. Methods Human liver hepatocellular carcinoma cells HepG2 were transfected with recombinant eukaryotic plasmid pcDNA3.1/myc-His-core, NS3 or NS5A to overexpress these proteins, and the expression of IFN-βwere detected by qRT-PCR, Western blotting and ELISA. Luc2P reporter plasmids pGL4.10-IFNβ-P were constructed and transfected into HepG2 cells, and the activity of IFN-βpromoter were determined through luciferase assay for regulation mechanism study. Results Both mRNA level and protein expression of IFN-β were significantly decreased (P Conclusions HCV protein NS3 and NS5A could inhibit innate IFN-β expression and thus escape immune selection and hinder the host immune responses.

  9. Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A.

    Science.gov (United States)

    Lai, Chao-Kuen; Jeng, King-Song; Machida, Keigo; Lai, Michael M C

    2008-09-01

    The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.

  10. Small heterodimer partner 1 directly interacts with NS5A viral protein and has a key role in HCV related liver cell transformation.

    Science.gov (United States)

    Conti, Beatrice; Porcu, Cristiana; Viscomi, Carmela; Minutolo, Antonella; Costantini, Susan; Corazzari, Marco; Iannucci, Gino; Barbaro, Barbara; Balsano, Clara

    2016-12-20

    HCV life cycle is strictly correlated with the hepatocyte lipid metabolism; moreover, the progression of HCV chronic hepatitis is accelerated by the presence of liver steatosis. Among the steatogenic genes deregulated during the HCV infection one of the most attractive is the Small Heterodimer Protein 1 (SHP1; NR0B2), that is involved in a remarkable number of metabolic functions. HCV NS5A is an essential and integral component of the HCV membranous-web replicon complex (RC) and plays an essential role to transfer the viral genome from the RCs to the surface of the lipid droplets (LDs) that, in turn, play a key function during HCV life cycle.With the help of a HCV infection model, we demonstrate a functional interaction between SHP1 and HCV NS5A protein. SHP1 silencing (siSHP1) reversed the pro-oncogenic effects of HCV infection, inducing a significant decrease in liver lipid accumulation and in NS5A protein expression. Moreover, siSHP1 causes a strong modulation of some genes involved in HCV-related EMT, such as: HNF4, a central regulators of hepatocyte differentiation, E-Cadherin, SNAILs.Our data suggest that SHP1 results not only to be strictly connected to the pathogenesis of HCV-related liver steatosis, but also to its progression towards the liver transformation.

  11. TRIM14 inhibits hepatitis C virus infection by SPRY domain-dependent targeted degradation of the viral NS5A protein

    Science.gov (United States)

    Wang, Shanshan; Chen, Yongzhi; Li, Chunfeng; Wu, Yaoxing; Guo, Lei; Peng, Changwei; Huang, Yueping; Cheng, Genhong; Qin, F. Xiao-Feng

    2016-01-01

    Tripartite motif 14 (TRIM14) was reported to function as a mitochondrial signaling adaptor in mediating innate immune responses. However, the involvement of TRIM14 in host defense against viral infection and molecular mechanisms remain unclear. Here, we demonstrated that enforced expression of TRIM14 could potently inhibit the infection and replication of HCV in hepatocytes, whereas TRIM14 knockout cells became more susceptible to HCV infection. Interestingly, further experiments revealed that such anti-HCV activity was independent of activating the NF-κB or interferon pathways but required the C-terminal SPRY domain of no signaling capacity. In searching for mechanisms how TRIM14 exerts its antiviral function we found that TRIM14 interacted with HCV encoded non-structural protein NS5A and could strongly induce its degradation dependent on the NS5A1 subdomain. Interestingly extensive domain mapping analyses revealed that NS5A degradation was mediated by the highly conserved SPRY domain of TRIM14, which might involve the K48 ubiquitination pathway. Collectively, our work uncovered a new mechanism responsible for host defense against HCV infection, and could potentially aid the development of novel anti-HCV therapeutics. PMID:27578425

  12. Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

    Science.gov (United States)

    Mandal, Anirban; Ganta, Krishna Kumar

    2016-01-01

    Hepatitis C virus is major cause of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Presently available direct-acting antiviral drugs have improved success rate; however, high cost limits their utilization, especially in developing countries like India. In the present study, we evaluated anti-HCV potential of several siRNAs targeted against the HCV RNA-dependent RNA polymerase NS5B and cellular factors, La autoantigen, PSMA7, and human VAMP-associated protein to intercept different steps of viral life cycle. The target genes were downregulated individually as well as in combinations and their impact on viral replication was evaluated. Individual downregulation of La autoantigen, PSMA7, hVAP-A, and NS5B resulted in inhibition of HCV replication by about 67.2%, 50.7%, 39%, and 52%, respectively. However, antiviral effect was more pronounced when multiple genes were downregulated simultaneously. Combinations of siRNAs against La autoantigen with NS5B or hVAP-A resulted in greater inhibition in HCV replication. Our findings indicate that siRNA is a potential therapeutic tool for inhibiting HCV replication and simultaneously targeting multiple viral steps with the combination of siRNAs is more effective than silencing a single target. PMID:27446609

  13. 华南HCV1b亚型NS5A插入式复制子重组质粒的构建和序列分析%Construction and sequence analysis of recombinant HCV-1b replicon by replacing NS5A region

    Institute of Scientific and Technical Information of China (English)

    李京涛; 周斌; 周元平; 曾艳丽; 李威; 王俊洁; 张健; 张浩; 刘叔文

    2012-01-01

    Objective To construct the recombinant HCV-lb replicon by replacing NS5A region using serum samples from patients with chronic hepatitis C (CHC) in South China and explore the biological characteristics of NS5A protein in response to antiviral therapy. Methods The on-off plasmid containing the cutting sites of the restriction endonudeases Miu I and Bel I was designed based on the backbone of robust HCV lb replicon. The full-length fragments of HCV NS5A were amplified from different CHC patients by RT-PCR and cloned into pMD-18 vector, followed by sequence analysis of amino acid mutation of ISDR, PKRBD, V3 and IRRDR within the NS5A region. If the amplicon obtained contained no Miu I or Bel I cutting sites, the NS5A fragment was re-amplified using primers containing the cutting sites and inserted into the replicon for replacement. Results The full-length fragments of NS5A were obtained successfully from CHC patients. The core region of ISDR-V3 of NS5A was replaced in the HCV replicon plasmid and showed correct sequences. The amino acid mutations of ISDR and PKRBD within NS5A were more frequent in patients with sustained viral response (SVR) than those without SVR. A high variability in the amino acid sequence was observed in both IRRDR and V3 regions. Conclusion The plug-in type recombinant HCV replicon for replacement of NS5A region in the virus from CHC patients has been successfully constructed, which provides a basis for further investigation of the biological characteristics of NS5A protein, the mechanisms of interferon-resistance, and antiviral therapy of difficult-to-treat CHC.%目的 构建来自华南不同慢性丙型肝炎(CHC)患者的HCV NS5A的复制子重组质粒并测序分析,探索HCV NS5A在抗病毒应答中生物学特性.方法 以能够高效复制的1b型HCV复制子质粒为骨架,构成带有MIuⅠ和BclⅠ双酶切位点的开关质粒,采用逆转录-聚合酶链反应从不同CHC患者血清中扩增获得HCV 1b亚型NS5A

  14. A novel immuno-competitive capture mass spectrometry strategy for protein-protein interaction profiling reveals that LATS kinases regulate HCV replication through NS5A phosphorylation.

    Science.gov (United States)

    Meistermann, Hélène; Gao, Junjun; Golling, Sabrina; Lamerz, Jens; Le Pogam, Sophie; Tzouros, Manuel; Sankabathula, Sailaja; Gruenbaum, Lore; Nájera, Isabel; Langen, Hanno; Klumpp, Klaus; Augustin, Angélique

    2014-11-01

    Mapping protein-protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein-protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein-protein interactions with high specificity.

  15. Phylogenetic analysis of NS5B gene of classical swine fever virus isolates indicates plausible Chinese origin of Indian subgroup 2.2 viruses.

    Science.gov (United States)

    Patil, S S; Hemadri, D; Veeresh, H; Sreekala, K; Gajendragad, M R; Prabhudas, K

    2012-02-01

    Twenty-three CSFV isolates recovered from field outbreaks in various parts of India during 2006-2009 were used for genetic analysis in the NS5B region (409 nts). Seventeen of these were studied earlier [16] in the 5'UTR region. Phylogenetic analysis indicated the continued dominance of subgroup 1.1 strains in the country. Detailed analysis of a subgroup 2.2 virus indicated the plausible Chinese origin of this subgroup in India and provided indirect evidence of routes of CSFV movement within South East Asia region.

  16. Discovery of benzimidazole-diamide finger loop (Thumb Pocket I) allosteric inhibitors of HCV NS5B polymerase: Implementing parallel synthesis for rapid linker optimization.

    Science.gov (United States)

    Goulet, Sylvie; Poupart, Marc-André; Gillard, James; Poirier, Martin; Kukolj, George; Beaulieu, Pierre L

    2010-01-01

    Previously described SAR of benzimidazole-based non-nucleoside finger loop (Thumb Pocket I) inhibitors of HCV NS5B polymerase was expanded. Prospecting studies using parallel synthesis techniques allowed the rapid identification of novel cinnamic acid right-hand sides that provide renewed opportunities for further optimization of these inhibitors. Novel diamide derivatives such as 44 exhibited comparable potency (enzymatic and cell-based HCV replicon) as previously described tryptophan-based inhibitors but physicochemical properties (e.g., aqueous solubility and lipophilicity) have been improved, resulting in molecules with reduced off-target liabilities (CYP inhibition) and increased metabolic stability.

  17. Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Sanne B; Li, Yi-Ping;

    2013-01-01

    to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations...... against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared...

  18. Cell-death-inducing DFFA-like Effector B Contributes to the Assembly of Hepatitis C Virus (HCV) Particles and Interacts with HCV NS5A

    Science.gov (United States)

    Cai, Hua; Yao, Wenxia; Li, Leike; Li, Xinlei; Hu, Longbo; Mai, Runming; Peng, Tao

    2016-01-01

    Hepatitis C virus (HCV) uses components of the very-low-density lipoprotein (VLDL) pathway for assembly/release. We previously reported that hepatocyte nuclear factor 4α (HNF4α) participates in HCV assembly/release through downstream factors those participate in VLDL assembly/secretion. Cell-death-inducing DFFA-like effector B (CIDEB) is an important regulator of the VLDL pathway. CIDEB is required for entry of HCV particles from cell culture (HCVcc), but the effects of CIDEB on the post-entry steps of the HCV lifecycle are unclear. In the present study, we determined that CIDEB is required for HCV assembly in addition to HCVcc entry. Furthermore, CIDEB interacts with the HCV NS5A protein, and the N terminus of CIDEB and the domain I of NS5A are involved in this interaction. Moreover, CIDEB silencing impairs the association of apolipoprotein E (ApoE) with HCV particles. Interestingly, CIDEB is also required for the post-entry stages of the dengue virus (DENV) life cycle. Collectively, these results indicate that CIDEB is a new host factor that is involved in HCV assembly, presumably by interacting with viral protein, providing new insight into the exploitation of the VLDL regulator CIDEB by HCV. PMID:27282740

  19. 酵母双杂合系统在丙型肝炎病毒NS5A研究中的应用%IDENTIFICATION OF THE NOVEL HEPATITIS C VIRUS NS5A-INTERACTING PROTEINS IN YEAST TWO-HYBRID SYSTEM

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    酵母双杂合系统是在酵母细胞中研究蛋白-蛋白相互作用的新技术.应用该技术,以丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)为"诱饵",筛检了人肝癌细胞HepG2来源的cDNA文库,获得了7个NS5A结合蛋白的基因克隆.报道了其中两个粜钥寺?#2、#16)的结果,并对"人核小体组装蛋白相关蛋白"(Human nucleosome assembly protein-related protein,hNRP)和"载脂蛋白A-I"(Apolipoprotein A-I)与NS5A结合的生物学意义进行了讨论.

  20. A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

    Directory of Open Access Journals (Sweden)

    Lucile Warter

    Full Text Available GB virus B (GBV-B is closely related to hepatitis C virus (HCV, infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs of these viruses fold into four similar structured domains (I-IV, with domains II-III-IV comprising the viral internal ribosomal entry site (IRES. We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC genome (within the 3'NTR, upstream of the poly(U tract, and NS5A coding sequence are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.

  1. Genotypic and phenotypic analyses of hepatitis C virus variants observed in clinical studies of VX-222, a nonnucleoside NS5B polymerase inhibitor.

    Science.gov (United States)

    Jiang, Min; Zhang, Eileen Z; Ardzinski, Andrzej; Tigges, Ann; Davis, Andrew; Sullivan, James C; Nelson, Michelle; Spanks, Joan; Dorrian, Jennifer; Nicolas, Olivier; Bartels, Doug J; Rao, B Govinda; Rijnbrand, Rene; Kieffer, Tara L

    2014-09-01

    VX-222, a thiophene-2-carboxylic acid derivative, is a selective nonnucleoside inhibitor of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In phase 1 and 2 clinical studies, VX-222 demonstrated effective antiviral efficacy, with substantial reductions in plasma HCV RNA in patients chronically infected with genotype 1 HCV. To characterize the potential for selection of VX-222-resistant variants in HCV-infected patients, the HCV NS5B gene was sequenced at baseline and during and after 3 days of VX-222 dosing (monotherapy) in a phase 1 study. Variants with the substitutions L419C/I/M/P/S/V, R422K, M423I/T/V, I482L/N/T, A486S/T/V, and V494A were selected during VX-222 dosing, and their levels declined over time after the end of dosing. Phenotypic analysis of these variants was conducted using HCV replicons carrying site-directed mutations. Of the 17 variants, 14 showed reduced susceptibility to VX-222 compared with the wild type, with the L419C/S and R422K variants having higher levels of resistance (>200-fold) than the rest of the variants (6.8- to 76-fold). The M423I and A486S variants remained susceptible to VX-222. The 50% effective concentration (EC50) for the L419P variant could not be obtained due to the poor replication of this replicon. The majority of the variants (15/17) were less fit than the wild type. A subset of the variants, predominately the L419S and R422K variants, were observed when the efficacy and safety of VX-222- and telaprevir-based regimens given for 12 weeks were investigated in genotype 1 HCV-infected patients in a phase 2 study. The NS3 and NS5B variants selected during the dual combination therapy showed reduced susceptibility to both telaprevir and VX-222 and had a lower replication capacity than the wild type. The phase 1b study has the ClinicalTrials.gov identifier NCT00911963, and the phase 2a study has ClinicalTrials.gov identifier NCT01080222.

  2. Intracellular expression of the proliferative marker Ki-67 and viral proteins (NS3, NS5A and C in chronic, long lasting hepatitis C virus (HCV infection.

    Directory of Open Access Journals (Sweden)

    Karolina Olejniczak

    2008-01-01

    Full Text Available Hepatitis C virus (HCV continues to represent the main causative agent of the hepatitis, which leads to chronic transformation of the process in 60-80% individuals. It remains unclear how far cellular expression of HCV proteins in vivo may represent an index of progression of the disease and of proliferative activity in the liver in chronic hepatitis C. Aim of the studies included detection and subcellular localization of three HCV proteins (NS3, NS5A and C in liver biopsies from adults (n=19 with chronic, long lasting hepatitis C as related to hepatocyte proliferative activity. The immunocytochemical ABC (avidin biotin-peroxidase complex technique was applied, alone or associated with the ImmunoMax technique. Results of the immunocytochemical tests were compared to histological alterations in liver biopsies, proliferation index and with selected clinical data. A significantly higher expression of NS3 protein was noted, as compared to expressions of NS5A and C proteins. In all the patients, cytoplasmic localization of all proteins dominated over nuclear localization (p0.05. At the level of electron microscopy, protein localization in endoplasmic reticulum (ER membranes, mitochondria, perinuclear region and/or in hepatocyte cell nucleus was observed. No direct relationships could be demonstrated between expressions of HCV proteins and of Ki-67 antigen. No correlations could also be demonstrated between cellular expression of any HCV protein on one hand and grading or staging, alanine transaminase (ALT, serum level of HCV RNA or alpha-fetoprotein (AFP on the other. However, positive correlations were disclosed between proliferative activity of hepatocytes on one hand and patient's age, grading and staging on the other. Advanced hepatic fibrosis correlated also with serum levels of AFP. The studies were supplemented with data on subcellular localization of HCV proteins. Moreover, they indicated that in HCV infection grading and staging

  3. Molecular modeling and residue interaction network studies on the mechanism of binding and resistance of the HCV NS5B polymerase mutants to VX-222 and ANA598.

    Science.gov (United States)

    Xue, Weiwei; Jiao, Pingzu; Liu, Huanxiang; Yao, Xiaojun

    2014-04-01

    Hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) with essential functions in viral genome replication and represents a promising therapeutic target to develop direct-acting antivirals (DAAs). Multiple nonnucleoside inhibitors (NNIs) binding sites have been identified within the polymerase. VX-222 and ANA598 are two NNIs targeting thumb II site and palm I site of HCV NS5B polymerase, respectively. These two molecules have been shown to be very effective in phase II clinical trials. However, the emergence of resistant HCV replicon variants (L419M, M423T, I482L mutants to VX-222 and M414T, M414L, G554D mutants to ANA598) has significantly decreased their efficacy. To elucidate the molecular mechanism about how these mutations influenced the drug binding mode and decreased drug efficacy, we studied the binding modes of VX-222 and ANA598 to wild-type and mutant polymerase by molecular modeling approach. Molecular dynamics (MD) simulations results combined with binding free energy calculations indicated that the mutations significantly altered the binding free energy and the interaction for the drugs to polymerase. The further per-residue binding free energy decomposition analysis revealed that the mutations decreased the interactions with several key residues, such as L419, M423, L474, S476, I482, L497, for VX-222 and L384, N411, M414, Y415, Q446, S556, G557 for ANA598. These were the major origins for the resistance to these two drugs. In addition, by analyzing the residue interaction network (RIN) of the complexes between the drugs with wild-type and the mutant polymerase, we found that the mutation residues in the networks involved in the drug resistance possessed a relatively lower size of topology centralities. The shift of betweenness and closeness values of binding site residues in the mutant polymerase is relevant to the mechanism of drug resistance of VX-222 and ANA598. These results can provide an atomic-level understanding about

  4. D p-branes, NS5-branes and U-duality from nonabelian (2,0) theory with Lie 3-algebra

    Science.gov (United States)

    Honma, Yoshinori; Ogawa, Morirou; Shiba, Shotaro

    2011-04-01

    We derive the super Yang-Mills action of D p-branes on a torus T p-4 from the nonabelian (2, 0) theory with Lie 3-algebra [1]. Our realization is based on Lie 3-algebra with pairs of Lorentzian metric generators. The resultant theory then has negative norm modes, but it results in a unitary theory by setting VEV's of these modes. This procedure corresponds to the torus compactification, therefore by taking a transformation which is equivalent to T-duality, the D p-brane action is obtained. We also study type IIA/IIB NS5brane and Kaluza-Klein monopole systems by taking other VEV assignments. Such various compactifications can be realized in the nonabelian (2, 0) theory, since both longitudinal and transverse directions can be compactified, which is different from the BLG theory. We finally discuss U-duality among these branes, and show that most of the moduli parameters in U-duality group are recovered. Especially in D5-brane case, the whole U-duality relation is properly reproduced.

  5. Using an Old Drug to Target a New Drug Site: Application of Disulfiram to Target the Zn-Site in HCV NS5A Protein.

    Science.gov (United States)

    Lee, Yu-Ming; Duh, Yulander; Wang, Shih-Ting; Lai, Michael M C; Yuan, Hanna S; Lim, Carmay

    2016-03-23

    In viral proteins, labile Zn-sites, where Zn(2+) is crucial for maintaining the native protein structure but the Zn-bound cysteines are reactive, are promising drug targets. Here, we aim to (i) identify labile Zn-sites in viral proteins using guidelines established from our previous work and (ii) assess if clinically safe Zn-ejecting agents could eject Zn(2+) from the predicted target site and thus inhibit viral replication. As proof-of-concept, we identified a labile Zn-site in the hepatitis C virus (HCV) NS5A protein and showed that the antialcoholism drug, disulfiram, could inhibit HCV replication to a similar extent as the clinically used antiviral agent, ribavirin. The discovery of a novel viral target and a new role for disulfiram in inhibiting HCV replication will enhance the therapeutic armamentarium against HCV. The strategy presented can also be applied to identify labile sites in other bacterial or viral proteins that can be targeted by disulfiram or other clinically safe Zn-ejectors.

  6. Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.

    Directory of Open Access Journals (Sweden)

    Sarah E Boyce

    Full Text Available Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B--the C-terminal tail and β-loop--in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor-NS5B complex are absent in the inhibitor-bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.

  7. Optimizing choice of oral interferon-free treatment for genotype 1 hepatitis C virus using testing for NS5A resistance: a cost-utility analysis from the perspective of the Italian National Health Service

    Science.gov (United States)

    Westerhout, Kirsten Y; Bouwmeester, Walter; Duchesne, Inge; Pisini, Marta; Piena, Marjanne A; Damele, Francesco; Gueron, Beatrice; Treur, Maarten; Belsey, Jonathan

    2017-01-01

    Background Patients with genotype-1 hepatitis C virus infection who have failed to respond to standard therapy or who relapse following treatment may be considered for an interferon-free regimen incorporating a nonstructural protein 5A (NS5A) inhibitor. Sustained virologic response (SVR) with these regimens is typically >90%, but this is reduced in patients with NS5A resistance. European Association for Study of the Liver guidelines recommend simeprevir + sofosbuvir ± ribavirin (SMV+SOF±R) for re-treating patients failing an NS5A inhibitor-containing regimen. An alternative strategy would be to test for NS5A resistance prior to treatment, with therapy optimized based on the results. This study investigates the cost-effectiveness of this strategy. Materials and methods A Markov model was used to estimate disease progression for treatment-experienced genotype 1 patients with severe fibrosis or compensated cirrhosis. Targeted treatment with either SMV+SOF±R or sofosbuvir + ledipasvir ± ribavirin (SOF+LDV±R) based on pretreatment NS5A resistance testing was compared to routine SOF+LDV±R without testing. Treatment duration was 12 or 24 weeks for patients with severe fibrosis or compensated cirrhosis (Metavir F3/F4). SVR data for the treatment options were based on the results of published clinical trials. The analysis was carried out from the perspective of the Italian National Health Service. Results Optimized treatment using NS5A resistance testing yielded 0.163 additional QALYs and increased costs of €2,789 per patient versus no testing. The incremental cost-effectiveness ratio (ICER) was €17,078/QALY. Sensitivity analysis identified the SVR attributable to each of the treatment regimens as the most sensitive determinant of ICER (range: €10,055/QALY–€43,501/QALY across plausible range). Probabilistic sensitivity analysis demonstrated that, at a willingness-to-pay threshold of €30,000/QALY, the probability that NS5A-directed treatment will be cost

  8. Structure-based design, synthesis, and biological evaluation of 1,1-dioxoisothiazole and benzo[b]thiophene-1,1-dioxide derivatives as novel inhibitors of hepatitis C virus NS5B polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Hee; Tran, Martin T.; Ruebsam, Frank; Xiang, Alan X.; Ayida, Benjamin; McGuire, Helen; Ellis, David; Blazel, Julie; Tran, Chinh V.; Murphy, Douglas E.; Webber, Stephen E.; Zhou, Yuefen; Shah, Amit M.; Tsan, Mei; Showalter, Richard E.; Patel, Rupal; Gobbi, Alberto; LeBrun, Laurie A.; Bartkowski, Darian M.; Nolan, Thomas G.; Norris, Daniel A.; Sergeeva, Maria V.; Kirkovsky, Leo; Zhao, Qiang; Han, Qing; Kissinger, Charles R. (Anadys)

    2008-07-28

    A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS5B inhibitors with nanomolar IC{sub 50} values. The most potent compound exhibited IC{sub 50} less than 10 nM against the genotype 1b HCV polymerase and EC{sub 50} of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.

  9. Dengue virus-specific CD4+ and CD8+ T lymphocytes target NS1, NS3 and NS5 in infected Indian rhesus macaques.

    Science.gov (United States)

    Mladinich, Katherine M; Piaskowski, Shari M; Rudersdorf, Richard; Eernisse, Christopher M; Weisgrau, Kim L; Martins, Mauricio A; Furlott, Jessica R; Partidos, Charalambos D; Brewoo, Joseph N; Osorio, Jorge E; Wilson, Nancy A; Rakasz, Eva G; Watkins, David I

    2012-02-01

    Every year, Dengue virus (DENV) infects approximately 100 million people. There are currently several vaccines undergoing clinical studies, but most target the induction of neutralizing antibodies. Unfortunately, DENV infection can be enhanced by subneutralizing levels of antibodies that bind virions and deliver them to cells of the myeloid lineage, thereby increasing viral replication (termed antibody-dependent enhancement [ADE]). T lymphocyte-based vaccines may offer an alternative that avoids ADE. The goal of our study was to describe the cellular immune response generated after primary DENV infection in Indian rhesus macaques. We infected eight rhesus macaques with 10⁵ plaque-forming units (PFU) of DENV serotype 2 (DENV2) New Guinea C (NGC) strain, and monitored viral load and the cellular immune response to the virus. Viral replication peaked at day 4 post-infection and was resolved by day 10. DENV-specific CD4+ and CD8+ T lymphocytes targeted nonstructural (NS) 1, NS3 and NS5 proteins after resolution of peak viremia. DENV-specific CD4+ cells expressed interferon-gamma (IFN-γ) along with tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and macrophage inflammatory protein-1 beta (MIP-1β). In comparison, DENV-specific CD8+ cells expressed IFN-γ in addition to MIP-1β and TNF-α and were positive for the degranulation marker CD107a. Interestingly, a fraction of the DENV-specific CD4+ cells also stained for CD107a, suggesting that they might be cytotoxic. Our results provide a more complete understanding of the cellular immune response during DENV infection in rhesus macaques and contribute to the development of rhesus macaques as an animal model for DENV vaccine and pathogenicity studies.

  10. Randomized, placebo-controlled, single-ascending-dose study of BMS-791325, a hepatitis C virus (HCV) NS5B polymerase inhibitor, in HCV genotype 1 infection.

    Science.gov (United States)

    Sims, Karen D; Lemm, Julie; Eley, Timothy; Liu, Menping; Berglind, Anna; Sherman, Diane; Lawitz, Eric; Vutikullird, Apinya B; Tebas, Pablo; Gao, Min; Pasquinelli, Claudio; Grasela, Dennis M

    2014-06-01

    BMS-791325 is a nonnucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase with low-nanomolar potency against genotypes 1a (50% effective concentration [EC50], 3 nM) and 1b (EC50, 7 nM) in vitro. BMS-791325 safety, pharmacokinetics, and antiviral activity were evaluated in a double-blind, placebo-controlled, single-ascending-dose study in 24 patients (interferon naive and experienced) with chronic HCV genotype 1 infection, randomized (5:1) to receive a single dose of BMS-791325 (100, 300, 600, or 900 mg) or placebo. The prevalence and phenotype of HCV variants at baseline and specific posttreatment time points were assessed. Antiviral activity was observed in all cohorts, with a mean HCV RNA decline of ≈2.5 log10 copies/ml observed 24 h after a single 300-mg dose. Mean plasma half-life among cohorts was 7 to 9 h; individual 24-hour levels exceeded the protein-adjusted EC90 for genotype 1 at all doses. BMS-791325 was generally well tolerated, with no serious adverse events or discontinuations. Enrichment for resistance variants was not observed at 100 to 600 mg. At 900 mg, variants (P495L/S) associated with BMS-791325 resistance in vitro were transiently observed in one patient, concurrent with an observed HCV RNA decline of 3.4 log10 IU/ml, but were replaced with wild type by 48 h. Single doses of BMS-791325 were well tolerated; demonstrated rapid, substantial, and exposure-related antiviral activity; displayed dose-related increases in exposure; and showed viral kinetic and pharmacokinetic profiles supportive of once- or twice-daily dosing. These results support its further development in combination with other direct-acting antivirals for HCV genotype 1 infection. (This trial has been registered at ClinicalTrials.gov under registration no. NCT00664625.).

  11. Prediction of prognostic biomarkers for Interferon-based therapy to Hepatitis C Virus patients: a metaanalysis of the NS5A protein in subtypes 1a, 1b, and 3a

    Directory of Open Access Journals (Sweden)

    Zada Suher

    2010-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is a worldwide health problem with no vaccine and the only approved therapy is Interferon-based plus Ribavarin. Response prediction to treatment has health and economic impacts, and is a multi-factorial problem including both host and viral factors (e.g: age, sex, ethnicity, pre-treatment viral load, and dynamics of the HCV non-structural protein NS5A quasispecies. We implement a novel approach for extracting features including informative markers from mutations in the non-structural 5A protein (NS5A, specifically its Interferon sensitivity determining region (ISDR and V3 regions, and use a novel bioinformatics approach for pattern recognition on the NS5A protein and its motifs to find biomarkers for response prediction using class association rules and comparing the predictability of the different features. Results A total of 58 sequences from sustained responders and 94 from non-responders were downloaded from the HCV LANL database. Site-specific signatures for response prediction from the NS5A protein were extracted from the alignments. Class association rules were generated (e.g.: sustained response is associated with position A2368T in subtype 1a (support 100% and confidence 52.19%; in subtype 1b, response is associated with E2356G/D/K (support 76.3% and confidence 67.3%. Conclusion The V3 region was a more accurate biomarker than the ISDR region. Subtype-specific class association rules gave better support and confidence than profile hidden Markov models HMMs scores, genetic distances or number of variable sites, and would thus aid in the prediction of prognostic biomarkers and improve the accuracy of prognosis. Sites-specific class association rules in the V3 region of the NS5A protein have given the best support and confidence.

  12. Optimizing choice of oral interferon-free treatment for genotype 1 hepatitis C virus using testing for NS5A resistance: a cost-utility analysis from the perspective of the Italian National Health Service

    Directory of Open Access Journals (Sweden)

    Westerhout KY

    2017-02-01

    Full Text Available Kirsten Y Westerhout,1 Walter Bouwmeester,1 Inge Duchesne,2 Marta Pisini,2 Marjanne A Piena,1 Francesco Damele,3 Beatrice Gueron,2 Maarten Treur,1 Jonathan Belsey4 1Pharmerit BV, Marten Meesweg, Rotterdam, the Netherlands; 2Janssen EMEA, Turnhoutseweg, Beerse, Belgium; 3Janssen-Cilag SpA, Via Michelangelo Buonarroti, Cologno Monzese, Italy; 4JB Medical Ltd, Old Brickworks, Little Cornard, United Kingdom Background: Patients with genotype-1 hepatitis C virus infection who have failed to respond to standard therapy or who relapse following treatment may be considered for an interferon-free regimen incorporating a nonstructural protein 5A (NS5A inhibitor. Sustained virologic response (SVR with these regimens is typically >90%, but this is reduced in patients with NS5A resistance. European Association for Study of the Liver guidelines recommend simeprevir + sofosbuvir ± ribavirin (SMV+SOF±R for re-treating patients failing an NS5A inhibitor-containing regimen. An alternative strategy would be to test for NS5A resistance prior to treatment, with therapy optimized based on the results. This study investigates the cost-effectiveness of this strategy.Materials and methods: A Markov model was used to estimate disease progression for treatment-experienced genotype 1 patients with severe fibrosis or compensated cirrhosis. Targeted treatment with either SMV+SOF±R or sofosbuvir + ledipasvir ± ribavirin (SOF+LDV±R based on pretreatment NS5A resistance testing was compared to routine SOF+LDV±R without testing. Treatment duration was 12 or 24 weeks for patients with severe fibrosis or compensated cirrhosis (Metavir F3/F4. SVR data for the treatment options were based on the results of published clinical trials. The analysis was carried out from the perspective of the Italian National Health Service.Results: Optimized treatment using NS5A resistance testing yielded 0.163 additional QALYs and increased costs of €2,789 per patient versus no testing. The

  13. Frequency of Natural Resistance within NS5a Replication Complex Domain in Hepatitis C Genotypes 1a, 1b: Possible Implication of Subtype-Specific Resistance Selection in Multiple Direct Acting Antivirals Drugs Combination Treatment

    Directory of Open Access Journals (Sweden)

    Sabrina Bagaglio

    2016-03-01

    Full Text Available Different HCV subtypes may naturally harbor different resistance selection to anti-NS5a inhibitors. 2761 sequences retrieved from the Los Alamos HCV database were analyzed in the NS5a domain 1, the target of NS5a inhibitors. The NS5a resistance-associated polymorphisms (RAPs were more frequently detected in HCV G1b compared to G1a. The prevalence of polymorphisms associated with cross-resistance to compounds in clinical use (daclatasvir, DCV, ledipasvir, LDV, ombitasvir, and OMV or scheduled to come into clinical use in the near future (IDX719, elbasvir, and ELV was higher in G1b compared to G1a (37/1552 (2.4% in 1b sequences and 15/1209 (1.2% in 1a isolates, p = 0.040. Interestingly, on the basis of the genotype-specific resistance pattern, 95 (6.1% G1b sequences had L31M RAP to DCV/IDX719, while 6 sequences of G1a (0.5% harbored L31M RAP, conferring resistance to DCV/LDV/IDX719/ELV (p < 0.0001. Finally, 28 (2.3% G1a and none of G1b isolates harbored M28V RAP to OMV (p < 0.0001. In conclusion, the pattern of subtype-specific resistance selection in the naturally occurring strains may guide the treatment option in association with direct acting antivirals (DAAs targeting different regions, particularly in patients that are difficult to cure, such as those with advanced liver disease or individuals who have failed previous DAAs.

  14. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K

    2011-01-01

    To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A...... domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (¿40) or 25-aa (¿25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. ¿40 conferred efficient growth characteristics...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...

  15. Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

    Science.gov (United States)

    Dibenedetto, Silvia; Cluet, David; Stebe, Pierre-Nicolas; Baumle, Véronique; Léault, Jérémie; Terreux, Raphaël; Bickle, Marc; Chassey, Benoit D E; Mikaelian, Ivan; Colas, Pierre; Spichty, Martin; Zoli, Michele; Rudkin, Brian B

    2013-07-01

    We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two binding proteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2 and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A

  16. Selection of replicon variants resistant to ACH-806, a novel hepatitis C virus inhibitor with no cross-resistance to NS3 protease and NS5B polymerase inhibitors.

    Science.gov (United States)

    Yang, Wengang; Zhao, Yongsen; Fabrycki, Joanne; Hou, Xiaohong; Nie, Xingtie; Sanchez, Amy; Phadke, Avinash; Deshpande, Milind; Agarwal, Atul; Huang, Mingjun

    2008-06-01

    We have discovered a novel class of compounds active against hepatitis C virus (HCV), using a surrogate cellular system, HCV replicon cells. The leading compound in the series, ACH-806 (GS-9132), is a potent and specific inhibitor of HCV. The selection of resistance replicon variants against ACH-806 was performed to map the mutations conferring resistance to ACH-806 and to determine cross-resistance profiles with other classes of HCV inhibitors. Several clones emerged after the addition of ACH-806 to HCV replicon cells at frequencies and durations similar to that observed with NS3 protease inhibitors and NS5B polymerase inhibitors. Phenotypic analyses of these clones revealed that they are resistant to ACH-806 but remain sensitive to other classes of HCV inhibitors. Moreover, no significant change in the susceptibility to ACH-806 was found when the replicon cellular clones resistant to NS3 protease inhibitors and NS5B polymerase inhibitors were examined. Sequencing of the entire coding region of ACH-806-resistant replicon variants yielded several consensus mutations. Reverse genetics identified two single mutations in NS3, a cysteine-to-serine mutation at amino acid 16 and an alanine-to-valine mutation at amino acid 39, that are responsible for the resistance of the replicon variants to ACH-806. Both mutations are located at the N terminus of NS3 where extensive interactions with the central hydrophobic region of NS4A exist. These data provide evidence that ACH-806 inhibits HCV replication by a novel mechanism.

  17. A retrospective case-control study of hepatitis C virus infection and oral lichen planus in Japan: association study with mutations in the core and NS5A region of hepatitis C virus

    Directory of Open Access Journals (Sweden)

    Nagao Yumiko

    2012-04-01

    Full Text Available Abstract Background The aims of this study were to assess the prevalence of hepatitis C virus (HCV infection in Japanese patients with oral lichen planus and identify the impact of amino acid (aa substitutions in the HCV core region and IFN-sensitivity-determining region (ISDR of nonstructural protein 5A (NS5A associated with lichen planus. Methods In this retrospective study, 59 patients (group 1-A with oral lichen planus among 226 consecutive patients who visited our hospital and 85 individuals (group 1-B, controls with normal oral mucosa were investigated for the presence of liver disease and HCV infection. Risk factors for the presence of oral lichen planus were assessed by logistic regression analysis. We compared aa substitutions in the HCV core region (70 and/or 91 and ISDR of NS5A of 12 patients with oral lichen planus (group 2-A and 7 patients who did not have oral lichen planus (group 2-B among patients (high viral loads, genotype 1b who received interferon (IFN therapy in group1-A. Results The prevalence of anti-HCV and HCV RNA was 67.80% (40/59 and 59.32% (35/59, respectively, in group 1-A and 31.76% (27/85 and 16.47% (14/85, respectively, in group 1-B. The prevalence of anti-HCV (P P Conclusion We observed a high prevalence of HCV infection in patients with oral lichen planus. Longstanding HCV infection, hypoalbuminemia, and smoking were significant risk factors for the presence of oral lichen planus in patients. It is advisable for Japanese patients with lichen planus to be tested for HCV infection during medical examination.

  18. Hepatitis C Genotype Prevalence in Monastir Region, Tunisia: Correlation between 5' Untranslated Region (5'UTR), Non-structural 5B (NS5B), and Core Sequences in HCV Subtyping.

    Science.gov (United States)

    Souii, Amira; Elargoubi, Aida; Fallecker, Catherine; Mastouri, Maha; Drouet, Emmanuel

    2016-09-01

    Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. It constitutes a major public health around the world. There is no vaccine available against HCV, and current therapies are effective in only small percentage of patients. HCV has wide population-specific genotype variability. Genotype knowledge and viral load assessment are equally important for designing therapeutic strategies. Taking into account that the molecular epidemiology of HCV variants circulating in Tunisia is not yet well elucidated, and that, at present, little is known about the distribution pattern of HCV in Monastir region (Tunisia), we aimed, herein, to evaluate the prevalence of HCV genotypes in Monastir and to identify risk-related factors. For this purpose, 50 anti-HCV antibody-positive cases were diagnosed and subjected to viral RNA extraction, amplification, genotyping, and viral load quantification. Molecular epidemiology was studied by 5' untranslated region (5' UTR) sequencing as compared with the non-structural 5B (NS5B) and core region sequences. Overall concordance between 5' UTR, core, and NS5B sequencing was 100 %. The highest prevalent genotype was 1b (50 %) followed by genotypes 1a (16 %), 4a (12 %), 2a (10 %), 2c (8 %), and 3a (4 %). Interestingly, the subtype 1b had a statistically significant higher viral load than the other genotypes followed by subtype 1a. Based on these data, this study revealed a high prevalence of HCV genotype 1 (subtypes 1b and 1a) compared to other genotypes. A continued monitoring of HCV and knowledge of circulating genotypes could impact on future vaccine formulations.

  19. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) of thiazolone derivatives as hepatitis C virus NS5B polymerase allosteric inhibitors

    Science.gov (United States)

    Lei, Beilei; Du, Juan; Li, Shuyan; Liu, Huanxiang; Ren, Yueying; Yao, Xiaojun

    2008-08-01

    Three-dimensional quantitative structure-activity relationship (3D-QSAR) models for a series of thiazolone derivatives as novel inhibitors bound to the allosteric site of hepatitis C virus (HCV) NS5B polymerase were developed based on CoMFA and CoMSIA analyses. Two different conformations of the template molecule and the combinations of different CoMSIA field/fields were considered to build predictive CoMFA and CoMSIA models. The CoMFA and CoMSIA models with best predictive ability were obtained by the use of the template conformation from X-ray crystal structures. The best CoMFA and CoMSIA models gave q 2 values of 0.621 and 0.685, and r 2 values of 0.950 and 0.940, respectively for the 51 compounds in the training set. The predictive ability of the two models was also validated by using a test set of 16 compounds which gave r pred 2 values of 0.685 and 0.822, respectively. The information obtained from the CoMFA and CoMSIA 3D contour maps enables the interpretation of their structure-activity relationship and was also used to the design of several new inhibitors with improved activity.

  20. Multi-parameter optimization of aza-follow-ups to BI 207524, a thumb pocket 1 HCV NS5B polymerase inhibitor. Part 2: Impact of lipophilicity on promiscuity and in vivo toxicity.

    Science.gov (United States)

    Beaulieu, Pierre L; Bolger, Gordon; Deon, Dan; Duplessis, Martin; Fazal, Gulrez; Gagnon, Alexandre; Garneau, Michel; LaPlante, Steven; Stammers, Timothy; Kukolj, George; Duan, Jianmin

    2015-03-01

    We describe our efforts to identify analogs of thumb pocket 1 HCV NS5B inhibitor 1 (aza-analog of BI 207524) with improved plasma to liver partitioning and a predicted human half-life consistent with achieving a strong antiviral effect at a reasonable dose in HCV-infected patients. Compounds 3 and 7 were identified that met these criteria but exhibited off-target promiscuity in an in vitro pharmacology screen and in vivo toxicity in rats. High lipophilicity in this class was found to correlate with increased probability for promiscuous behavior and toxicity. The synthesis of an 8×11 matrix of analogs allowed the identification of C3, an inhibitor that displayed comparable potency to 1, improved partitioning to the liver and reduced lipophilicity. Although C3 displayed reduced propensity for in vitro off-target inhibition and the toxicity profile in rats was improved, the predicted human half-life of this compound was short, resulting in unacceptable dosing requirements to maintain a strong antiviral effect in patients.

  1. Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

    Directory of Open Access Journals (Sweden)

    Jiandong Shi

    Full Text Available Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB, CD8+ T-cell epitopes of the dengue virus (DENV NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21,-B (53, and-C (42 had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01, 515NS5525 (98.71%, A*24:02, 225NS5232 (99.35%, A*33:03, 516NS5523 (98.71%, A*33:03, and 284NS5291 (98.06%, A*33:03, were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01, 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01, 262NS5269 (92.90%, B*38:02, and 538NS5547 (90.97%, B*51:01, were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02, 514NS5524 (98.71%, C*01:02 and C*14:02, 92NS599 (98.06%, C*03:02 and C*15:02, 362NS5369 (44.84%, C*03:04 and C*08:01, 225NS5232 (99.35%, C*04:01, 234NS5241(96.77%, C*04:01, 361NS5369 (94.84%, C*04:01, 515NS5522 (98.71%, C*14:02, 515NS5524 (98.71%, C*14:02, were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02, 200NS5210 (A*11:01, 362NS5369 (C*03:04, C*08:01, and 514NS5524 (C*01:02, C*14:02 could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

  2. Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

    Science.gov (United States)

    Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Li, Jianfan; Li, Yanhan; Wang, Haixuan; Hu, Yunzhang

    2015-01-01

    Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

  3. Evaluation of Resistance-Associated Substitutions in NS5A Using Direct Sequence and Cycleave Method and Treatment Outcome with Daclatasvir and Asunaprevir for Chronic Hepatitis C Genotype 1

    Science.gov (United States)

    Ishii, Kunihide; Morita, Masaru; Morita, Yasuyo; Sugiyama, Gen; Fukushima, Hirofumi; Yano, Yoichi; Noguchi, Kazunori; Nakamura, Hiroki; Hisatomi, Junjiro; Kumemura, Hiroto; Shirachi, Miki; Iwane, Shinji; Okada, Michiaki; Honma, Yuichi; Arinaga-Hino, Teruko; Miyajima, Ichiro; Ogata, Kei; Kuwahara, Reiichiro; Amano, Keisuke; Kawaguchi, Toshihiro; Kuromatsu, Ryoko; Torimura, Takuji

    2016-01-01

    Background The aim of this study was to evaluate the efficacy of daclatasvir plus asunaprevir therapy in patients infected with hepatitis C virus and determine its relevance to resistant variants. Methods A total of 629 consecutive patients infected with hepatitis C virus genotype 1 were assessed. Daclatasvir (60 mg/day) plus asunaprevir (200 mg/day) was given for 24 weeks. The virological responses and resistance-associated substitutions of hepatitis C virus mutants were examined by the direct sequence and cycleave methods were evaluated. Results Overall, 89.4% (555/621) of patients exhibited a sustained virological response (SVR). The SVR rates in the patients with wild type, mixed, and mutant type Y93 by direct sequencing were 92.5% (520/562), 70.3% (26/37), and 42.9% (9/21), respectively. The SVR rates in the patients with 100%, 90%, 80%-30%, and 20%-0% Y93 wild by the cycleave method were 93.4% (456/488), 88.2%(30/34), 56.0%(14/25), and 36.8%(7/19), respectively. In contrast, the SVR rates for the wild type and mixed/mutant type L31 by direct sequencing were 90.2% (534/592) and 72.4% (21/29), respectively. In the multivariate analyses, the wild type Y93, no history of simeprevir therapy, the wild type L31, and low HCV RNA level were independent factors of SVR. Conclusion NS5A resistance-associated substitutions, especially Y93H, were major factors predicting the SVR. Although direct sequencing can predict the SVR rate, the cycleave method is considered to be more useful for predicting the SVR when used in combination. PMID:27684567

  4. Assessing ubiquitination of viral proteins: lessons from flavivirus NS5

    OpenAIRE

    Taylor, R. Travis; Best, Sonja M.

    2011-01-01

    Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of ...

  5. G-Structures and Wrapped NS5-Branes

    CERN Document Server

    Gauntlett, J P; Pakis, S; Waldram, D; Gauntlett, Jerome P.; Martelli, Dario; Pakis, Stathis; Waldram, Daniel

    2002-01-01

    We analyse the geometrical structure of supersymmetric solutions of type II supergravity of the form R^{1,9-n} x M_n with non-trivial NS flux and dilaton. Solutions of this type arise naturally as the near-horizon limits of wrapped NS fivebrane geometries. We concentrate on the case n=7, preserving two or four supersymmetries, corresponding to branes wrapped on associative or SLAG three-cycles. Given the existence of Killing spinors, we show that M_7 admits a G_2-structure or an SU(3)-structure, respectively, of specific type. We also prove the converse result. We use the existence of these geometric structures as a new technique to derive some known and new explicit solutions, as well as a simple theorem implying that we have vanishing NS three-form and constant dilaton whenever M_7 is compact with no boundary. The analysis extends simply to other type II examples and also to type I supergravity.

  6. Immunogenicity of HGV NS5 protein expressed from Sf9 insect cells

    Institute of Scientific and Technical Information of China (English)

    Hao Ren; Fen Lu Zhu; Shi Ying Zhu; Yan Bin Song; Zhong Tian Qi

    2001-01-01

    @@ INTRODUCTIONAlthough reliable assays for the detection ofhepatitis C virus and E virus became available, still10% 20% hepatitis are not caused byhepatitis A-E virus[1-3]. In 1996, two research groups isolatedthis agent independently and almost simultaneouslyand named hepatitis G virus and GB virus C,respectively[4-7].

  7. Characterization of forced degradation products and in silico toxicity prediction of Sofosbuvir: A novel HCV NS5B polymerase inhibitor.

    Science.gov (United States)

    Swain, Debasish; Samanthula, Gananadhamu; Bhagat, Shweta; Bharatam, P V; Akula, Venkatakrishna; Sinha, Barij N

    2016-02-20

    Sofosbuvir is a direct acting antiviral medication used to treat Hepatitis C viral infection. The present study focuses on the degradation behavior of the drug under various stress conditions (hydrolysis, oxidative, thermal and photolytic) as per International Conference on Harmonization (ICH Q1A (R2)) guidelines. A high performance liquid chromatographic system (HPLC) was used to develop a selective, precise and accurate method for separating all the degradation products. The separation was achieved on a Sunfire™ C18 (150mm×4.6mm×5μm) stationary phase with a mobile phase of 10mM ammonium acetate (pH 5.0) buffer and acetonitrile in gradient elution mode. A quadrupole-time of flight mass analyzer equipped with an electrospray ionization technique was used to propose the structural information based on the MS/MS and accurate mass measurements. Seven degradation products were identified and characterised by LC-ESI-QTOF-MS/MS. In silico toxicity of the drug and its degradation products was determined using TOPKAT and DEREK toxicity prediction softwares. The proposed method was validated as per the ICH Q2 guidelines.

  8. Mutations induced in the NS5B gene of bovine viral diarrhea virus by antiviral treatment convey resistance to the compound

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV) is a widespread bovine pathogen for which there is no specific therapeutic agent. A previous study using 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) to treat calves persistently infected with BVDV resulted in a decrease in the vira...

  9. Variabilidad clonal de la polimerasa NS5b del virus de la hepatitis C en aislados de muestras clínicas

    OpenAIRE

    Salvatierra, Karina Alejandra

    2010-01-01

    La infección crónica provocada por el virus de la hepatitis C (VHC) sigue siendo un gran problema de salud pública a nivel mundial. Se estima que 200 millones de personas (3% de la población mundial) se encuentran infectadas por este virus.El tratamiento actual basado en la combinación de dos fármacos no específicos, el interferón-α pegilado y la ribavirina; provoca numerosos efectos secundarios, el tratamiento es de larga duración y además es costoso y tiene poco éxito en pacientes infectado...

  10. Increased Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression and Steatosis During Hepatitis C Virus (HCV) Subgenome Replication: Role of Nonstructural Component-5A (NS5A) and CCAAT/Enhancer Binding Protein ß (C/EBPß)

    Science.gov (United States)

    Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated tra...

  11. Discovery of (7R)-14-cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl) amino}-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid (MK-3281), a potent and orally bioavailable finger-loop inhibitor of the hepatitis C virus NS5B polymerase.

    Science.gov (United States)

    Narjes, Frank; Crescenzi, Benedetta; Ferrara, Marco; Habermann, Jörg; Colarusso, Stefania; Ferreira, Maria del Rosario Rico; Stansfield, Ian; Mackay, Angela Claire; Conte, Immacolata; Ercolani, Caterina; Zaramella, Simone; Palumbi, Maria-Cecilia; Meuleman, Philip; Leroux-Roels, Geert; Giuliano, Claudio; Fiore, Fabrizio; Di Marco, Stefania; Baiocco, Paola; Koch, Uwe; Migliaccio, Giovanni; Altamura, Sergio; Laufer, Ralph; De Francesco, Raffaele; Rowley, Michael

    2011-01-13

    Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

  12. Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase.

    Science.gov (United States)

    Benmansour, Fatiha; Eydoux, Cécilia; Querat, Gilles; de Lamballerie, Xavier; Canard, Bruno; Alvarez, Karine; Guillemot, Jean-Claude; Barral, Karine

    2016-02-15

    Using a functional high-throughput screening (HTS) and subsequent SAR studies, we have discovered a novel series of non-nucleoside dengue viral polymerase inhibitors. We report the elaboration of SAR around hit compound 1 as well as the synthesis and antiviral evaluation of 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole and 5-phenyl-2-[2-(2-thienyl)ethenyl]-1,3,4-oxadiazole analogues derived from a rapid and easily accessible chemical pathway. A large number of compounds prepared by this method were shown to possess in vitro activity against the polymerase of dengue virus. The most potent inhibitors were tested against Dengue virus clinical isolates on infected cells model and exhibit submicromolar activity on the four dengue virus serotypes.

  13. Affinity-Based Screening Technology and HCV Drug Discovery

    Institute of Scientific and Technical Information of China (English)

    LI Bin

    2003-01-01

    @@ NS5A is one of the non-structural gene products encoded by Hepatitis C virus (HCV) and related viruses that are essential for viral replication. The amino acid sequence of NS5A is conserved between different HCV genotypes and the primary amino acid sequence of NS5A is unique to HCV and closely related viruses. Importantly, NS5A is unrelated to any human protein. This indicates that drugs designed to block the actions of NS5A could inhibit the replication of HCV without showing toxic side effects in human host cells, thus making NS5A inhibitors ideal anti-viral drugs. However, there are presently no functional assays for this essential viral protein. Therefore, conventional high throughput screening (HTS) approaches can not be used to discover antiviral drugs against NS5A.

  14. Nonstructural 5A protein of hepatitis C virus regulates heat shock protein 72 for its own propagation.

    Science.gov (United States)

    Lim, Y S; Shin, K S; Oh, S H; Kang, S M; Won, S J; Hwang, S B

    2012-05-01

    We identified heat shock protein 72 (Hsp72) as a host factor that was differentially expressed in cells expressing nonstructural 5A (NS5A) protein. To investigate how NS5A modulates Hsp72 in hepatitis C virus (HCV) life cycle, we examined the role of Hsp72 in HCV replication and virus production. NS5A specifically interacted with Hsp72. Both Hsp72 and nuclear factor of activated T cells 5 (NFAT5) levels were increased in cells expressing NS5A protein. Treatments of N-acetylcysteine and glutathione markedly reduced protein levels of both NFAT5 and Hsp72. Knockdown of NFAT5 resulted in decrease in Hsp72 level in cells expressing NS5A. Importantly, silencing of Hsp72 expression resulted in decrease in both RNA replication and virus production in HCV-infected cells. These data indicate that NS5A modulates Hsp72 via NFAT5 and reactive oxygen species activation for HCV propagation.

  15. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  16. Molecular simulations illuminate the role of regulatory components of the RNA polymerase from the hepatitis C virus in influencing protein structure and dynamics.

    Science.gov (United States)

    Davis, Brittny C; Thorpe, Ian F

    2013-07-02

    The RNA polymerase (gene product NS5B) from the hepatitis C virus is responsible for replication of the viral genome and is a validated drug target for new therapeutic agents. NS5B has a structure resembling an open right hand (containing the fingers, palm, and thumb subdomains), a hydrophobic C-terminal region, and two magnesium ions coordinated in the palm domain. Biochemical data suggest that the magnesium ions provide structural stability and are directly involved in catalysis, while the C-terminus plays a regulatory role in NS5B function. Nevertheless, the molecular mechanisms by which these two features regulate polymerase activity remain unclear. To answer this question, we performed molecular dynamics simulations of NS5B variants with different C-terminal lengths in the presence or absence of magnesium ions to determine the impact on enzyme properties. We observed that metal binding increases both the magnitude and the degree of correlated enzyme motions. In contrast, we observed that the C-terminus restricts enzyme dynamics. Under certain conditions, our simulations revealed a fully closed conformation of NS5B that may facilitate de novo initiation of RNA replication. This knowledge is important because it fosters the development of a comprehensive description of RNA replication by NS5B and is relevant to understanding the functional properties of a broad class of related RNA polymerases such as 3D-pol from poliovirus. Ultimately, this information may also be pertinent to designing novel NS5B therapeutics.

  17. Hepatitis C virus nonstructural protein 5B is involved in virus morphogenesis.

    Science.gov (United States)

    Gouklani, Hamed; Bull, Rowena A; Beyer, Claudia; Coulibaly, Fasséli; Gowans, Eric J; Drummer, Heidi E; Netter, Hans J; White, Peter A; Haqshenas, Gholamreza

    2012-05-01

    The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.

  18. De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.

    Directory of Open Access Journals (Sweden)

    Pilar Clemente-Casares

    Full Text Available Hepatitis C virus (HCV shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V(max/K(m ratios were statistically different between genotype 1 and the other genotypes (p<0.001, mainly due to differences in V(max values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.

  19. Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

    Energy Technology Data Exchange (ETDEWEB)

    Khunchai, Sasiprapa [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Junking, Mutita [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Suttitheptumrong, Aroonroong; Yasamut, Umpa [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Sawasdee, Nunghathai [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Morchang, Atthapan [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Chaowalit, Prapaipit [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); and others

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer For the first time how DENV NS5 increases RANTES production. Black-Right-Pointing-Pointer DENV NS5 physically interacts with human Daxx. Black-Right-Pointing-Pointer Nuclear localization of NS5 is required for Daxx interaction and RANTES production. -- Abstract: Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.

  20. Investigation of the mode of binding of a novel series of N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamides to the hepatitis C virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Gentles, Robert G.; Sheriff, Steven; Beno, Brett R.; Wan, Changhong; Kish, Kevin; Ding, Min; Zheng, Xiaofan; Chupak, Louis; Poss, Michael A.; Witmer, Mark R.; Morin, Paul; Wang, Ying-Kai; Rigat, Karen; Lemm, Julie; Voss, Stacey; Liu, Mengping; Pelosi, Lenore; Roberts, Susan B.; Gao, Min; Kadow, John F. (BMS)

    2013-11-20

    Structure based rationales for the activities of potent N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamide inhibitors of the hepatitis C viral polymerase are described herein. These compounds bind to the hepatitis C virus non-structural protein 5B (NS5B), and co-crystal structures of select examples from this series with NS5B are reported. Comparison of co-crystal structures of a potent analog with both NS5B genotype 1a and genotype 1b provides a possible explanation for the genotype-selectivity observed with this compound class and suggests opportunities for the further optimization of the series.

  1. Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte

    2015-01-01

    efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a...

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

    Science.gov (United States)

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

    2016-03-01

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

  3. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

    Directory of Open Access Journals (Sweden)

    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  4. Inhibition of hepatitis C virus protein expression by RNA interference.

    Science.gov (United States)

    Sen, Adrish; Steele, Robert; Ghosh, Asish K; Basu, Arnab; Ray, Ranjit; Ray, Ratna B

    2003-10-01

    Hepatitis C virus (HCV) is a serious human pathogen and an estimated 170 million people are infected worldwide. Current therapeutic regimens have shown limited efficacy against selected genotypes of the virus. The phenomenon of RNA interference can be used to selectively block homologous genes post-transcriptionally, and has revolutionized approaches to study gene function. In this report, we have demonstrated that small interfering RNAs (siRNAs) targeted against NS5A of HCV genotype 1a specifically inhibit NS5A RNA and protein expression in a human hepatoma (HepG2) cell line. Expression of endogenous alpha-actin and the ds-RNA activated serine/threonine kinase-PKR were unaltered, demonstrating that the inhibitory effect observed from siRNA was specific to the HCV NS5A protein. We next examined whether siRNA directed against NS5A could inhibit core protein expression, the first gene product synthesized in virus infected cells due to its localization at the 5' end of the HCV polyprotein. For this purpose, a full-length cDNA clone from HCV (H77, genotype 1a) was used, and results indicated that the introduction of NS5A targeted siRNA resulted in an inhibition of NS5A and core protein expression. Moreover, we observed that this siRNA effectively inhibited NS5A mediated activation of the IL-8 promoter. Taken together, our results demonstrated that siRNA was effective in inhibiting HCV protein expression, and may have therapeutic potential to limit HCV replication in chronically infected patients.

  5. Complex Pattern of Resistance-Associated Substitutions of Hepatitis C Virus after Daclatasvir/Asunaprevir Treatment Failure

    Science.gov (United States)

    Hasebe, Chitomi; Osaki, Yukio; Joko, Kouji; Yagisawa, Hitoshi; Sakita, Shinya; Okushin, Hiroaki; Satou, Takashi; Hisai, Hiroyuki; Abe, Takehiko; Tsuji, Keiji; Tamada, Takashi; Kobashi, Haruhiko; Mitsuda, Akeri; Ide, Yasushi; Ogawa, Chikara; Tsuruta, Syotaro; Takaguchi, Kouichi; Murakawa, Miyako; Asahina, Yasuhiro; Enomoto, Nobuyuki; Izumi, Namiki

    2016-01-01

    Backgrounds & Aims We aimed to clarify the characteristics of resistance-associated substitutions (RASs) after treatment failure with NS5A inhibitor, daclatasvir (DCV) in combination with NS3/4A inhibitor, asunaprevir (ASV), in patients with chronic hepatitis C virus genotype 1b infection. Methods This is a nationwide multicenter study conducted by the Japanese Red Cross Liver Study Group. The sera were obtained from 68 patients with virological failure after 24 weeks of DCV/ASV treatment. RASs in NS5A and NS3 were determined by population sequencing. Results The frequency of signature RASs at position D168 of NS3 was 68%, and at positions L31 and Y93 of NS5A was 79 and 76%, respectively. The frequency of dual signature RASs in NS5A (L31-RAS and Y93-RAS) was 63%. RASs at L28, R30, P32, Q54, P58, and A92 in addition to dual signature RAS were detected in 5, 5, 1, 22, 2, and 0 patients, respectively. In total, triple, quadruple, and quintuple RASs in combination with dual signature RAS were detected in 35, 10, and 1.5% patients, respectively. These RASs were detected in patients without baseline RASs or who prematurely discontinued therapy. Co-existence of D168 RAS in NS3 and L31 and/or Y93 RAS in NS5A was observed in 62% of patients. Conclusion Treatment-emergent RASs after failure with DCV/ASV combination therapy are highly complex in more than 50% of the patients. The identification of complex RAS patterns, which may indicate high levels of resistance to NS5A inhibitors, highlights the need for RAS sequencing when considering re-treatment with regimens including NS5A inhibitors. PMID:27776192

  6. Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization.

    Science.gov (United States)

    Valero, María Llanos; Sabariegos, Rosario; Cimas, Francisco J; Perales, Celia; Domingo, Esteban; Sánchez-Prieto, Ricardo; Mas, Antonio

    2016-06-01

    Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options.

  7. Heat shock protein 70 is associated with replicase complex of Japanese encephalitis virus and positively regulates viral genome replication.

    Directory of Open Access Journals (Sweden)

    Jing Ye

    Full Text Available Japanese encephalitis virus (JEV is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. The NS5 protein of JEV is a key component of the viral replicase complex, which plays a crucial role in viral pathogenesis. In this study, tandem affinity purification (TAP followed by mass spectrometry analysis was performed to identify novel host proteins that interact with NS5. Heat shock protein 70 (Hsp70, eukaryotic elongation factor 1-alpha (eEF-1α and ras-related nuclear protein (Ran were demonstrated to interact with NS5. In addition to NS5, Hsp70 was also found to interact with NS3 which is another important member of the replicase complex. It was observed that the cytoplasmic Hsp70 partially colocalizes with the components of viral replicase complex including NS3, NS5 and viral dsRNA during JEV infection. Knockdown of Hsp70 resulted in a significantly reduced JEV genome replication. Further analysis reveals that Hsp70 enhances the stability of viral proteins in JEV replicase complex. These results suggest an important role for Hsp70 in regulating JEV replication, which provides a potential target for the development of anti-JEV therapies.

  8. Flaviviral Replication Complex: Coordination between RNA Synthesis and 5'-RNA Capping.

    Science.gov (United States)

    Klema, Valerie J; Padmanabhan, Radhakrishnan; Choi, Kyung H

    2015-08-13

    Genome replication in flavivirus requires (-) strand RNA synthesis, (+) strand RNA synthesis, and 51-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (-) RNA synthesis, (+) RNA synthesis, and 51-RNA capping and methylation.

  9. Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir

    DEFF Research Database (Denmark)

    Ramirez Almeida, Santseharay; Mikkelsen, Lotte S.; Gottwein, Judith M.

    2016-01-01

    barrier to resistance for sofosbuvir, whereas resistance to this DAA can be induced in genotype 3a. We therefore isolated HCV genotype 3a variants with reduced sensitivity to sofosbuvir, with increased fitness and with cross-resistance to other NS5B inhibitors. These findings indicate that sofosbuvir......Background & Aims Direct-acting antivirals (DAAs) effectively eradicate chronic hepatitis C virus (HCV) infection, although HCV genotype 3a is less responsive to these drugs. We aimed to develop genotype 3a infectious cultures and study the effects of inhibitors of NS5A and NS5B and resistance...... with increasing concentrations of sofosbuvir was used to promote selection of HCV-resistant variants. Results We engineered a DBN3a variant with 17 substitutions (DBN3acc) that had replication and propagation kinetics in Huh7.5 cells comparable with prototype J6/JFH1. The adaptive mutations also produced culture...

  10. Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver

    Science.gov (United States)

    Okushin, Kazuya; Enooku, Kenichiro; Fujinaga, Hidetaka; Moriya, Kyoji; Yotsuyanagi, Hiroshi; Aizaki, Hideki; Suzuki, Tetsuro; Matsuura, Yoshiharu; Koike, Kazuhiko

    2017-01-01

    The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS) in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2’-5’ oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity. PMID:28107512

  11. Direct-acting Antiviral Agents Resistance-associated Polymorphisms in Chinese Treatment-na(i)ve Patients Infected with Genotype 1b Hepatitis C Virus

    Institute of Scientific and Technical Information of China (English)

    Ye Wang; Hui-Ying Rao; Xing-Wang Xie; Lai Wei

    2015-01-01

    Background:It has been reported that several baseline polymorphisms of direct-acting antivirals (DAAs) agents resistance-associated variants (RAVs) would affect the treatment outcomes of patients chronically infected with hepatitis C virus (CHC).The aim of this study is to investigate the prevalence of DAAs RAVs in treatment-na(i)ve GT1b CHC patients.Methods:Direct sequencing and ultra-deep sequencing of the HCV NS3,NS5A,and NS5B gene were performed in baseline serum samples of treatment-ha(i)ve patients infected with genotype lb hepatitis C virus (HCVs).Results:One hundred and sixty CHC patients were studied.Complete sequence information was obtained for 145 patients (NS3),148 patients (NS5A),and 137 patients (NS5B).Treatment-failure associated variants of DAAs were detected:56.6% (82/145) of the patients presented S122G for simeprevir (NS3 protease inhibitor);10.1% (14/148) of the patients presented Y93H for daclatasvir and ledipasvir (NS5A protein inhibitors);94.2% (129/137) of the patients presented C316N for sofosbuvir (NS5B polymerase inhibitor).Nearly,all of the DAAs RAVs detected by ultra-deep sequencing could be detected by direct sequencing.Conclusions:The majority of genotype lb CHC patients in China present a virus population carrying HCV DAAs RAVs.Pretreatment sequencing of HCV genome might need to be performed when patients infected with GTlb HCV receiving DAAs-containing regimens in China.Population sequencing would be quite quantified for the work.

  12. Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

    Directory of Open Access Journals (Sweden)

    Eliana F Castro

    Full Text Available Bovine viral diarrhea virus (BVDV is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC is a non-nucleoside polymerase inhibitor (NNI of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5 present an N264D mutation in the NS5B gene (RdRp whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5 remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

  13. Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

    OpenAIRE

    Park, Chorong; Min, Saehong; Park, Eun-Mee; Lim, Yun-Sook; Kang, Sangmin; Suzuki, Tetsuro; Shin, Eui-Cheol; Hwang, Soon B.

    2015-01-01

    The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of th...

  14. Hagedorn Behavior of Little String Theories

    CERN Document Server

    Harmark, T

    2000-01-01

    We examine the Hagedorn behavior of little string theory using its conjectured duality with near-horizon NS5-branes. In particular, by studying the string-corrected NS5-brane supergravity solution, it is shown that tree-level corrections to the temperature vanish, while the leading one-loop string correction generates the correct temperature dependence of the entropy near the Hagedorn temperature. Finally, the Hagedorn behavior of ODp-brane theories, which are deformed versions of little string theory, is considered via their supergravity duals.

  15. Daclatasvir with sofosbuvir and ribavirin for hepatitis C virus infection with advanced cirrhosis or post‐liver transplantation recurrence

    OpenAIRE

    Poordad, Fred; Schiff, Eugene R.; Vierling, John M.; Landis, Charles,A.; Fontana, Robert J.; Yang, Rong; McPhee, Fiona; Hughes, Eric A.; Noviello, Stephanie; Swenson, Eugene S.

    2016-01-01

    Chronic hepatitis C virus (HCV) infection with advanced cirrhosis or post‐liver transplantation recurrence represents a high unmet medical need with no approved therapies effective across all HCV genotypes. The open‐label ALLY‐1 study assessed the safety and efficacy of a 60‐mg once‐daily dosage of daclatasvir (pan‐genotypic NS5A inhibitor) in combination with sofosbuvir at 400 mg once daily (NS5B inhibitor) and ribavirin at 600 mg/day for 12 weeks with a 24‐week follow‐up in two cohorts of p...

  16. Supergravity and Light-Like Non-commutativity

    CERN Document Server

    Alishahiha, M; Russo, Jorge G; Alishahiha, Mohsen; Oz, Yaron; Russo, Jorge G.

    2000-01-01

    We construct dual supergravity descriptions of field theories and little string theories with light-like non-commutativity. The field theories are realized on the world-volume of Dp branes with light-like NS $B$ field and M5 branes with light-like $C$ field. The little string theories are realized on the world-volume of NS5 branes with light-like RR $A$ fields. The supergravity backgrounds are closely related to the $A=0,B=0,C=0$ backgrounds. We discuss the implications of these results. We also construct dual supergravity descriptions of ODp theories realized on the worldvolume of NS5 branes with RR backgrounds.

  17. Structural modeling and analysis of dengue-mediated inhibition of interferon signaling pathway.

    NARCIS (Netherlands)

    Aslam, B; Ahmad, J; Ali, a; Paracha, R Z; Tareen, S H K; Khusro, S; Ahmad, T; Muhammad, S a; Niazi6 And V Azevedo, U

    2015-01-01

    Dengue virus (DENV) belongs to the family Flaviviridae and can cause major health problems worldwide, including dengue fever and dengue shock syndrome. DENV replicon in human cells inhibits interferon alpha and beta with the help of its non-structural proteins. Non-structural protein 5 (NS5) of DENV

  18. Metastable Supersymmetry Breaking Vacua on Abelian Brane Models

    CERN Document Server

    Halyo, Edi

    2009-01-01

    We construct Abelian brane models with metastable vacua which are obtained from deformations of ${\\cal N}=2$ supersymmetric brane configurations. One such model lives on a D4 brane stretched between two displaced and rotated NS5 branes. Another one lives on a D5 brane wrapped on a deformed and fibered $A_2$ singularity.

  19. Soil Compaction Investigation. Report No. 3: Compaction Studies on Sand Subgrade

    Science.gov (United States)

    1949-10-01

    TRACKING After Com.J2actlon ~Dr~) After Com:12action ~Wet) After Grading Prior to Com11action Water Dry Water Dry Water Dry Compaction Number of...Water Dry Water Dry Depth Content Density , Depth Content Density , Ft ; Lb/CuFt Cam;paction Ft ! Lb/CuFt Compaction Before SoeJ.d.ns 5-Min Soaking

  20. Efficacy and Safety of Daclatasvir in Hepatitis C: An Overview.

    Science.gov (United States)

    Gamal, Nesrine; Gitto, Stefano; Andreone, Pietro

    2016-12-28

    Hepatitis C virus (HCV) infection is a growing public health concern, with 184 million people infected worldwide. During the past decade, interferon has been the backbone of HCV treatment, even though it remains far from ideal. The latest development of the new direct antivirals has drastically changed the treatment approach for chronic hepatitis C (CHC). Inhibitors of the HCV NS5A region have garnered remarkable interest among treating physicians, due to their high potency and favourable safety profile. In particular, treatment with daclatasvir (DCV) has yielded high rates of vriologic response in patients infected with genotype (Gt) 1 and Gt 3, when used in combination with other antivirals of a different class, such as sofosbuvir. Although few data are available for DCV treatment of the other Gts, the results in patients with Gt 2 and Gt 4 infection appear promising, as do those for unique patient populations. NS5A-resistant viral variants can pre-exist or emerge after treatment failure for the HCV NS5A inhibitors. Nonetheless, DCV-resistant viral variants continue to be sensitive to interferon and other classes of antivirals such as NS3/4A and NS5B inhibitors. Herein, we aimed to provide an overview of the current knowledge about DCV in the treatment of CHC.

  1. Retreatment efficacy and predictors of ledipasvir plus sofosbuvir to HCV genotype 1 in Japan.

    Science.gov (United States)

    Akuta, Norio; Sezaki, Hitomi; Suzuki, Fumitaka; Fujiyama, Shunichiro; Kawamura, Yusuke; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Saitoh, Satoshi; Suzuki, Yoshiyuki; Arase, Yasuji; Ikeda, Kenji; Kumada, Hiromitsu

    2017-02-01

    Predictors of treatment efficacy with ledipasvir plus sofosbuvir as direct-acting antiviral (DAA) regimen for HCV infection are still unclear. Retreatment efficacy of ledipasvir plus sofosbuvir for failures to prior DAA regimens, including NS5A inhibitors, are also unknown because resistance-associated variants (RAVs) in NS5A have been shown to persist up to the long-term of post-treatment. One hundred seventy-five patients with chronic HCV genotype 1 infection, without decompensated liver cirrhosis and hepatocellular carcinoma, were evaluated SVR12 by ledipasvir 90 mg plus sofosbuvir 400 mg once-daily for 12 weeks. Overall, SVR12 were 92%, based on intention to treat analysis. In failures to daclatasvir plus asunaprevir, SVR12 were 71%. The study using ultra-deep sequencing showed that ledipasvir plus sofosbuvir was effective to one case of failures to daclatasvir plus asunaprevir with multidrug RAVs (triple mutation in NS3-D168/NS5A-L31/NS5A-Y93). Multivariate analysis identified FIB4 index (plus sofosbuvir for HCV genotype 1 infection, including multidrug RAVs in Japan. Treatment efficacy could be predicted by the combination of viral and host factors. J. Med. Virol. 89:284-290, 2017. © 2016 Wiley Periodicals, Inc.

  2. Kaluza-Klein monopole and 5-brane effective actions

    NARCIS (Netherlands)

    Eyras, E; Lozano, Y

    2000-01-01

    We review the construction of the Kaluza-Klein monopole of the Type IIA theory in the most general case of a massive background, as well as its relation via T-duality with the Type IIB NS-5-brane. This last effective action is shown to be related by S-duality to the D5-brane effective action. [GRAPH

  3. Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

    Institute of Scientific and Technical Information of China (English)

    Ning-Shao Xia; Hai-Jie Yang; Jun Zhang; Chang-Qing Lin; Ying-Bin Wang; Juan Wang; Mei-YunZhan; MH Ng

    2001-01-01

    AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.``

  4. POTENSI PROBIOTIK BAKTERI ASAM LAKTAT ASAL BEKASAM IKAN NILA [Probiotic Potential of Bekasam Lactic Acid Bacteria of Tilapia Fish

    Directory of Open Access Journals (Sweden)

    Astri Nurnaafi

    2015-07-01

    Full Text Available Bekasam is well known in Indonesia as one of fermented fish product. Several fermented products generate lactic acid bacteria (LAB which has probiotic potential with beneficial effects on human health. However, In Indonesia, the research on LAB isolated from fermented fish product, including bekasam, is still rarely conducted. The aim of this study was to evaluate the probiotic potential of LAB isolated from bekasam. Two LAB isolates namely NS(5 and NS(6 were selected based on their resistance to gastric pH (pH 2.0, intestinal pH (pH 7.2 and bile salts (0.5% oxgal. Pathogenic test, antimicrobial activity test, characterization and identification of the isolats were also performed respectively. The result showed that NS(5 isolate survived at pH 2.0, pH 7.2 and bile salts (oxgal. It was obtained that NS(5 isolate was non pathogenic bacteria which exhibited antimicrobial activity against Salmonella Typhimurium ATCC 14028 and Escherichia coli. The characterization result showed that NS(5 isolate was Gram-positive bacteria, rod-shaped, non-endospore producer, negative catalase, homofermentative, non motile, having an amilolitik as well as lipolitik activity and able to grow at 30-37°C, NaCl 2-7% dan pH 4.4-9.6. Isolate NS(5 isolate was then identified as Lactobacillus plantarum 1 strain with 99.9% of similarity. Meanwhile, NS(6 isolate was not able to survive in the medium containing bile salts (oxgal, therefore it was not categorized as a probiotic candidate.

  5. Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

    Directory of Open Access Journals (Sweden)

    Tsubasa Munakata

    2007-09-01

    Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

  6. Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture.

    Science.gov (United States)

    Yu, Mei; Peng, Betty; Chan, Katie; Gong, Ruoyu; Yang, Huiling; Delaney, William; Cheng, Guofeng

    2014-05-01

    Genotype 6 (GT6) hepatitis C virus (HCV) is prevalent in Southeast Asia and southern China, where it can constitute up to 50% of HCV infections. Despite this, no direct-acting antivirals are approved to treat GT6 HCV infection, and no cell culture systems have been described. In this study, we aimed to develop a GT6 HCV subgenomic replicon to facilitate the identification and development of new HCV therapies with pan-genotype activity. A subgenomic replicon cDNA encoding a GT6a consensus sequence plus an NS5A amino acid substitution (S232I) was synthesized. Electroporation of RNA encoding the GT6a replicon into Huh-7-derived cells consistently yielded 20 to 100 stable replicon colonies. Genotypic analyses of individual replicon colonies revealed new adaptive mutations across multiple viral nonstructural proteins. The E30V and K272R mutations in NS3 and the K34R mutation in NS4A were observed most frequently and were confirmed to enhance GT6a replicon replication in the presence of the NS5A amino acid substitution S232I. These new adaptive mutations allowed establishment of robust luciferase-encoding GT6a replicons for reproducible quantification of HCV replication, and the luciferase-encoding replicons enabled efficient determinations of antiviral activity for HCV inhibitors in a 384-well assay format. While nucleoside/nucleotide NS5B inhibitors and cyclophilin A inhibitors had similar antiviral activities against both GT6a and GT1b replicons, some nonnucleoside NS5B inhibitors, NS3 protease inhibitors, and NS5A inhibitors had less antiviral activity against GT6a replicons. In conjunction with other genotype replicons, this robust GT6a replicon system will aid in the development of pan-genotypic HCV regimens.

  7. Cellular immune responses and occult infection in seronegative heterosexual partners of chronic hepatitis C patients.

    Science.gov (United States)

    Roque-Cuéllar, M C; Sánchez, B; García-Lozano, J R; Praena-Fernández, J M; Núñez-Roldán, A; Aguilar-Reina, J

    2011-10-01

    It is unknown whether hepatitis C virus (HCV)-specific cellular immune responses can develop in seronegative sexual partners of chronically HCV-infected patients and whether they have occult infection. Thirty-one heterosexual partners of patients with chronic HCV were studied, fifteen of them with HCV transmission risks. Ten healthy individuals and 17 anti-HCV seropositive patients, without viremia, were used as controls. Virus-specific CD4+ and CD8+ T-cell responses were measured by flow cytometry against six HCV peptides, situated within the nonstructural (NS) proteins NS3, NS4 and NS5, through intracellular detection of gamma interferon (IFN-γ) or interleukin 4 (IL-4) production and CD69 expression. Sexual partners had a higher production of IFN-γ and IL-4 by CD4+ cells against NS3-p124 (P = 0.003), NS5b-p257 (P = 0.005) and NS5b-p294 (P = 0.012), and CD8+ cells against NS3-p124 (P = 0.002), NS4b-p177 (P = 0.001) and NS3-p294 (P = 0.004) as compared with healthy controls. We observed elevated IFN-γ production by CD4+ T cells against NS5b-p257 (P = 0.042) and NS5b-p294 (P = 0.009) in the sexual partners with HCV transmission risks (sexual, professional and familial altogether) than in those without risks. RNA was extracted from peripheral blood mononuclear cells (PBMC), and detection of HCV-RNA positive and replicative (negative) strands was performed by strand-specific real-time PCR. In four sexual partners, the presence of positive and negative HCV- RNA strands in PBMC was confirmed. Hence, we found an HCV-specific cellular immune response as well as occult HCV infection in seronegative and aviremic sexual partners of chronically HCV-infected patients.

  8. Dicty_cDB: Contig-U15693-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ixed Tissue and... 111 1e-19 1 ( AZ917266 ) 4911.fd64h15.x1 Saccharomyces bayanus MCYC 623-6C... 100 3e-19 2 ( EF650282 ) Diogm...EF650282_1( EF650282 |pid:none) Diogmites grossus alanyl-tRNA synt... 273 1e-71 (B1IJG7) RecName: Full=Alany

  9. Characterization of the Anti-HCV Activities of the New Cyclophilin Inhibitor STG-175

    Science.gov (United States)

    Gallay, Philippe A.; Chatterji, Udayan; Bobardt, Michael D.; Long, Zhengyu; Zhang, Shengli; Su, Zhuang

    2016-01-01

    Shortened current direct-acting antiviral (DAA) therapies while less expensive, have not provided satisfactory efficacy in naïve cirrhotics, treatment experienced non-cirrhotics or even genotype-3 (GT3)-infected patients. Since DAA regimens consist of the same classes of inhibitors—NS5A (NS5Ai) and NS5B (NS5Bi) +/- NS3 (NS3i) inhibitors—it is likely that their costs will be high and will provide similar degrees of protection. Integrating drugs with distinct mechanisms of action (MoA) into DAA regimens could provide the solution for shortening the period of treatment. One such class of agents is the cyclophilin inhibitors (CypI), which has shown efficacy in patients. Resistance-associated variants persist for years post-treatment in patients exposed to NS5Ai or NS5Bi who fail to achieve a sustained virologic response, impairing their chance for cure on retreatment with existing DAA combinations. Because of their high barrier to resistance, CypI may be particularly useful as a rescue therapy for patients who have relapsed with DAA resistance-associated variants. In this study, we analyzed the anti-HCV properties of the novel cyclosporine A (CsA) derivate—STG-175. The non-immunosuppressive STG-175 possesses a high (EC50 11.5–38.9 nM) multi-genotypic (GT1a to 4a) anti-HCV activity. STG-175 clears cells from HCV since no viral replication rebound was observed after cessation of drug treatment. It presents a higher barrier to resistance than other CypI or selected DAAs. HCV variants, which emerged under STG-175 pressure, are only ~2-fold resistant to the drug. No cross-resistance was observed with DAAs STG-175 was efficacious against DAA-resistant HCV variants. Drug combination studies revealed that STG-175 provides additive and synergistic effects against GT1a to 4a. STG-175 inhibits the infection of HCV, HIV-1 and HBV in mono-, dual- and triple-infection settings. Altogether these results suggest that the new CypI STG-175 represents an attractive drug partner

  10. Similar prevalence of low-abundance drug-resistant variants in treatment-naive patients with genotype 1a and 1b hepatitis C virus infections as determined by ultradeep pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Severine Margeridon-Thermet

    Full Text Available Hepatitis C virus (HCV variants that confer resistance to direct-acting-antiviral agents (DAA have been detected by standard sequencing technology in genotype (G 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.A total of 3.5 million high-quality reads of ≥ 200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region. The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.

  11. Vectores recombinantes basados en el virus modificado de Ankara (MVA) com vacunas preventivas y terapeúticas contra la hepatitis C

    OpenAIRE

    Esteban, Mariano; Gómez, Carmen E.; Perdiguero, Beatriz

    2014-01-01

    [ES] Los virus recombinantes de la invención contienen secuencias que se encuentran insertadas en el mismo sitio de inserción del MVA y que permiten la expresión simultáneamente de varios antígenos del VHC, concretamente las proteínas maduras estructurales (Core, E1, E2 y p7) y no estructurales (NS2, NS3, NS4A, NS4B, NS5A más los 201 aminoácidos de la región N-terminal de NS5B). Con ello se consiguen virus recombinantes estables, que permiten el desencadenamiento de una respuesta inmune contr...

  12. Efficient cell culture system for hepatitis C virus genotype 7a

    DEFF Research Database (Denmark)

    2013-01-01

    by the corresponding genes of the genotype 7a strain QC69 and characterized them in Huh7.5 cells. Sequence analysis of 7a/JFH1 recombinants recovered after viral passage in Huh7.5 cells following 4 independent transfection experiments revealed adaptive mutations in Core, E2, NS2, NS5A and NS5B. In reverse genetic...... studies the importance of these mutations for improved growth kinetics was shown. Adapted 7a/JFH1 viruses showed growth kinetics, infectivity and RNA titers comparable to a previously developed 3a/JFH1 reference virus. Conclusion: The developed 7a/JFH1 viruses provide a robust in vitro tool for research...

  13. New direct-acting antivirals in hepatitis C therapy: a review of sofosbuvir, ledipasvir, daclatasvir, simeprevir, paritaprevir, ombitasvir and dasabuvir.

    Science.gov (United States)

    McConachie, Sean M; Wilhelm, Sheila M; Kale-Pradhan, Pramodini B

    2016-01-01

    Hepatitis C is a chronic infection associated with considerable morbidity and mortality. In recent years, there has been a shift in treatment paradigm with the discovery and approval of agents that target specific proteins vital for hepatitis C replication. The NS3/4A inhibitors simeprevir and paritaprevir, the NS5A inhibitors ombitasvir, ledipasvir, and daclatasvir, and the NS5B inhibitors sofosbuvir and dasabuvir have been newly FDA approved and incorporated as first-line agents into the latest IDSA-AASLD guidelines for Hepatitis C treatment. Used in combination, these agents produce higher rates of sustained virologic response and less adverse effects than historical options, along with limited rates of resistance. Pertinent clinical data, pharmacology, and pharmacokinetics are reviewed for these new direct acting antiviral agents.

  14. Three-dimensional N=4 Linear Quivers and non-Abelian T-duals

    CERN Document Server

    Lozano, Yolanda; Montero, Jesus; Nunez, Carlos

    2016-01-01

    In this paper we construct a new Type IIB background with an $AdS_4$ factor that preserves ${\\cal N}=4$ Supersymmetry. This solution is obtained using a non-Abelian T-duality transformation on the Type IIA reduction of the $AdS_4\\times S^7$ background. We interpret our configuration as a patch of a more general background with localised sources, dual to the renormalisation fixed point of a $T_{\\rho}^{\\hat{\\rho}} (SU(N))$ quiver field theory. This relates explicitly the $AdS_4$ geometry to a D3-D5-NS5 brane intersection, illuminating what seems to be a more general phenomenon, relating $AdS_{p+1}$ backgrounds generated by non-Abelian T-duality to Dp-D(p+2)-NS5 branes intersections.

  15. Little strings and T-duality

    CERN Document Server

    Kim, Jungmin; Lee, Kimyeong

    2015-01-01

    We study the 2d N = 4 gauge theory descriptions of little strings on type II NS5- branes. The IIB strings on N NS5-branes are described by the N = (4,4) gauge theories, whose Higgs branch CFTs on U(N) instanton moduli spaces are relevant. The IIA strings are described by N = (4,4) circular A_{N-1} quiver theories, whose Coulomb branch CFTs are relevant. We study new N = (0,4) quiver gauge theories for the IIA strings, which make it easier to study some infrared observables. In particular, we show that the elliptic genera of the IIA / IIB strings precisely map to each other by T-duality.

  16. Divergent antiviral effects of bioflavonoids on the hepatitis C virus life cycle

    Energy Technology Data Exchange (ETDEWEB)

    Khachatoorian, Ronik, E-mail: RnKhch@ucla.edu [Molecular Biology Interdepartmental Ph.D. Program (MBIDP), Molecular Biology Institute, David Geffen School of Medicine at University of California, Los Angeles, California, CA (United States); Molecular Biology Institute, David Geffen School of Medicine at University of California, Los Angeles, California, CA (United States); Arumugaswami, Vaithilingaraja, E-mail: VArumugaswami@mednet.ucla.edu [Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California, Los Angeles, California, CA (United States); Department of Surgery, Regenerative Medicine Institute at Cedars-Sinai Medical Center, Los Angeles, California, CA (United States); Raychaudhuri, Santanu, E-mail: SRaychau@ucla.edu [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles, California, CA (United States); Yeh, George K., E-mail: GgYeh@ucla.edu [Molecular Biology Interdepartmental Ph.D. Program (MBIDP), Molecular Biology Institute, David Geffen School of Medicine at University of California, Los Angeles, California, CA (United States); Molecular Biology Institute, David Geffen School of Medicine at University of California, Los Angeles, California, CA (United States); Maloney, Eden M., E-mail: EMaloney@ucla.edu [Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California, CA (United States); Wang, Julie, E-mail: JulieW1521@ucla.edu [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles, California, CA (United States); and others

    2012-11-25

    We have previously demonstrated that quercetin, a bioflavonoid, blocks hepatitis C virus (HCV) proliferation by inhibiting NS5A-driven internal ribosomal entry site (IRES)-mediated translation of the viral genome. Here, we investigate the mechanisms of antiviral activity of quercetin and six additional bioflavonoids. We demonstrate that catechin, naringenin, and quercetin possess significant antiviral activity, with no associated cytotoxicity. Infectious virion secretion was not significantly altered by these bioflavonoids. Catechin and naringenin demonstrated stronger inhibition of infectious virion assembly compared to quercetin. Quercetin markedly blocked viral translation whereas catechin and naringenin demonstrated mild activity. Similarly quercetin completely blocked NS5A-augmented IRES-mediated translation in an IRES reporter assay, whereas catechin and naringenin had only a mild effect. Moreover, quercetin differentially inhibited HSP70 induction compared to catechin and naringenin. Thus, the antiviral activity of these bioflavonoids is mediated through different mechanisms. Therefore combination of these bioflavonoids may act synergistically against HCV.

  17. Dynamic evolution of hepatitis C virus resistance-associated substitutions in the absence of antiviral treatment

    Science.gov (United States)

    Eltahla, Auda A.; Leung, Preston; Pirozyan, Mehdi R.; Rodrigo, Chaturaka; Grebely, Jason; Applegate, Tanya; Maher, Lisa; Luciani, Fabio; Lloyd, Andrew R.; Bull, Rowena A.

    2017-01-01

    Resistance against new hepatitis C virus (HCV) antivirals is an area of increasing interest. Resistance-associated substitutions (RASs) have been identified in treatment-naïve individuals, but pressures driving treatment-independent RAS emergence are poorly understood. We analysed the longitudinal evolution of RASs in twelve participants with early acute HCV infections. Full-genome deep sequences were analysed for changes in RAS frequency within NS3, NS5A and NS5B-coding regions over the course of the infection. Emergence of RASs relevant only to the polymerase non-nucleoside inhibitors (NNI) was detected, and these lay within CD8+ T-cell epitopes. Conversely, the loss of NNI RASs over time appeared likely to be driven by viral fitness constraints. These results highlight the importance of monitoring CD8+ T cell epitope-associated RASs in populations with dominant HLA types. PMID:28139734

  18. Sofosbuvir/velpatasvir regimen promises an effective pan-genotypic hepatitis C virus cure

    Science.gov (United States)

    Mir, Fazia; Kahveci, Alp S; Ibdah, Jamal A; Tahan, Veysel

    2017-01-01

    Hepatitis C virus (HCV) is a global pandemic, with nearly 200 million infected patients worldwide. HCV is the most common blood-borne infection in the US with numerous health implications including liver fibrosis, cirrhosis, and hepatocellular cancer. Traditional genotype-based HCV therapies with interferon resulted in moderate success in the sustained elimination of viral genome. Recent clinical trials of the once-daily combination tablet of sofosbuvir, a nonstructural (NS) 5B polymerase inhibitor, and velpatasvir, an NS5A inhibitor, demonstrate sustained virologic response rates of about 95%, regardless of prior treatment experience or presence of cirrhosis across all HCV genotypes. Patients reported improvements in general health, fatigue, and emotional and mental well-being after completing combination therapy. The combination treatment is effective, but does need to be administered with caution in patients receiving certain medications or with certain diseases. Herein, we review the safety and efficacy of sofosbuvir/velpatasvir combination regimen for all HCV genotypes.

  19. Observations on fluxes near anti-branes

    CERN Document Server

    Cohen-Maldonado, Diego; Van Riet, Thomas; Vercnocke, Bert

    2015-01-01

    We revisit necessary conditions for gluing local (anti)-D3 throats into flux throats with opposite charge. These consistency conditions typically reveal singularities in the 3-form fluxes whose meaning is being debated. In this note we prove, under well-motivated assumptions, that singularities remain even when the anti-D3 branes are puffed up into spherical NS5 branes. It does not seem possible to ascribe the singular flux to the self-energy of the 5-branes but rather to the singular clumping of the background fluxes. We furthermore comment on the gluing conditions at finite temperature and point out that one specific assumption of a recent no-go theorem can be broken if anti-branes are to polarise into spherical NS5 branes at zero temperature. Our first result, however, casts some doubt on whether this gap in the no-go theorem can be successfully employed to construct finite temperature solutions.

  20. Three-dimensional {N}=4 linear quivers and non-Abelian T-duals

    Science.gov (United States)

    Lozano, Yolanda; Macpherson, Niall T.; Montero, Jesús; Núñez, Carlos

    2016-11-01

    In this paper we construct a new Type IIB background with an AdS 4 factor that preserves {N}=4 Supersymmetry. This solution is obtained using a non-Abelian T-duality transformation on the Type IIA reduction of the AdS 4 × S 7 background. We interpret our configuration as a patch of a more general background with localised sources, dual to the renormalisation fixed point of a {T}_{ρ}^{widehat{ρ}}(SU(N)) quiver field theory. This relates explicitly the AdS 4 geometry to a D3-D5-NS5 brane intersection, illuminating what seems to be a more general phenomenon, relating AdS p+1 backgrounds generated by non-Abelian T-duality to Dp- D( p + 2)-NS5 branes intersections.

  1. Screening of cellular proteins that interact with the classical swine fever virus non-structural protein 5A by yeast two-hybrid analysis

    Indian Academy of Sciences (India)

    Chengcheng Zhang; Lei He; Kai Kang; Heng Chen; Lei Xu; Yanming Zhang

    2014-03-01

    Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell’s function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A’s function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.

  2. Hepatitis C Drugs: The End of the Pegylated Interferon Era and the Emergence of All-Oral, Interferon-Free Antiviral Regimens: A Concise Review

    Directory of Open Access Journals (Sweden)

    Alan Hoi Lun Yau

    2014-01-01

    Full Text Available Between 2001 and 2011, the standard of care for chronic hepatitis C virus (HCV infection was a combination of pegylated interferon (PEGIFN and ribavirin (RBV. In May 2011, boceprevir and telaprevir, two first-generation NS3/4A protease inhibitors, were approved in combination with PEG-IFN and RBV for 24 to 48 weeks in hepatitis C virus genotype 1 infections. In December 2013, simeprevir, a second-generation NS3/4A protease inhibitor, was approved for use with PEG-IFN and RBV for 12 weeks in genotype 1, while sofosbuvir, a NS5B nucleotide polymerase inhibitor, was approved for use with PEG-IFN and RBV for 12 weeks in genotypes 1 and 4, as well as with RBV alone for 12 weeks in genotype 2 and for 24 weeks in genotype 3. Sofosbuvir combined with simeprevir or an NS5A replication complex inhibitor (ledipasvir or daclatasvir with or without RBV for 12 weeks in genotype 1 resulted in a sustained virological response >90%, irrespective of previous treatment history or presence of cirrhosis. Similarly impressive sustained virological response rates have been shown with ABT-450/r (ritonavir-boosted NS3/4A protease inhibitor-based regimens in combination with other direct-acting antiviral agent(s with or without RBV for 12 weeks in genotype 1. The optimal all-oral interferon-free antiviral regimen likely entails a combination of an NS5B nucleotide polymerase inhibitor with either a second-generation NS3/4A protease inhibitor or an NS5A replication complex inhibitor with or without RBV. Further research is needed to determine the role of resistance testing, clarify the optimal follow-up duration post-treatment, and evaluate the antiviral efficacy and safety in difficult-to-cure patient populations.

  3. The Black Hole Interior and a Curious Sum Rule

    CERN Document Server

    Giveon, Amit; Troost, Jan

    2013-01-01

    We analyze the Euclidean geometry near non-extremal NS5-branes in string theory, including regions beyond the horizon and beyond the singularity of the black brane. The various regions have an exact description in string theory, in terms of cigar, trumpet and negative level minimal model conformal field theories. We study the worldsheet elliptic genera of these three superconformal theories, and show that their sum vanishes. We speculate on the significance of this curious sum rule for black hole physics.

  4. The black hole interior and a curious sum rule

    Energy Technology Data Exchange (ETDEWEB)

    Giveon, Amit [Racah Institute of Physics, The Hebrew University,Jerusalem, 91904 (Israel); Itzhaki, Nissan [Physics Department, Tel-Aviv University,Ramat-Aviv, 69978 (Israel); Troost, Jan [Laboratoire de Physique Théorique,Unité Mixte du CRNS et de l’École Normale Supérieure,associée à l’Université Pierre et Marie Curie 6,UMR 8549 École Normale Supérieure,24 Rue Lhomond Paris 75005 (France)

    2014-03-12

    We analyze the Euclidean geometry near non-extremal NS5-branes in string theory, including regions beyond the horizon and beyond the singularity of the black brane. The various regions have an exact description in string theory, in terms of cigar, trumpet and negative level minimal model conformal field theories. We study the worldsheet elliptic genera of these three superconformal theories, and show that their sum vanishes. We speculate on the significance of this curious sum rule for black hole physics.

  5. Successful achievement of sustained virological response to triple combination therapy containing simeprevir in two patients with chronic hepatitis C who had failed asunaprevir:Daclatasvir combination therapy.

    Science.gov (United States)

    Ozeki, Itaru; Nakajima, Tomoaki; Yamaguchi, Masakatsu; Kimura, Mutsuumi; Arakawa, Tomohiro; Kuwata, Yasuaki; Ohmura, Takumi; Sato, Takahiro; Hige, Shuhei; Karino, Yoshiyasu; Toyota, Joji

    2016-10-01

    Patients 1 and 2 were treatment-naive women who had genotype 1b chronic hepatitis C. Both had IL-28B genotype TT, and amino acid substitutions of core 70 and 91 were both wild type. Search for the presence of resistance-associated variants (RAV) in non-structural (NS)3 and NS5A regions confirmed wild-type D168 and L31, along with Y93H, in both patients. These patients participated in a Japanese phase III clinical study of asunaprevir and daclatasvir at the age of 52 and 67 years, respectively, and were treated with the combination regimen for 24 weeks. However, both experienced post-treatment relapse, and then treated with triple combination therapy with simeprevir, pegylated interferon (IFN) and ribavirin at the age of 53 and 68 years, respectively, and achieved sustained virological response. A search for RAV prior to simeprevir treatment identified multiple resistance including D168E, Y93H and L31V in both patients. It has been demonstrated that, in many cases, a treatment failure with a combination of asunaprevir and daclatasvir results in acquisition of RAV in NS3 and NS5A regions and that drug-resistant mutants, particularly those in the NS5A region, survive for a long time. In these cases, direct-acting antivirals targeted towards the NS5A region may have a limited efficacy. The present case report is based on an idea that a regimen containing IFN with simeprevir could be a therapeutic option particularly for those who are likely to be highly sensitive and tolerable to IFN.

  6. Pharmacoinformatics approach for investigation of alternative potential hepatitis C virus nonstructural protein 5B inhibitors

    Directory of Open Access Journals (Sweden)

    Mirza MU

    2015-03-01

    Full Text Available Muhammad Usman Mirza,1 Noor-Ul-Huda Ghori,2 Nazia Ikram,3 Abdur Rehman Adil,4 Sadia Manzoor3 1Centre for Research in Molecular Medicine (CRiMM, The University of Lahore, Lahore, 2Atta-ur-Rehman School of Applied Biosciences (ASAB, National University of Science and Technology, Islamabad, 3Institute of Molecular Biology and Biotechnology (IMBB, The University of Lahore, Lahore, Pakistan; 4Centre for Excellence in Molecular Biology (CEMB, The University of Punjab, Lahore, Pakistan Abstract: Hepatitis C virus (HCV is one of the major viruses affecting the world today. It is a highly variable virus, having a rapid reproduction and evolution rate. The variability of genomes is due to hasty replication catalyzed by nonstructural protein 5B (NS5B which is also a potential target site for the development of anti-HCV agents. Recently, the US Food and Drug Administration approved sofosbuvir as a novel oral NS5B inhibitor for the treatment of HCV. Unfortunately, it is much highlighted for its pricing issues. Hence, there is an urgent need to scrutinize alternate therapies against HCV that are available at affordable price and do not have associated side effects. Such a need is crucial especially in underdeveloped countries. The search for various new bioactive compounds from plants is a key part of pharmaceutical research. In the current study, we applied a pharmacoinformatics-based approach for the identification of active plant-derived compounds against NS5B. The results were compared to docking results of sofosbuvir. The lead compounds with high-binding ligands were further analyzed for pharmacokinetic and pharmacodynamic parameters based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET profile. The results showed the potential alternative lead compounds that can be developed into commercial drugs having high binding energy and promising ADMET properties. Keywords: hepatitis C, NS5B inhibitors, molecular docking, Auto

  7. A complement-microglial axis drives synapse loss during virus-induced memory impairment.

    Science.gov (United States)

    Vasek, Michael J; Garber, Charise; Dorsey, Denise; Durrant, Douglas M; Bollman, Bryan; Soung, Allison; Yu, Jinsheng; Perez-Torres, Carlos; Frouin, Arnaud; Wilton, Daniel K; Funk, Kristen; DeMasters, Bette K; Jiang, Xiaoping; Bowen, James R; Mennerick, Steven; Robinson, John K; Garbow, Joel R; Tyler, Kenneth L; Suthar, Mehul S; Schmidt, Robert E; Stevens, Beth; Klein, Robyn S

    2016-06-22

    Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

  8. TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

    OpenAIRE

    Taylor, R. Travis; Lubick, Kirk J.; Robertson, Shelly J.; Broughton, James P.; Bloom, Marshall E.; Bresnahan, Wade A.; Best, Sonja M.

    2011-01-01

    In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential f...

  9. Cyclophilin Inhibitors Remodel the Endoplasmic Reticulum of HCV-Infected Cells in a Unique Pattern Rendering Cells Impervious to a Reinfection.

    Directory of Open Access Journals (Sweden)

    Udayan Chatterji

    Full Text Available The mechanisms of action by which cyclophilin inhibitors (CypI interfere with the HCV life cycle remain poorly understood. We reported that CypI and NS5A inhibitors (NS5Ai, but not other classes of anti-HCV agents, prevent assembly of double membrane vesicles (DMVs, which protect replication complexes. We demonstrated that both NS5A and the isomerase cyclophilin A (CypA are required for DMV formation. Here, we examined whether CypI mediate an additional antiviral effect that could further explain the high efficacy of CypI. We identified a unique action of CypI. CypI remodel the organization of the endoplasmic reticulum (ER of HCV-infected cells, but not of uninfected cells. This effect is specific since it was not observed for other classes of anti-HCV agents including NS5Ai, and has no effect on the viability of CypI-treated cells. Since ER serves as platform for the establishment of HCV replication complexes, we asked whether the ER reorganization by CypI would prevent cells from being newly infected. Remarkably, CypI-treated HCV-pre-infected cells remain totally impervious to a reinfection, suggesting that the CypI-mediated ER reorganization prevents a reinfection. This block is not due to residual CypI since CypI-resistant HCV variants also fail to infect these cells. The ER reorganization by CypI is rapid and reversible. This study provides the first evidence that CypI trigger a unique ER reorganization of infected cells, rendering cells transiently impervious to a reinfection. This study further suggests that the HCV-induced ER rearrangement represents a key target for the development of new therapies.

  10. Molecular genotyping of HCV infection in seropositive blood donor

    Science.gov (United States)

    Zarin, Siti Noraziah Abu; Ibrahim, Nazlina

    2013-11-01

    This study is to investigate the prevalence of hepatitis C virus infection in seropositive blood donor. RNA was extracted from 32 positive samples in National Blood Centre and Melaka Hospital. The core and NS5B sequences were obtained from 23 samples. Genotype 3a is most prevalent in this study followed by genotype 1a. Evidence of mixed-genotypes (3a and 1b) infections was found in 5 subjects.

  11. Molecular mechanism of hepatitis C virus replicon variants with reduced susceptibility to a benzofuran inhibitor, HCV-796.

    Science.gov (United States)

    Howe, Anita Y M; Cheng, Huiming; Johann, Stephen; Mullen, Stanley; Chunduru, Srinivas K; Young, Dorothy C; Bard, Joel; Chopra, Rajiv; Krishnamurthy, Girija; Mansour, Tarek; O'Connell, John

    2008-09-01

    HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log(10) HCV copies/mug total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 A of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.

  12. Naturally occurring mutations in the nonstructural region 5B of hepatitis C virus (HCV from treatment-naive Korean patients chronically infected with HCV genotype 1b.

    Directory of Open Access Journals (Sweden)

    Dong-Won Kim

    Full Text Available The nonstructural 5B (NS5B protein of the hepatitis C virus (HCV with RNA-dependent RNA polymerase (RdRp activity plays a pivotal role in viral replication. Therefore, monitoring of its naturally occurring mutations is very important for the development of antiviral therapies and vaccines. In the present study, mutations in the partial NS5B gene (492 bp from 166 quasispecies of 15 genotype-1b (GT treatment-naïve Korean chronic patients were determined and mutation patterns and frequencies mainly focusing on the T cell epitope regions were evaluated. The mutation frequency within the CD8+ T cell epitopes was significantly higher than those outside the CD8+ T cell epitopes. Of note, the mutation frequency within predicted CD4+ T cell epitopes, a particular mutational hotspot in Korean patients was significantly higher than it was in patients from other areas, suggesting distinctive CD4+ T cell-mediated immune pressure against HCV infection in the Korean population. The mutation frequency in the NS5B region was positively correlated with patients with carrier-stage rather than progressive liver disease (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Furthermore, the mutation frequency in four codons (Q309, A333, V338 and Q355 known to be related to the sustained virological response (SVR and end-of treatment response (ETR was also significantly higher in Korean patients than in patients from other areas. In conclusion, a high degree of mutation frequency in the HCV GT-1b NS5B region, particularly in the predicted CD4+ T cell epitopes, was found in Korean patients, suggesting the presence of distinctive CD4+ T cell pressure in the Korean population. This provides a likely explanation of why relatively high levels of SVR after a combined therapy of pegylated interferon (PEG-IFN and ribavirin (RBV in Korean chronic patients with GT-1b infections are observed.

  13. (p,q)-Five Brane and (p,q)-String Solutions, their Bound State and its Near Horizon Limit

    CERN Document Server

    Kluson, J

    2016-01-01

    We determine (p,q)-string and (p,q)-five brane solutions of type IIB supergravity using SL(2,Z)-symmetry of the full type IIB superstring theory. We also determine SL(2,Z)-transformed solution corresponding to the bound state of NS5-branes and fundamental strings. Then we analyze its near horizon limit and we show that it leads to the AdS(3)xS(3) with mixed fluxes.

  14. Cyclophilin Inhibitors Remodel the Endoplasmic Reticulum of HCV-Infected Cells in a Unique Pattern Rendering Cells Impervious to a Reinfection

    Science.gov (United States)

    Chatterji, Udayan; Bobardt, Michael; Schaffer, Lana; Wood, Malcolm; Gallay, Philippe A.

    2016-01-01

    The mechanisms of action by which cyclophilin inhibitors (CypI) interfere with the HCV life cycle remain poorly understood. We reported that CypI and NS5A inhibitors (NS5Ai), but not other classes of anti-HCV agents, prevent assembly of double membrane vesicles (DMVs), which protect replication complexes. We demonstrated that both NS5A and the isomerase cyclophilin A (CypA) are required for DMV formation. Here, we examined whether CypI mediate an additional antiviral effect that could further explain the high efficacy of CypI. We identified a unique action of CypI. CypI remodel the organization of the endoplasmic reticulum (ER) of HCV-infected cells, but not of uninfected cells. This effect is specific since it was not observed for other classes of anti-HCV agents including NS5Ai, and has no effect on the viability of CypI-treated cells. Since ER serves as platform for the establishment of HCV replication complexes, we asked whether the ER reorganization by CypI would prevent cells from being newly infected. Remarkably, CypI-treated HCV-pre-infected cells remain totally impervious to a reinfection, suggesting that the CypI-mediated ER reorganization prevents a reinfection. This block is not due to residual CypI since CypI-resistant HCV variants also fail to infect these cells. The ER reorganization by CypI is rapid and reversible. This study provides the first evidence that CypI trigger a unique ER reorganization of infected cells, rendering cells transiently impervious to a reinfection. This study further suggests that the HCV-induced ER rearrangement represents a key target for the development of new therapies. PMID:27442520

  15. Allosteric inhibitors of hepatitis C polymerase: discovery of potent and orally bioavailable carbon-linked dihydropyrones.

    Science.gov (United States)

    Li, Hui; Linton, Angelica; Tatlock, John; Gonzalez, Javier; Borchardt, Allen; Abreo, Mel; Jewell, Tanya; Patel, Leena; Drowns, Matthew; Ludlum, Sarah; Goble, Mike; Yang, Michele; Blazel, Julie; Rahavendran, Ravi; Skor, Heather; Shi, Stephanie; Lewis, Cristina; Fuhrman, Shella

    2007-08-23

    The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. Further structure-activity relationship (SAR) studies led to the identification of compounds, exemplified by 23 and 24, with significantly improved antiviral activities in the cell-based replicon assay and favorable pharmacokinetic profiles.

  16. AGOR 28

    Science.gov (United States)

    2014-10-23

    the end to the run, the temperature shot up to over 130-degrees very rapidly and the shaft was stopped. J1 requires self- aligning bearings, which...Sally Ride. iv. Working on NS5 Hierarchy 4. Operator Concerns: • Dodge Shaft Bearings – For the Starboard shaft , the temperature on the...stopped the shaft before going to 200-rpm. I was on the dock observing when they shut down. For the Port Shaft , the temperatures remained below 100

  17. Exotic branes in Double Field Theory

    Directory of Open Access Journals (Sweden)

    Musaev Edvard

    2016-01-01

    Full Text Available The non-geometric Q- and R-monopole are shown to be a particular case of the DFT-monopole solution. The notion of magnetic charge for the solutions is defined and shown to be equal to the magnetic charge of the NS5-brane solution. This is a talk presented by the author at the conference QUARKS’16 in St.-Petersburg.

  18. Serendipitous identification of natural intergenotypic recombinants of hepatitis C in Ireland.

    LENUS (Irish Health Repository)

    Moreau, Isabelle

    2006-01-01

    BACKGROUND: Recombination between hepatitis C single stranded RNA viruses is a rare event. Natural viable intragenotypic and intergenotypic recombinants between 1b-1a, 1a-1c and 2k-1b, 2i-6p, respectively, have been reported. Diagnostically recombinants represent an intriguing challenge. Hepatitis C genotype is defined by interrogation of the sequence composition of the 5\\' untranslated region [5\\'UTR]. Occasionally, ambiguous specimens require further investigation of the genome, usually by interrogation of the NS5B region. The original purpose of this study was to confirm the existence of a suspected mixed genotype infection of genotypes 2 and 4 by clonal analysis at the NS5B region of the genome in two specimens from two separate individuals. This initial identification of genotype was based on analysis of the 5\\'UTR of the genome by reverse line probe hybridisation [RLPH]. RESULTS: The original diagnosis of a mixed genotype infection was not confirmed by clonal analysis of the NS5B region of the genome. The phylogenetic analysis indicated that both specimens were natural intergenotypic recombinant forms of HCV. The recombination was between genotypes 2k and 1b for both specimens. The recombination break point was identified as occurring within the NS2 region of the genome. CONCLUSION: The viral recombinants identified here resemble the recombinant form originally identified in Russia. The RLPH pattern observed in this study may be a signature indicative of this particular type of intergenotype recombinant of hepatitis C meriting clonal analysis of NS2.

  19. A Multiplex Microsphere Immunoassay for Zika Virus Diagnosis.

    Science.gov (United States)

    Wong, Susan J; Furuya, Andrea; Zou, Jing; Xie, Xuping; Dupuis, Alan P; Kramer, Laura D; Shi, Pei-Yong

    2017-02-01

    Rapid and accurate diagnosis of infectious agents is essential for patient care, disease control, and countermeasure development. The present serologic diagnosis of Zika virus (ZIKV) infection relies mainly on IgM-capture ELISA which is confounded with the flaw of cross-reactivity among different flaviviruses. In this communication, we report a multiplex microsphere immunoassay (MIA) that captures the diagnostic power of viral envelope protein (that elicits robust, yet cross-reactive antibodies to other flaviviruses) and the differential power of viral nonstructural proteins NS1 and NS5 (that induce more virus-type specific antibodies). Using 153 patient specimens with known ZIKV and/or dengue virus (DENV; a closely related flavivirus) infections, we showed that (i) ZIKV envelope-based MIA is equivalent or more sensitive than IgM-capture ELISA in diagnosing ZIKV infection, (ii) antibody responses to NS1 and NS5 proteins are more ZIKV-specific than antibody response to envelope protein, (iii) inclusion of NS1 and NS5 in the MIA improves the diagnostic accuracy when compared with the MIA that uses envelope protein alone. The multiplex MIA achieves a rapid diagnosis (turnaround time<4h) and requires small specimen volume (10μl) in a single reaction. This serologic assay could be developed for use in clinical diagnosis of ZIKV infection and for monitoring immune responses in vaccine trials.

  20. A giant graviton genealogy

    CERN Document Server

    Lozano, Yolanda; Prinsloo, Andrea

    2013-01-01

    In this article we extend the construction of giant gravitons from holomorphic surfaces [arXiv:hep-th/0010206] to the ABJM correspondence. We construct a new class of 1/6-BPS M5-branes wrapping 5-manifolds in S^7/Z_k and supported by a large angular momentum in the orbifold space. These orbifold giant gravitons undergo a supersymmetry enhancement to 1/3-BPS and 1/2-BPS configurations in special cases. The compactification of M-theory on AdS_4 x S^7/Z_k to type IIA superstring theory on AdS_4 x CP^3 then gives rise to another new class of 1/6-BPS D4 and NS5-branes wrapping 4 and 5-manifolds in CP^3. The D4-branes carry a combination of D0-brane charge and angular momentum in the complex projective space, while the NS5-branes are supported only by D0-brane charge. Finally, we present a detailed analysis of a one-parameter family of 1/2-BPS M5-brane orbifold giant gravitons, and their D4 and NS5-brane CP^3 descendants.

  1. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses

    DEFF Research Database (Denmark)

    Gottwein, Judith; Scheel, Troels; Høgh, Mette;

    2007-01-01

    , which has a high prevalence worldwide. METHODS: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression...... (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies. RESULTS: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable...... growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants...

  2. Heads or Tails: Genotyping of Hepatitis C Virus Concerning the 2k/1b Circulating Recombinant Form

    Science.gov (United States)

    Schuermans, Wim; Orlent, Hans; Desombere, Isabelle; Descheemaeker, Patrick; Van Vlierberghe, Hans; Geerts, Anja; Verhelst, Xavier; Reynders, Marijke; Padalko, Elizaveta

    2016-01-01

    As different hepatitis C virus (HCV) genotypes respond differently to initiated therapy, correct HCV genotyping is essential. A potential risk for misclassification of the intergenotypic HCV circulating recombinant form (CRF) 2k/1b strains exists, depending on the genotyping method used. The aim was to investigate the differences in HCV genotyping methods with regard to CRF 2k/1b and to gain insight in the prevalence of the CRF 2k/1b. Genotyping results by Versant HCV Genotype Assay were compared with nonstructural protein 5B (NS5B) sequencing. In total, from November 2001 until March 2015, 3296 serum samples were analyzed by Versant HCV Genotype Assay. As misclassified CRF is harbored among HCV genotype 2, we further focused our search on 142 (4.3%) samples positive for HCV genotype 2. On 116 (81.7%) retrieved samples, the NS5B sequencing was performed. Twelve out of the 116 retrieved samples (10.3%) were classified as CRF 2k/1b by sequencing of the NS5B region. Ten of these 12 samples were originally misclassified as genotype 2a or 2c, while 2 of them were misclassified as genotype 2. Our results show that the current prevalence of CRF 2k/1b is underestimated. The importance of correct HCV genotyping is emphasized, considering the tailored choice of treatment regimen and overall prognosis. PMID:27563879

  3. Construction of a dengue virus type 4 reporter replicon and analysis of temperature-sensitive mutations in non-structural proteins 3 and 5.

    Science.gov (United States)

    Alcaraz-Estrada, Sofia L; Manzano, Mark Irvin M; Del Angel, Rosa M; Levis, Robin; Padmanabhan, R

    2010-11-01

    Replicon systems have been useful to study mechanisms of translation and replication of flavivirus RNAs. In this study, we constructed a dengue virus 4 replicon encoding a Renilla luciferase (R(luc)) reporter, and six single-residue substitution mutants were generated: L128F and S158P in the non-structural protein (NS) 3 protease domain gene, and N96I, N390A, K437R and M805I in the NS5 gene. The effects of these substitutions on viral RNA translation and/or replication were examined by measuring R(luc) activities in wild-type and mutant replicon RNA-transfected Vero cells incubated at 35, 37 and 39 °C. Our results show that none of the mutations affected translation of replicon RNAs; however, L128F and S158P of NS3 at 39°C, and N96I of NS5 at 37 and 39°C, presented temperature-sensitive (ts) phenotypes for replication. Furthermore, using in vitro methyltransferase assays, we identified that the N96I mutation in NS5 exhibited a ts phenotype for N7-methylation, but not for 2'-O-methylation.

  4. Sofosbuvir plus ledipasvir in combination for the treatment of hepatitis C infection.

    Science.gov (United States)

    Bourlière, Marc; Adhoute, Xavier; Ansaldi, Christelle; Oules, Valérie; Benali, Souad; Portal, Isabelle; Castellani, Paul; Halfon, Philippe

    2015-01-01

    Sofsobuvir is the first-in-class NS5B nucleotide inhibitor to be launched as a treatment for the hepatitis C virus (HCV). Its viral potency, pan genotypic activity and high barrier to resistance make it the ideal candidate to become a backbone for several IFN-free regimens. Ledipasvir is a NS5A inhibitor with multi genotypic activity but modest barrier to resistance. The once-daily fixed-dose combination of sofosbuvir plus ledipasvir is the first-in-market single-tablet regimen for the treatment of hepatitis C infection. Recent data demonstrated that this FDC alone, or in combination with ribavirin, is able to achieve HCV cure of at least 90% or more among genotype 1,4, 5 and 6 patients. This combination appears to be suboptimal in genotype 3 patients and other direct acting antiviral combinations with sofosbuvir will help to fulfill this gap in the near future. The safety profile of the fixed dose combination is good. Resistance is not an issue with sofosbuvir but may be a significant issue with regards to ledipasvir for those rare individuals who harbor baseline HCV NS5A resistance-associated variants that conferred a high resistance level. The rational for using FDCs and the available clinical data are reviewed.

  5. Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

    Institute of Scientific and Technical Information of China (English)

    Xiao-quan Li; Shu-lin Zhang; Li-hua Zhong; Jun Cheng; Yuan Hong; Meng-dong Lan; Xiao-bin Chen; Cheng-fu Sun

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coil BL21, the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG), and it was analyzed with sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyelonai antibody, with which we studied the function of NSSATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results Recombinant NS5ATP5 (molecular weight: 65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person, and it was detected mainly in the cytoplasm. Conclusion The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.

  6. Hepatitis C virus non-structural 5B protein interacts with cyclin A2 and regulates viral propagation

    DEFF Research Database (Denmark)

    Pham, Long; Ngo, HT; Lim, YS

    2012-01-01

    Background & Aims Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc......, in vitro and in vivo protein binding assays, luciferase reporter gene assay, and immunoblot assay. Results We showed that siRNA-mediated depletion of CycA2 significantly inhibited HCV replication in both HCV subgenomic replicon cells and HCVcc-infected cells. Furthermore, HCV non-structural 5B (NS5B......) specifically interacted with CycA2 in vitro and in vivo. Protein interaction was mediated through the cyclin box of CycA2 and the palm domain of NS5B. We further showed that R/HxL motif in the palm domain of HCV NS5B mediated protein interaction with CycA2 and this interaction was necessary for HCV replication...

  7. Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV particle assembly and maturation.

    Directory of Open Access Journals (Sweden)

    MinKyung Yi

    2009-05-01

    Full Text Available Recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis C virus (HCV indicate that several nonstructural viral proteins, including NS2, NS3, and NS5A, are involved in the process of viral assembly and release. Other recent work suggests that Ser-168 of NS2 is a target of CK2 kinase-mediated phosphorylation, and that this controls the stability of the genotype 1a NS2 protein. Here, we show that Ser-168 is a critical determinant in the production of infectious virus particles. Substitution of Ser-168 with Ala (or Gly ablated production of infectious virus by cells transfected with a chimeric viral RNA (HJ3-5 containing core-NS2 sequences from the genotype 1a H77 virus within the background of genotype 2a JFH1 virus. An S168A substitution also impaired production of virus by cells transfected with JFH1 RNA. This mutation did not alter polyprotein processing or genome replication. This defect in virus production could be rescued by expression of wt NS2 in trans from an alphavirus replicon. The trans-complementing activities of NS2 from genotypes 1a and 2a demonstrated strong preferences for rescue of the homologous genotype. Importantly, the S168A mutation did not alter the association of core or NS5A proteins with host cell lipid droplets, nor prevent the assembly of core into particles with sedimentation and buoyant density properties similar to infectious virus, indicating that NS2 acts subsequent to the involvement of core, NS5A, and NS3 in particle assembly. Second-site mutations in NS2 as well as in NS5A can rescue the defect in virus production imposed by the S168G mutation. In aggregate, these results indicate that NS2 functions in trans, in a late-post assembly maturation step, perhaps in concert with NS5A, to confer infectivity to the HCV particle.

  8. Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease

    Institute of Scientific and Technical Information of China (English)

    Gui-Xin Du; Li-Hua Hou; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang

    2002-01-01

    AIM: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors. METHODS: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coliwas induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease.RESULTS: The single-chain recombinant protease was overexpressed as soluble protein when the E. coliwas induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15℃). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %).The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not.CONCLUSION: A simple

  9. Chronic hepatitis C: future treatment

    Directory of Open Access Journals (Sweden)

    Wendt A

    2014-01-01

    Full Text Available Astrid Wendt, Xavier Adhoute, Paul Castellani, Valerie Oules, Christelle Ansaldi, Souad Benali, Marc BourlièreDepartment of Hepato-Gastroenterology, Hôpital Saint-Joseph, Marseille, FranceAbstract: The launch of first-generation protease inhibitors (PIs is a major step forward in HCV treatment. However, the major advance is up to now restricted to genotype 1 (GT-1 patients. The development of second-wave and second-generation PIs yields higher antiviral potency through plurigenotypic activity, more convenient daily administration, fewer side effects and, for the second-generation PIs, potential activity against resistance-associated variants. NS5B inhibitors include nucleoside/nucleotide inhibitors (NIs and non-nucleotide inhibitors (NNIs. NIs have high efficacy across all genotypes. Sofosbuvir has highly potent antiviral activity across all genotypes in association with pegylated interferon and ribavirin (PR, thus allowing shortened treatment duration. NS5A inhibitors (NS5A.I have highly potent antiviral activity. It has recently been shown for the first time that NS5A.I in combination with protease inhibitors can cure GT-1b null responders in an interferon-free regimen. Besides, several studies demonstrate that interferon (IFN-free regimens with direct-acting antiviral agent combinations are able to cure a large number of either naïve or treatment-experienced GT-1 patients. Moreover, quadruple regimen with PR is able to cure almost all GT-1 null responders. The development of pan-genotypic direct-acting antiviral agents (NIs or NS5A.I allows new combinations with or without PR that increase the rate of sustained virological response for all patients, even for those with cirrhosis and independently of the genotype. Therefore, the near future of HCV treatment looks promising. The purpose of this article is to provide an overview of the clinical results recently reported for HCV treatment.Keywords: SVR, direct antiviral agents, host

  10. Anti-hepatitis C virus drugs:research advances%抗丙型肝炎病毒药物研究进展

    Institute of Scientific and Technical Information of China (English)

    王靖; 何小羊

    2015-01-01

    慢性丙型肝炎病毒(HCV)感染是全球性的公共卫生问题之一,可导致严重的肝病,包括肝硬化及肝癌. 传统的标准治疗方案利巴韦林+聚乙二醇化干扰素对病毒响应率低,副作用大,难以有效治疗HCV感染. 近年来,多种新类型HCV抑制剂得以开发,包括直接抗病毒药物(DAA)以及宿主靶向药物. 这些药物针对病毒生命周期中的必需蛋白,主要包括NS3/4A蛋白酶、NS5B聚合酶、NS5A蛋白或病毒复制相关宿主因子,具有较高的抗病毒活性及良好的安全性和耐受性. 核苷类NS5B聚合酶抑制剂sofosbuvir的成功上市,使HCV的治疗取得了突破性进展.本文综述了近年来抗HCV药物的发展情况,总结了各类抗HCV药物的结构、作用机制及相关抗病毒性质.%Chronic hepatitis C virus (HCV) infection is one of global public health problems, and predominantly results in severe hepatic diseases including hepatic cirrhosis and hepatocellular carcinoma. The traditional standard of care for chronic HCV is the combination of ribavirin and pegylated interferon, which is insufficient to cure the HCV infection due to the suboptimal sustained viral response and severe side effects. Recently new therapeutic approaches have been developed, including direct-acting antiviral agents (DAA) and host-targeting agents, which target essential proteins or host factors in HCV life cycle, such as NS3/4A protease, NS5B polymerase and NS5A protein. These agents exhibit high efficacy, improving safety and tolerability. Thus, the approval of the nucleotide polymerase inhibitor sofosbuvir provides a breakthrough therapy against chronic hepatitis C infection. This review describes the current different classes of HCV inhibitors during the last decades, and their chemical structures, mechanisms and related anti-viral property.

  11. In Vitro Activity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor

    Science.gov (United States)

    Koev, Gennadiy; Irvin, Michelle; Beyer, Jill; Liu, Yaya; Krishnan, Preethi; Reisch, Thomas; Mondal, Rubina; Wagner, Rolf; Molla, Akhteruzzaman; Maring, Clarence; Collins, Christine

    2014-01-01

    Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections. PMID:25534735

  12. Natural Polymorphisms Conferring Resistance to HCV Protease and Polymerase Inhibitors in Treatment-Naive HIV/HCV Co-Infected Patients in China.

    Directory of Open Access Journals (Sweden)

    Kali Zhou

    Full Text Available The advent of direct-acting agents (DAAs has improved treatment of HCV in HIV co-infection, but may be limited by primary drug resistance. This study reports the prevalence of natural polymorphisms conferring resistance to NS3/4A protease inhibitors and NS5B polymerase inhibitors in treatment-naïve HIV/HCV co-infected individuals in China.Population based NS3/4A sequencing was completed for 778 treatment-naïve HIV/HCV co-infected patients from twelve provinces. NS3 sequences were amplified by nested PCR using in-house primers for genotypes 1-6. NS5B sequencing was completed for genotyping in 350 sequences. Resistance-associated variants (RAVs were identified in positions associated with HCV resistance.Overall, 72.8% (566/778 of all HCV sequences had at least one RAV associated with HCV NS3/4A protease inhibitor resistance. Variants were found in 3.6% (7/193 of genotype 1, 100% (23/23 of genotype 2, 100% (237/237 of genotype 3 and 92% (299/325 of genotype 6 sequences. The Q80K variant was present in 98.4% of genotype 6a sequences. High-level RAVs were rare, occurring in only 0.8% of patients. 93% (64/69 patients with genotype 1b also carried the C316N variant associated with NS5B low-level resistance.The low frequency of high-level RAVs associated with primary HCV DAA resistance among all genotypes in HIV/HCV co-infected patients is encouraging. Further phenotypic studies and clinical research are needed.

  13. Serendipitous identification of natural Intergenotypic recombinants of hepatitis C in Ireland

    Directory of Open Access Journals (Sweden)

    Kenny-Walsh Elizabeth

    2006-11-01

    Full Text Available Abstract Background Recombination between hepatitis C single stranded RNA viruses is a rare event. Natural viable intragenotypic and intergenotypic recombinants between 1b-1a, 1a-1c and 2k-1b, 2i-6p, respectively, have been reported. Diagnostically recombinants represent an intriguing challenge. Hepatitis C genotype is defined by interrogation of the sequence composition of the 5' untranslated region [5'UTR]. Occasionally, ambiguous specimens require further investigation of the genome, usually by interrogation of the NS5B region. The original purpose of this study was to confirm the existence of a suspected mixed genotype infection of genotypes 2 and 4 by clonal analysis at the NS5B region of the genome in two specimens from two separate individuals. This initial identification of genotype was based on analysis of the 5'UTR of the genome by reverse line probe hybridisation [RLPH]. Results The original diagnosis of a mixed genotype infection was not confirmed by clonal analysis of the NS5B region of the genome. The phylogenetic analysis indicated that both specimens were natural intergenotypic recombinant forms of HCV. The recombination was between genotypes 2k and 1b for both specimens. The recombination break point was identified as occurring within the NS2 region of the genome. Conclusion The viral recombinants identified here resemble the recombinant form originally identified in Russia. The RLPH pattern observed in this study may be a signature indicative of this particular type of intergenotype recombinant of hepatitis C meriting clonal analysis of NS2.

  14. Genetic vaccination with Flt3-L and GM-CSF as adjuvants:Enhancement of cellular and humoral immune responses that results in protective immunity in a murine model of hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    Jens Encke; Jomo Bernardin; Jasmin Geib; Gocha Barbakadze; Raymond Bujdoso; Wolfgang Stremmel

    2006-01-01

    AIM: To investigate whether transfection of plasmid DNA encoding these cytokines enhances both humoral and cellular immune responses to hepatitis C virus (HCV) in a murine model.METHODS: We established a tumor model of HCV infection using syngenic mouse myeloma cells stably transfected with NS5. Co-vaccination of DNA encoding granulocyte macrophage colony-stimulating factor (GMCSF) and Fit-3 ligand together with a plasmid encoding for the HCV NS5 protein was carried out. Mice were sacrificed 14 d after the last immunization event with collection of spleen cells and serum to determine humoral and cellular immune responses.RESULTS: Co-vaccination of DNA encoding GM-CSF and Flt-3 ligand together with a plasmid encoding for the HCV NS5 protein induced increased antibody responses and CD4+ T cell proliferation to this protein. Vaccination with DNA encoding GM-CSF and Flt-3L promoted protection against tumor formation and/or reduction in mice coimmunized with cytokine-encoding DNA constructs. This suggests this strategy is capable of generating cytotoxic T lymphocyte activity in vivo. Following inoculation with plasmid DNA encoding Flt-3L, no increase in spleen size or in dendritic cell (DC) and natural killer cell numbers was observed. This was in contrast to a dramatic increase of both cell types after administration of recombinant Flt3-L in vivo. This suggests that vaccination with plasmid DNA encoding cytokines that regulate DC generation and mobilization may not promote unwanted side effects, such as autoimmunity, splenic fibrosis or hematopoietic malignancies that may occur with administration of recombinant forms of these proteins.CONCLUSION: Our data support the view that plasmid DNA vaccination is a promising approach for HCV immunization, and may provide a general adjuvant vaccination strategy against malignancies and other pathogens.

  15. 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.

  16. Systematic analysis of enhancer and critical cis-acting RNA elements in the protein-encoding region of the hepatitis C virus genome.

    Science.gov (United States)

    Chu, Derrick; Ren, Songyang; Hu, Stacy; Wang, Wei Gang; Subramanian, Aparna; Contreras, Deisy; Kanagavel, Vidhya; Chung, Eric; Ko, Justine; Amirtham Jacob Appadorai, Ranjit Singh; Sinha, Sanjeev; Jalali, Ziba; Hardy, David W; French, Samuel W; Arumugaswami, Vaithilingaraja

    2013-05-01

    Hepatitis C virus (HCV) causes chronic hepatitis, cirrhosis, and liver cancer. cis-acting RNA elements of the HCV genome are critical for translation initiation and replication of the viral genome. We hypothesized that the coding regions of nonstructural proteins harbor enhancer and essential cis-acting replication elements (CRE). In order to experimentally identify new cis RNA elements, we utilized an unbiased approach to introduce synonymous substitutions. The HCV genome coding for nonstructural proteins (nucleotide positions 3872 to 9097) was divided into 17 contiguous segments. The wobble nucleotide positions of each codon were replaced, resulting in 33% to 41% nucleotide changes. The HCV genome containing one of each of 17 mutant segments (S1 to S17) was tested for genome replication and infectivity. We observed that silent mutations in segment 13 (S13) (nucleotides [nt] 7457 to 7786), S14 (nt 7787 to 8113), S15 (nt 8114 to 8440), S16 (nt 8441 to 8767), and S17 (nt 8768 to 9097) resulted in impaired genome replication, suggesting CRE structures are enriched in the NS5B region. Subsequent high-resolution mutational analysis of NS5B (nt 7787 to 9289) using approximately 51-nucleotide contiguous subsegment mutant viruses having synonymous mutations revealed that subsegments SS8195-8245, SS8654-8704, and SS9011-9061 were required for efficient viral growth, suggesting that these regions act as enhancer elements. Covariant nucleotide substitution analysis of a stem-loop, JFH-SL9098, revealed the formation of an extended stem structure, which we designated JFH-SL9074. We have identified new enhancer RNA elements and an extended stem-loop in the NS5B coding region. Genetic modification of enhancer RNA elements can be utilized for designing attenuated HCV vaccine candidates.

  17. Identification of conserved and HLA-A*2402-restricted epitopes in Dengue virus serotype 2.

    Science.gov (United States)

    Duan, Zhi-Liang; Liu, Hui-Fang; Huang, Xi; Wang, Si-Na; Yang, Jin-Lin; Chen, Xin-Yu; Li, De-Zhou; Zhong, Xiao-Zhi; Chen, Bo-Kun; Wen, Jin-Sheng

    2015-01-22

    In this study, we set out to identify dengue virus serotype 2 (DENV-2)-specific HLA-A*2402-restricted epitopes and determine the characteristics of T cells generated to these epitopes. We screened the full-length amino-acid sequence of DENV-2 to find potential epitopes using the SYFPEITHI algorithm. Twelve putative HLA-A*2402-binding peptides conserved in hundreds of DENV-2 strains were synthesized, and the HLA restriction of peptides was tested in HLA-A*2402 transgenic mice. Nine peptides (NS4b(228-237), NS2a(73-81), E(298-306), M(141-149), NS4a(96-105), NS4b(159-168), NS5(475-484), NS1(162-171), and NS5(611-620)) induced high levels of peptide-specific IFN-γ-secreting cells in HLA-A*2402 transgenic mice. Apart from IFN-γ, NS4b(228-237-), NS2a(73-81-) and E(298-306)-specific CD8(+) cells produced TNF-α and IL-6 simultaneously, whereas M(141-149-) and NS5(475-484-) CD8(+) cells produced only IL-6. Moreover, splenic mononuclear cells (SMCs) efficiently recognized and killed peptide-pulsed splenocytes. Furthermore, each of nine peptides could be recognized by splenocytes from DENV-2-infected HLA-A*2402 transgenic mice. The SMCs from HLA-A*2402 transgenic mice immunized with nine immunogenic peptides efficiently killed DENV-2-infected splenic monocytes. The present identified epitopes have the potential to be new diagnostic tools for characterization of T-cell immunity in DENV infection and may serve as part of a universal epitope-based vaccine.

  18. Evolution of viral RNA in a Chinese patient to interferon/ribavirin therapy for hepatitis C

    Institute of Scientific and Technical Information of China (English)

    Xian-Zi Wen; Zhi-Hai Chen; Ya-Zhi Wei; Jia-Fu Ji

    2012-01-01

    Objective:The combination of interferon (IFN) and ribavirin (RBV) is the standard therapy for hepatitis C virus (HCV) infection.HCV genotype 2a has proved more amenable to the therapy,but its efficacy is yet limited.This study aimed to investigate the mechanism of the poor response in a case of HCV genotype 2a infection.Methods:We analyzed dynamic change of HCV RNA from a patient,infected with HCV genotype 2a,showing a poor virological response to IFN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing.Then we constructed subgenomic Japanese fulminant hepatitis-1 (JFH1) replicon and different chimeric replicons with humanized Gaussia luciferase gene.The chimeric replicons were derived from subgenomic JFH1 replicon,in which the NS5A region was replaced by the patient's sequence from the pre/post-treatment,and the chimeric replicons' susceptibility to IFN were evaluated by relative Gausia Luciferase activity.Results:The pretreatment HCV sequences appeared almost uniform,and the quasispecies variation was further more simplified after 12 weeks of therapy.Besides,the quasispecies variation seemed to be more diversified in the NS5A,relatively,a region crucial for IFN response,and each of chimeric replicons exhibited distinct response to IFN.Conclusions:During the course of the chronic infection,HCV population seems to be adapted to the patient's immunological system,and further to be selected by combination of IFN/RBV therapy,indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN.In addition,each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region (ISDR) of NS5A during chronic infection and IFN/RBV therapy.

  19. On hepatitis C virus evolution: the interaction between virus and host towards treatment outcome.

    Directory of Open Access Journals (Sweden)

    Cíntia Bittar

    Full Text Available BACKGROUND: Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV, the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses. METHODS: Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection. RESULTS: This analysis shows that the viral population that persists after treatment for most non-responder patients is present in before-treatment samples, suggesting it is adapted to evade treatment. In contrast, the population found in before treatment samples from most end-of-treatment responder patients either are selected out or appears in low frequency after relapse, therefore changing the population structure. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient. CONCLUSION: Although evolutionary behavior throughout treatment showed that each patient presented different population dynamics unrelated to therapy outcome, it seems that the viral population from non-responders that resists the treatment already had strains that could evade therapy before it started.

  20. Natural Polymorphisms Conferring Resistance to HCV Protease and Polymerase Inhibitors in Treatment-Naïve HIV/HCV Co-Infected Patients in China

    Science.gov (United States)

    Wang, Charles; Hu, Fengyu; Ning, Chuanyi; Lan, Yun; Tang, Xiaoping; Tucker, Joseph D.; Cai, Weiping

    2016-01-01

    Background The advent of direct-acting agents (DAAs) has improved treatment of HCV in HIV co-infection, but may be limited by primary drug resistance. This study reports the prevalence of natural polymorphisms conferring resistance to NS3/4A protease inhibitors and NS5B polymerase inhibitors in treatment-naïve HIV/HCV co-infected individuals in China. Methods Population based NS3/4A sequencing was completed for 778 treatment-naïve HIV/HCV co-infected patients from twelve provinces. NS3 sequences were amplified by nested PCR using in-house primers for genotypes 1–6. NS5B sequencing was completed for genotyping in 350 sequences. Resistance-associated variants (RAVs) were identified in positions associated with HCV resistance. Results Overall, 72.8% (566/778) of all HCV sequences had at least one RAV associated with HCV NS3/4A protease inhibitor resistance. Variants were found in 3.6% (7/193) of genotype 1, 100% (23/23) of genotype 2, 100% (237/237) of genotype 3 and 92% (299/325) of genotype 6 sequences. The Q80K variant was present in 98.4% of genotype 6a sequences. High-level RAVs were rare, occurring in only 0.8% of patients. 93% (64/69) patients with genotype 1b also carried the C316N variant associated with NS5B low-level resistance. Conclusions The low frequency of high-level RAVs associated with primary HCV DAA resistance among all genotypes in HIV/HCV co-infected patients is encouraging. Further phenotypic studies and clinical research are needed. PMID:27341031

  1. Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication.

    Science.gov (United States)

    Berger, Kristi L; Cooper, Jacob D; Heaton, Nicholas S; Yoon, Rosa; Oakland, Todd E; Jordan, Tristan X; Mateu, Guaniri; Grakoui, Arash; Randall, Glenn

    2009-05-05

    Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha co-localized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.

  2. Genetic Diversity and Selective Pressure in Hepatitis C Virus Genotypes 1–6: Significance for Direct-Acting Antiviral Treatment and Drug Resistance

    Directory of Open Access Journals (Sweden)

    Lize Cuypers

    2015-09-01

    Full Text Available Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1–6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity and an additional 24% as pan-genotypic conserved (≥95%. Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%–0.46% and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15, 33% (3/9, and 14% (2/14 of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%. NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may

  3. Preheating and Entropy Perturbations in Axion Monodromy Inflation

    CERN Document Server

    ,

    2016-01-01

    We study the preheating of gauge fields in a simple axion monodromy model and compute the induced entropy perturbations and their effect on the curvature fluctuations. We find that the correction to the spectrum of curvature perturbations has a blue spectrum with index $n_s = 5/2$. Hence, these induced modes are harmless for the observed structure of the universe. Since the spectrum is blue, there is the danger of overproduction of primordial black holes. However, we show that the observational constraints are easily satisfied.

  4. Brane Brick Models, Toric Calabi-Yau 4-Folds and 2d (0,2) Quivers

    CERN Document Server

    Franco, Sebastian; Seong, Rak-Kyeong

    2015-01-01

    We introduce brane brick models, a novel type of Type IIA brane configurations consisting of D4-branes ending on an NS5-brane. Brane brick models are T-dual to D1-branes over singular toric Calabi-Yau 4-folds. They fully encode the infinite class of 2d (generically) N=(0,2) gauge theories on the worldvolume of the D1-branes and streamline their connection to the probed geometries. For this purpose, we also introduce new combinatorial procedures for deriving the Calabi-Yau associated to a given gauge theory and vice versa.

  5. Sofosbuvir treatment and hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    Hepatitis C virus (HCV) infection is a serious problemworldwide. The use of interferon-based therapy hasmade HCV eradication challenging. The recent appearanceof direct-acting antiviral agents (DAAs) haschanged HCV therapy. Combining the use of DAAs withpeginterferon and ribavirin has improved treatmentefficacy. Furthermore, the combination of different orallyadministered DAAs has enabled interferon-free therapywith much higher efficacy and safety. In particular,sofosbuvir, a nucleotide-based NS5B inhibitor, preventsHCV RNA synthesis by acting as a "chain terminator".Treatment with sofosbuvir has attained an extremelyhigh rate of sustained virologic response. The currentreview summarizes the efficacy and safety of sofosbuvirtherapy.

  6. I,ROBOT

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    《少电》的读者们,大家好。我是来自未来的全能型机器人NS-5——如果大家觉得这十名字太生硬,可以叫我Robot(罗伯特),这是一个对所有机器人适用的名字,嘿嘿。相信大家都知道,个人计算机在上个世纪的八十年代得

  7. A Chemopreventive Trial to Study the Effects of High Tea Consumption on Smoking-Related Oxidative Stress

    Science.gov (United States)

    2010-03-01

    best outcome in lipid profile with an overall decrease in increase in HDL and decrease in LDL , triglycerides, and significant decrease in total...Males Cholesterol -11.1 (-16.4, 18.8) NS 5.9 (-5.9, 029.6) NS LDL -1.6...0.22, 0.28) NS -7.3 (-0.35, 0.17) NS HDL 2.1 (-0.08, 016) NS 1.2 (-0.07, 0.18) NS Triglycerides 12.2

  8. Baseband Eye Monitor Signal Discrimination and Identification Study Program

    Science.gov (United States)

    1980-06-01

    separation is complete. To identify an unclassified individual, use its X, anu X2 values used ir: the dato base. The individual is then considered in...S5IS0NNS4I~NI -SO0U 5.0~tSIS.Ns 5.-NO N voZ ’ *C. 0.. A 0 O * 1D ZIO N OA *~*s 9 11 ID 1,4 ** 0-~ *11A~A0 .L 7 07004’ J’N 500 *..SO.*0.S *US 5 0 475 oo *.. -0

  9. Genetic Diversity and Selective Pressure in Hepatitis C Virus Genotypes 1–6: Significance for Direct-Acting Antiviral Treatment and Drug Resistance

    Science.gov (United States)

    Cuypers, Lize; Li, Guangdi; Libin, Pieter; Piampongsant, Supinya; Vandamme, Anne-Mieke; Theys, Kristof

    2015-01-01

    Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1–6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity) and an additional 24% as pan-genotypic conserved (≥95%). Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%–0.46%) and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15), 33% (3/9), and 14% (2/14) of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%). NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may need to be

  10. Non-geometric branes are DFT monopoles

    CERN Document Server

    Bakhmatov, Ilya; Musaev, Edvard T

    2016-01-01

    The double field theory monopole solution by Berman and Rudolph is shown to reproduce non-geometric backgrounds with non-vanishing Q- and R-flux upon an appropriate choice of physical and dual coordinates. The obtained backgrounds depend non-trivially on dual coordinates and have only trivial monodromies. Upon smearing the solutions along the dual coordinates one reproduces the known $5^2_2$ solution for the Q-brane and co-dimension 1 solution for the R-brane. The T-duality invariant magnetic charge is explicitly calculated for all these backgrounds and is found to be equal to the magnetic charge of (unsmeared) NS5-brane.

  11. Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coli BL21,the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG),and it was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyclonal antibody,wi...

  12. A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

    Energy Technology Data Exchange (ETDEWEB)

    Yap, Thai Leong [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Chen, Yen Liang; Xu, Ting; Wen, Daying; Vasudevan, Subhash G. [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); Lescar, Julien, E-mail: julien@ntu.edu.sg [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2007-02-01

    Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program. Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 Å resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration.

  13. Mechanisms of Hepatitis C Viral Resistance to Direct Acting Antivirals

    Directory of Open Access Journals (Sweden)

    Asma Ahmed

    2015-12-01

    Full Text Available There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.

  14. Direct anti-HCV agents

    Directory of Open Access Journals (Sweden)

    Xingquan Zhang

    2016-01-01

    Full Text Available Unlike human immunodeficiency virus (HIV and hepatitis B virus (HBV, hepatitis C virus (HCV infection is a curable disease. Current direct antiviral agent (DAA targets are focused on HCV NS3/4A protein (protease, NS5B protein (polymerase and NS5A protein. The first generation of DAAs includes boceprevir and telaprevir, which are protease inhibitors and were approved for clinical use in 2011. The cure rate for genotype 1 patients increased from 45% to 70% when boceprevir or telaprevir was added to standard PEG-IFN/ribavirin. More effective and less toxic second generation DAAs supplanted these drugs by 2013. The second generation of DAAs includes sofosbuvir (Sovaldi, simeprevir (Olysio, and fixed combination medicines Harvoni and Viekira Pak. These drugs increase cure rates to over 90% without the need for interferon and effectively treat all HCV genotypes. With these drugs the “cure HCV” goal has become a reality. Concerns remain about drug resistance mutations and the high cost of these drugs. The investigation of new HCV drugs is progressing rapidly; fixed dose combination medicines in phase III clinical trials include Viekirax, asunaprevir+daclatasvir+beclabuvir, grazoprevir+elbasvir and others.

  15. Cloning of RNA-dependent RNA Polymerase (RdRp Gene from Genotype Dengue Type-2 (New Guinea-C Strain

    Directory of Open Access Journals (Sweden)

    Samian, R.

    2005-01-01

    Full Text Available Dengue virus causes febrile disease in human. Dengue infection causes dengue fever that is not life threatening. However, a severe form of the disease called dengue hemorrhagic fever (DH or dengue shock syndrome (DSS, proven to be fatal. A positive single stranded RNA virus genome encodes for a single polyprotein precursor and is arranged in the order of NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH. The purpose of this study was to clone NS5 gene that encodes for the RNA-dependent RNA polymerase (RdRp. This enzyme plays an important role in viral RNA replication. The RdRp associated by cofactors produce minus-strand single stranded RNA, which in turn, serves as a template for the production of new plus-strand single stranded genome. The virus RNA was extracted from Aedes albopictus cell line C6/36 that was infected with dengue virus type 2. Then, the extracted virus RNA was used as the template for RT-PCR. A 2.7 kb DNA fragment, representing the RNA-dependent RNA polymerase gene, wassuccessfully amplified using specific primers. The PCR product was then cloned into cloning vector (pGEM-T and transformed into E. coli JM109.

  16. Clonación de secuencias de alfavirus y flavivirus para su uso como controles positivos en el diagnóstico molecular

    Directory of Open Access Journals (Sweden)

    Daría Camacho

    Full Text Available El objetivo de la investigación fue obtener controles positivos para la validación de técnicas moleculares (RT-PCR utilizadas en diagnóstico e investigación de infecciones virales. A partir de cepas de CHIKV, Zika, DENV-1, DENV-2, DENV-3 y DENV-4, se extrajeron ARN virales para obtener por RT-PCR los ADN complementarios (ADNc de las secuencias nsP4 (CHIKV, NS5 (virus Zika, C/prM-M y 5´UTR-C (DENV-1, DENV-2, DENV-3, DENV-4 que fueron clonados en pGEM®-T Easy. La clonación se confirmó mediante PCR de colonias, de las cuales se extrajo el ADN plasmídico para la verificación de la clonación de los fragmentos. Se logró la clonación de ADNc correspondientes a nsP4, NS5, C/prM-M y 5´UTR-C de los distintos agentes virales. En conclusión se obtuvieron los plásmidos recombinantes con cada una de las secuencias especificadas para su posterior valoración como controles positivos en técnicas moleculares, evitando el uso de cultivos celulares que pueden resultar costosos, laboriosos y potencialmente peligrosos.

  17. A report on the outbreak of Zika virus on Easter Island, South Pacific, 2014.

    Science.gov (United States)

    Tognarelli, J; Ulloa, S; Villagra, E; Lagos, J; Aguayo, C; Fasce, R; Parra, B; Mora, J; Becerra, N; Lagos, N; Vera, L; Olivares, B; Vilches, M; Fernández, J

    2016-03-01

    Zika virus (ZIKV) is an emerging mosquito-borne flavivirus circulating in Asia and Africa. In 2013, a large outbreak was reported on the archipelago of French Polynesia. In this study, we report the detection and molecular characterization of Zika virus for the first time in Chile from an outbreak among the inhabitants of Easter Island. A total of 89 samples from patients suspected of having ZIKV infection were collected between the period from January to May, 2014. Molecular diagnosis of the virus was performed by RT-PCR followed by the sequencing of the region containing the NS5 gene. A comparison of the viral nucleic acid sequence with those of other strains of ZIKA virus was performed using the MEGA software. Fifty-one samples were found positive for ZIKV by RT-PCR analysis. Further analysis of the NS5 gene revealed that the ZIKV strains identified in Easter Island were most closely related to those found in French Polynesia (99.8 to 99.9% nt and 100% aa sequence identity). These results strongly suggest that the transmission pathway leading to the introduction of Zika virus on Easter Island has its origin in French Polynesia.

  18. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J. (Pharmasset); (Emerald)

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  19. Investigating Inflation in Type IIA

    Energy Technology Data Exchange (ETDEWEB)

    Hertzberg, Mark P.; /MIT; Kachru, Shamit; /Stanford U., Phys. Dept. /SLAC; Taylor, Washington; Tegmark, Max; /MIT, LNS

    2007-12-14

    We prove that inflation is forbidden in the most well understood class of semi-realistic type IIA string compactifications: Calabi-Yau compactifications with only standard NS-NS 3-form flux, R-R fluxes, D6-branes and O6-planes at large volume and small string coupling. With these ingredients, the first slow-roll parameter satisfies {epsilon} {ge} 27/13 whenever V > 0, ruling out both inflation (including brane/anti-brane inflation) and de Sitter vacua in this limit. Our proof is based on the dependence of the 4-dimensional potential on the volume and dilaton moduli in the presence of fluxes and branes. We also describe broader classes of IIA models which may include cosmologies with inflation and/or de Sitter vacua. The inclusion of extra ingredients, such as NS 5-branes and geometric or non-geometric NS-NS fluxes, evades the assumptions used in deriving the no-go theorem. We focus on NS 5-branes and outline how such ingredients may prove fruitful for cosmology, but we do not provide an explicit model. We contrast the results of our IIA analysis with the rather different situation in IIB.

  20. Genetic diversity of the hepatitis C virus: Impact and issues in the antiviral therapy

    Institute of Scientific and Technical Information of China (English)

    H Le Guillou-Guillemette; S Vallet; C Gaudy-Graffin; C Payan; A Pivert; A Goudeau; F Lunel-Fabiani

    2007-01-01

    The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The resuiting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies.This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the predictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic success. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antiviral therapy in patients infected by the same HCV genotype. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Interferon pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity.Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non-structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.

  1. Recurrent hepatitis C after liver transplant.

    Science.gov (United States)

    deLemos, Andrew S; Schmeltzer, Paul A; Russo, Mark W

    2014-08-21

    End stage liver disease from hepatitis C is the most common indication for liver transplantation in many parts of the world accounting for up to 40% of liver transplants. Antiviral therapy either before or after liver transplantation is challenging due to side effects and lower efficacy in patients with cirrhosis and liver transplant recipients, as well as from drug interactions with immunosuppressants. Factors that may affect recurrent hepatitis C include donor age, immunosuppression, IL28B genotype, cytomegalovirus infection, and metabolic syndrome. Older donor age has persistently been shown to have the greatest impact on recurrent hepatitis C. After liver transplantation, distinguishing recurrent hepatitis C from acute cellular rejection may be difficult, although the development of molecular markers may help in making the correct diagnosis. The advent of interferon free regimens with direct acting antiviral agents that include NS3/4A protease inhibitors, NS5B polymerase inhibitors and NS5A inhibitors holds great promise in improving outcomes for liver transplant candidates and recipients.

  2. Host cell kinases and the hepatitis C virus life cycle.

    Science.gov (United States)

    Colpitts, Che C; Lupberger, Joachim; Doerig, Christian; Baumert, Thomas F

    2015-10-01

    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

  3. Zebrafish as a potential model organism for drug test against hepatitis C virus.

    Directory of Open Access Journals (Sweden)

    Cun-Bao Ding

    Full Text Available Screening and evaluating anti- hepatitis C virus (HCV drugs in vivo is difficult worldwide, mainly because of the lack of suitable small animal models. We investigate whether zebrafish could be a model organism for HCV replication. To achieve NS5B-dependent replication an HCV sub-replicon was designed and created with two vectors, one with HCV ns5b and fluorescent rfp genes, and the other containing HCV's 5'UTR, core, 3'UTR and fluorescent gfp genes. The vectors containing sub-replicons were co-injected into zebrafish zygotes. The sub-replicon amplified in liver showing a significant expression of HCV core RNA and protein. The sub-replicon amplification caused no abnormality in development and growth of zebrafish larvae, but induced gene expression change similar to that in human hepatocytes. As the amplified core fluorescence in live zebrafish was detectable microscopically, it rendered us an advantage to select those with replicating sub-replicon for drug experiments. Ribavirin and oxymatrine, two known anti-HCV drugs, inhibited sub-replicon amplification in this model showing reduced levels of HCV core RNA and protein. Technically, this method had a good reproducibility and is easy to operate. Thus, zebrafish might be a model organism to host HCV, and this zebrafish/HCV (sub-replicon system could be an animal model for anti-HCV drug screening and evaluation.

  4. Adaptive mutations enhance assembly and cell-to-cell transmission of a high-titer hepatitis C virus genotype 5a Core-NS2 JFH1-based recombinant

    DEFF Research Database (Denmark)

    Mathiesen, Christian K; Prentoe, Jannick; Meredith, Luke W;

    2015-01-01

    -NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81......UNLABELLED: Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells......, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1orig mutant termed SA13/JFH1Core-NS5B...

  5. Identification of hepatitis C virus genotype 2a replicon variants with reduced susceptibility to ribavirin.

    Science.gov (United States)

    Hmwe, Su Su; Aizaki, Hideki; Date, Tomoko; Murakami, Kyoko; Ishii, Koji; Miyamura, Tatsuo; Koike, Kazuhiko; Wakita, Takaji; Suzuki, Tetsuro

    2010-03-01

    Ribavirin (RBV), a nucleoside analogue, is used in the treatment of hepatitis C virus (HCV) infection in combination with interferons. However, potential mechanisms of RBV resistance during HCV replication remain poorly understood. Serial passage of cells harboring HCV genotype 2a replicon in the presence of RBV resulted in the reduced susceptibility of the replicon to RBV. Transfection of fresh cells with RNA from RBV-resistant replicon cells demonstrated that the RBV resistance observed is largely replicon-derived. Four major amino acid substitutions: T1134S in NS3, P1969S in NS4B, V2405A in NS5A, and Y2471H in NS5B region, were identified. Site-directed mutagenesis of these mutations into the replicon indicated that Y2471H plays a role in the reduced susceptibility to RBV and leads to decrease in replication fitness. The results, in addition to analysis of sequence database, suggest that HCV variants with reduced susceptibility to RBV identified are preferential to genotype 2a.

  6. Evaluation of canonical siRNA and Dicer substrate RNA for inhibition of hepatitis C virus genome replication--a comparative study.

    Directory of Open Access Journals (Sweden)

    Bruno Carneiro

    Full Text Available Hepatitis C virus (HCV frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. The objective of this study was to test the ability of five Dicer substrate siRNAs (DsiRNA to inhibit HCV replication and to compare these molecules to canonical 21 nt siRNA. DsiRNA molecules were designed to target five distinct regions of the HCV genome - the 5' UTR and the coding regions for NS3, NS4B, NS5A or NS5B. These molecules were transfected into Huh7.5 cells that stably harboured an HCV subgenomic replicon expressing a firefly luciferase/neoR reporter (SGR-Feo-JFH-1 and were also tested on HCVcc-infected cells. All of the DsiRNAs inhibited HCV replication in both the subgenomic system and HCVcc-infected cells. When DsiRNAs were transfected prior to infection with HCVcc, the inhibition levels reached 99.5%. When directly compared, canonical siRNA and DsiRNA exhibited similar potency of virus inhibition. Furthermore, both types of molecules exhibited similar dynamics of inhibition and frequencies of resistant mutants after 21 days of treatment. Thus, DsiRNA molecules are as potent as 21 nt siRNAs for the inhibition of HCV replication and may provide future approaches for HCV therapy if the emergence of resistant mutants can be addressed.

  7. Differential in vivo clearance and response to secondary heterologous infections by H2(b)-restricted dengue virus-specific CD8+ T cells.

    Science.gov (United States)

    Beaumier, Coreen M; Jaiswal, Smita; West, Kim Y; Friberg, Heather; Mathew, Anuja; Rothman, Alan L

    2010-10-01

    Cytotoxic T lymphocytes (CTL) are hypothesized to play a role in clearance during primary dengue virus (DENV) infections, and contribute to immunopathology during secondary heterologous infections in humans. We previously reported skewed T-cell responses to secondary DENV infection in BALB/c (H-2(d)) mice, reproducing characteristics of human DENV infection. To set the stage for using widely available transgenic and knockout mice, we extended these studies to identify DENV-specific T-cell responses in C57BL/6 (H-2(b)) mice. We identified dominant CD8+ T-cell responses to H-2D(b)-restricted epitopes on the DENV NS4a (aa 249-265) and NS5 (aa 521-537) proteins. High frequencies of IFN-γ- and TNF-α-producing T cells directed at both epitopes were detected following primary infection with all four DENV serotypes, and were augmented by secondary DENV infections. In vivo cytotoxicity assays demonstrated rapid clearance of target cells pulsed with the NS4a peptide; in contrast, NS5 peptide-pulsed target cells were poorly cleared in vivo. These data characterize two H-2(b)-restricted T-cell epitopes displaying divergent in vivo function. These results should facilitate further studies of the in vivo effects of DENV-specific T cells, including the use of genetically modified mouse strains.

  8. Association of Hepatitis C Virus With Insulin Resistance: Evidences From Animal Studies and Clinical Studie

    Directory of Open Access Journals (Sweden)

    Sadaf Badar

    2012-01-01

    Full Text Available Context: HCV infection is strongly associated with development of insulin resistance and type-2 diabetes, however molecular mechanism of these associations is not known. The aim of this review was to conduct a comprehensive literature search to understand the nature of the association between hepatitis C virus (HCV infection and insulin resistance (IR. We also explored the role of HCV core protein and NS5a in modulating the course of the insulin-signaling pathway.Evidence Acquisitions: We searched Directory of Open Access Journals (DOAJ Google Scholar, Pubmed (NLM, LISTA (EBSCO, Web of Science (TS and PakMediNet.Results: Emerging evidence suggests an association between HCV infection and carotid/coronary vascular disease. IR appears to be a dominant underlying cause of accelerated atherosclerosis in patients with chronic hepatitis C (CHC. HCV can induce IR directly through the stimulation of SOCS3 and PPA2, and both of these molecules have been shown to inhibit interferon-α signaling. Improvement of insulin sensitivity may increase the response rate to antiviral treatment and prevent IR complications, including vascular diseases. The results of several clinical trials that have used insulin sensitizers (metformin and PPAR-γ agonists have been inconclusive.Conclusions: Beside the association between HCV and IR, the published data also have showed the possible association of HCV core and NS5A protein with IR.

  9. 3d N=2 mirror symmetry, pq-webs and monopole superpotentials

    CERN Document Server

    Benvenuti, Sergio

    2016-01-01

    D3 branes stretching between webs of (p,q) 5branes provide an interesting class of 3d N=2 theories. For generic pq-webs however the low energy field theory is not known. We use 3d mirror symmetry and Type IIB S-duality to construct Abelian gauge theories corresponding to D3 branes ending on both sides of a pq-web made of many coincident NS5's intersecting one D5. These theories contain chiral monopole operators in the superpotential and enjoy a non trivial pattern of global symmetry enhancements. In the special case of the pq-web with one D5 and one NS5, the 3d low energy SCFT admits three dual formulations. This triality can be applied locally inside bigger quiver gauge theories. We prove our statements using partial mirror symmetry \\`a la Kapustin-Strassler, showing the equality of the S^3_b partition functions and studying the quantum chiral rings.

  10. Genome-wide analysis for identification of adaptive diversification between hepatitis C virus subtypes 1a and 1b.

    Science.gov (United States)

    Li, Yan; Wang, Ruirui; Du, Xiaogang; Zhang, Mingwang; Xie, Meng

    2016-07-01

    Hepatitis C virus (HCV) is a major cause of liver disease and has been estimated to infect approximately 2%-3% of the world's population. HCV genotype 1 is the subject of intense research and clinical investigations because of its worldwide prevalence and poor access to treatment for patients in developing countries and marginalized populations. The predominant subtypes 1a and 1b of HCV genotype 1 present considerable differences in epidemiological features. However, the genetic signature underlying such phenotypic functional divergence is still an open question. Here, we performed a genome-wide evolutionary study on HCV subtypes 1a and 1b. The results show that adaptive selection has driven the diversification between these subtypes. Furthermore, the major adaptive divergence-related changes have occurred on proteins E1, NS4B, NS5A, and NS5B. Structurally, a number of adaptively selected sites cluster in functional regions potentially relevant to (i) membrane attachment and (ii) the interactions with viral and host cell factors and the genome template. These results might provide helpful hints about the molecular determinants of epidemiological divergence between HCV 1a and 1b.

  11. FDA approved hepatitis C virus drug Sovaldi (sofosbuvir)%FDA批准抗丙型肝炎新药索非布韦(sofosbuvir)上市

    Institute of Scientific and Technical Information of China (English)

    蔡巍; 陈斌; 田宁

    2014-01-01

    全球丙型肝炎病毒(HCV)的感染率高,且缺少有效的治疗药物.2013年12月6日,美国食品药物监督管理局(FDA)批准新分子实体药物索非布韦(sofosbuvir)片剂上市,商品名Sovaldi,用于慢性丙型肝炎的治疗.索非布韦是一种HCV聚合酶抑制剂,作用于病毒RNA复制的核苷酸类似物NS5B聚合酶位点,能中止病毒复制,是以NS5B聚合酶为靶点的唯一上市品种.本品与聚乙二醇干扰素/利巴韦林或单独与利巴韦林联用,与标准治疗方案相比治愈率更高且缩短给药时间,有广阔的应用前景.

  12. Ledipasvir/sofosbuvir fixed-dose combination for treatment of hepatitis C virus genotype 4 infection.

    Science.gov (United States)

    Nehra, V; Tan, E M; Rizza, S A; Temesgen, Z

    2016-02-01

    Hepatitis C virus (HCV) genotype 4 accounts for 8-13% of all chronic HCV infections worldwide. Patients with HCV genotype 4 have been reported to have poor treatment responses to PEGylated interferon and ribavirin regimens. Recently a single tablet, fixed-dose combination of sofosbuvir, an RNA-directed RNA polymerase (NS5B) inhibitor, and ledipasvir, a nonstructural protein 5A (NS5A) inhibitor, has been approved for treatment of chronic HCV infection. Two studies using the fixed-dose combination in chronic HCV genotype 4 for 12 weeks reported sustained virologic response rates at 12 weeks (SVR12) of 93-95%. Data also support the use of ledipasvir/sofosbuvir in chronic HCV genotype 4 and HIV co-infection. Administered as a single once-daily oral regimen, this ribavirin- and interferon-free regimen is well tolerated, with low potential for adverse effects and represents a significant advancement in the treatment of chronic HCV genotype 4 infection.

  13. Dual daclatasvir and sofosbuvir for treatment of genotype 3 chronic hepatitis C virus infection.

    Science.gov (United States)

    Sundaram, Vinay; Kowdley, Kris V

    2016-01-01

    Chronic hepatitis C virus (HCV) infection is one of the most common etiologies of liver-related mortality throughout the world. Traditionally, therapy has been focused on pegylated interferon in combination with ribavirin, with clinical trials demonstrating that HCV genotype 1 had the lowest response rate (40-50%), while genotype 3 had an intermediate response rate (60-70%). Recently, significant advances have been made with all-oral direct-acting antiviral (DAA) therapy, which have significantly improved cure rates for HCV genotype 1. Accordingly, HCV genotype 3 is now potentially the most difficult to treat. One of the most potent DAA medications is sofosbuvir, a pan-genotypic nucleotide analogue that inhibits the NS5B polymerase of HCV. Daclatasvir, a pan-genotypic inhibitor of the HCV NS5A replication complex, was recently approved in the United States for treatment of HCV genotype 3 in conjunction with sofosbuvir. This combination may provide a powerful tool in the treatment of HCV genotype 3.

  14. Sofosbuvir/velpatasvir: A promising combination

    Institute of Scientific and Technical Information of China (English)

    Aldo; Bonaventura; Fabrizio; Montecucco

    2016-01-01

    Hepatitis C virus(HCV) affects 3% of the world population. It represents the main cause of chronic liver disease and is responsible for extra-hepatic complications, such as type 2 diabetes and cardiovascular diseases. HCV includes 7 genotypes differing in the nucleotide sequence variability, the geographic distribution, the rates of viral clearance, the risk of progression to liver fibrosis and to hepatocellular carcinoma, and the response to therapy. Last years have seen remarkable advances in the field of HCV infection with the approval of direct antiviral agents(DAAs) targeting key viral proteins involved in the HCV replication. Several oral regimens combining DAAs from different families have been developed and these regimens showed increased and sustained virological response rates to above 90% reducing the treatment duration to 12 wk or less. In particular, sofosbuvir, a nucleotide analogue nonstructural(NS)5B polymerase inhibitor, and velpatasvir, a NS5 A inhibitor, have been tested in two phase 3 trials, the ASTRAL-2(against HCV genotype 2) and the ASTRAL-3(against HCV genotype 3), demonstrating to be effective, safe, and well tolerated in patients who were 18 years of age or older and had at least a 6-mo history of HCV infection with a compensated liver disease.

  15. Daclatasvir: potential role in hepatitis C

    Directory of Open Access Journals (Sweden)

    Lee C

    2013-10-01

    Full Text Available Choongho Lee College of Pharmacy, Dongguk University-Seoul, Goyang, Republic of Korea  Abstract: Chronic hepatitis C virus (HCV infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. It has been a tremendous burden on global health care systems. With the advent of a number of new direct-acting and host-targeting antiviral agents, current interferon-α- and ribavirin-based HCV therapy has started to move towards an interferon-sparing or even interferon-free strategy. In this regard, a recently identified NS5A inhibitor, daclatasvir, showed a great promise in clinical trials as another new class of direct-acting anti-HCV therapeutics, with a distinct mechanism of action. In this review, a variety of preclinical as well as clinical proof-of-concept studies of daclatasvir, including the studies of its discovery, mechanism of action, viral resistance, and host polymorphism profiles are reviewed. In addition, a role of daclatasvir in the future therapy for HCV patients is discussed briefly. Keywords: hepatitis C virus, nonstructural protein 5A, NS5A inhibitor, hepatitis C treatment

  16. [Molecular characterization of hepatitis C virus in patients referred to a reference laboratory of public health, University Hospital "Antonio Patricio de Alcalá", Cumani, Venezuela].

    Science.gov (United States)

    Sulbarán, María Zulay; Montaño, Lérida; Sulbarán, Yoneira; Loureiro, Carmen Luisa; Flores, Carmen Rosa; Farías, Yurviris; Maldonado, Antonio; Guillen, Genny; Rangel, Héctor; Pujol, Flor

    2016-03-01

    The World Health Organization estimates that approximately 170 million people are chronically infected with hepatitis C virus (HCV). This study evaluated the presence of antibodies against HCV by two immunoassays. HCV genotypes were analyzed by phylogenetic analysis of viral genome fragments amplified from the 5 'non-coding (5'NC) region and non-structural region 5b (NS5b), using reverse transcription and nested polymerase chain reaction (RT-PCR), in patients referred from January 2010 to February 2013 to the Reference Laboratory of Public Health, University Hospital "Antonio Patricio de Alcalá". The prevalence of anti-HCV antibodies was 0.57% (17/3005), being the group of patients older than 41 years the most affected (0.9%). A total of 16 samples were found positive for HCV RNA by RT-PCR in the 5'NC region (16/17, 94%). Phylogenetic analysis of the 5'NC region allowed to identify the circulation of genotypes 2 and 1, and one genotype 3 and one 4. By phylogenetic analysis of the NS5b region, diverse subtypes of HCV genotype 2 were identified (2a, 2j and 2s). This finding is in accordance with previous studies that indicate that this genotype is relatively diverse in our country.

  17. New hepatitis C therapies in clinical development

    Directory of Open Access Journals (Sweden)

    Vermehren Johannes

    2011-07-01

    Full Text Available Abstract With the current standard of care for the treatment of chronic hepatitis C, a combination of pegylated interferon alfa and ribavirin, sustained virologic response rates can be achieved in approximately 50% of patients only. Improved understanding of the viral life cycle has led to the identification of numerous potential targets for novel, direct-acting antiviral compounds. Inhibitors of the NS3/4A protease are currently the most advanced in clinical development. Recently completed phase 3 studies of the two protease inhibitors telaprevir and boceprevir, each given in combination with standard of care, yielded sustained virologic response rates in the range of 66-75% in treatment-naive patients and 59-66% in treatment-experienced patients with HCV genotype 1 infection. Studies of second-generation protease inhibitors, with the potential advantage of improved potency, drug metabolism and pharmacokinetics profile, are already underway. Inhibitors of the HCV NS5A protein and NS5B polymerase are potentially active across different HCV genotypes and have shown promising antiviral efficacy in early clinical studies. Other emerging mechanisms include silymarin components and inhibitors of cell proteins required for HCV replication. While improved formulations of current HCV therapies are also being developed, future hopes lie on the combination of direct-acting antivirals with the eventual possibility of interferon-free treatment regimens.

  18. Distribution and heterogeneity of hepatitis C genotypes in hepatitis patients in Cameroon.

    Science.gov (United States)

    Pasquier, Christophe; Njouom, Richard; Ayouba, Ahidjo; Dubois, Martine; Sartre, Michèle Tagni; Vessière, Aurelia; Timba, Isabelle; Thonnon, Jocelyn; Izopet, Jacques; Nerrienet, Eric

    2005-11-01

    Hepatitis C virus infects humans world-wide. The virus genome varies greatly and it has several genotypes. HCV infection is highly prevalent in Central Africa and Cameroon. Initial studies on the genetic variability of HCV showed infection with HCV genotypes 1, 2, and 4. We have now sequenced the NS5b and E2 regions of 156 HCV isolates collected from patients presenting for diagnosis in Yaounde and used the data to describe the distribution of HCV genotypes and subtypes in patients with hepatitis in Cameroon. Genotype 1 was more frequent than Genotypes 4 and 2. Genotypes 1 and 4 were highly heterogeneous, containing many subtypes described previously (1b, 1c, 1e, 1h, 1l, 4f, 4t, 4p, 4k) and unsubtyped groups. There was a systematic phylogenetic concordance between NS5b and E2 sequence clustering. The Genotype 2 sequences did not vary. Neither subject age nor gender influenced HCV distribution. HCV Genotypes 1 and 4 are very heterogeneous in Cameroon, perhaps due to ancient infections. The homogeneity of HCV Genotype 2 indicates its more recent introduction from western Africa.

  19. Innate immunity evasion by Dengue virus.

    Science.gov (United States)

    Morrison, Juliet; Aguirre, Sebastian; Fernandez-Sesma, Ana

    2012-03-01

    For viruses to productively infect their hosts, they must evade or inhibit important elements of the innate immune system, namely the type I interferon (IFN) response, which negatively influences the subsequent development of antigen-specific adaptive immunity against those viruses. Dengue virus (DENV) can inhibit both type I IFN production and signaling in susceptible human cells, including dendritic cells (DCs). The NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this function. DENV also encodes proteins that antagonize type I IFN signaling, including NS2A, NS4A, NS4B and NS5 by targeting different components of this signaling pathway, such as STATs. Importantly, the ability of the NS5 protein to bind and degrade STAT2 contributes to the limited host tropism of DENV to humans and non-human primates. In this review, we will evaluate the contribution of innate immunity evasion by DENV to the pathogenesis and host tropism of this virus.

  20. 广州地区人类白细胞抗原-Ⅰ类与丙型肝炎病毒-6a型感染相关性的研究%Study on association between hepatitis C-6a and HLA-Ⅰ allele in Guangzhou area

    Institute of Scientific and Technical Information of China (English)

    夏文杰; 叶欣; 付涌水; 王怡仲; 许茹; 徐秀章; 陈扬凯; 贝春花

    2011-01-01

    Objective :To study the association between hepatitis C-6a and HLA-Ⅰ allele in Guangzhou area .Methods :250 individuals of HCV antibodies positive and 621 health blood donors in Guangzhou area were enrolled in this study .Nested polymerase chain reaction (PCR ) was applied to detect HCV RNA in the sera .The gene sequences in E1 and NS5B area were detected for gene typing of HCV .LUMINEX SSO methods was used to detect the HLA-Ⅰ allele .The relative risk was evaluated in regular formular .Results :There were 69 samples as HCV-6a gene type in the 250 persons .RR value in A * 33 ,A * 30 was beyond 4 ,and RR value in B * 56 ,B * 54 ,B * 55 ,B * 58 ,B * 76 was also more than 4 .Conclusion :HLA-A19 and HLA-B22 are important genetic factor for infection of HCV-6a .%目的:广州地区HLA-Ⅰ类抗原和HCV-6a感染相关性的研究.方法:选取2002~2008年HCV抗体检测双试剂阳性无偿献血者250名,随机抽取621名广州地区无血缘关系的健康无偿献血者作为对照,用逆转录-套式聚合酶链反应扩增HCVE1和 NS5B基因,对E1和NS5B区扩增阳性的PCR产物核苷酸序列测定,将所测定的HCVE1、NS5B进行基因分型.LUMINEX-SSO方法检测HLA-A,B 检测样本HLA-A,B位点.根据公式对HLA型别相关性的相对危险性进行评估.结果:250名无偿献血者中有69个样本的HCVE1和NS5B区分型结果为HCV-6a,占到分析总数的27.6%.A*33、A*30与HCV-6a感染相关评估的RR值均大于4,B*56、B*54、B*55、B*58、B*76与HCV-6a感染相关评估的RR值均大于4,与HCV-6a感染具有强关联性.结论:广州地区HCV-6a的感染与A*19和B*22存在强关联性,对HCV-6a的地域性流行、治疗和预后评价有一定预见作用.

  1. Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication.

    Science.gov (United States)

    Wang, Chunfu; Pflugheber, Jill; Sumpter, Rhea; Sodora, Donald L; Hui, Daniel; Sen, Ganes C; Gale, Michael

    2003-04-01

    Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2alpha) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2alpha phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively

  2. Establishment of a SYBR Green Ⅰ-based real-time RT-PCR for rapid detection of duck Tembusu virus%鸭坦布苏病毒SYBR GreenⅠ实时荧光定量检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    万春和; 朱海侠; 施少华; 黄瑜; 程龙飞; 傅光华; 陈红梅

    2013-01-01

    A pair of specific primers targeted to non structure gene 5 (NS5) of duck Tembusu virus was designed and a SYBR Green Ⅰ fluorescent based real-time RT-PCR (RRT-PCR) was developed for the quantization of duck Tembusu virus.The detection limit of RRT-PCR was 2.74 × 102plasmid copies.The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak,a melting temperature (Tm) was (86.23±0.18) ℃,and no primer-dimers peak was observed.No amplification was detected from unrelated virus samples by this method,such as avian influenza virus,duck hepatitis virus type 1,avian paramyxovirus type 1,egg drop syndrome virus,duck reovirus.Fine reproducibility was obtained for detecting plasmid DNA with intra-assay of 0.52%1.48% and inter-assay of 0.71%-2.21%.The real-time PCR method developed in this study will be useful for rapid laboratory diagnosis and epidemiology investigation for duck Tembusu virus.%根据鸭坦布苏病毒(Duck Tembusu virus,DFV) NS5基因序列特征设计引物,建立基于SYBR Green Ⅰ检测模式的实时荧光定量RT-PCR(real-time RT-PCR,RRT-PCR),该方法检测DFV NS5基因2.74×103~2.74×10 7拷贝/μL反应范围内有很好的线性关系.扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为(86.23±0.18)℃,对禽流感病毒、鸭肝炎病毒、鸭源禽1型副黏病毒、鸭减蛋综合征病毒、番鸭呼肠孤病毒核酸均无阳性信号扩增,可重复性好,组内变异系数为0.52%~1.48%,组间变异系数0.71%~2.21%.检测速度快,从样本处理到报告结果仅需4h.

  3. 干扰素γ对丙型肝炎病毒复制子的抑制作用%The direct antiviral effect of interferon-γ on hepatitis C virus replicon

    Institute of Scientific and Technical Information of China (English)

    贾因棠; 魏来; 蒋栋; 丛旭; 费然

    2006-01-01

    目的研究干扰素(IFN)γ直接抑制丙型肝炎病毒(HCV)复制子的作用及其与IFN α联合用药的疗效,并对可能介导IFN γ抗HCV作用的干扰素刺激基因(ISG)的表达水平进行探讨.方法建立HCV复制子细胞模型,用IFN γ或IFN γ联合IFN α处理H CV复制子细胞,通过半定量逆转录聚合酶链反应(RT-PCR)及实时定量PCR检测细胞中HCV RNA水平;western blot检测细胞中HCV非结构蛋白5A(NS5A).结果 IFN γ对HCV RNA的复制及NS5A的表达有明显抑制作用,10 U/ml IFN γ可使HCV RNA较对照组减少36%,25 U/ml减少80%; IFN γ对HCV RNA及NS5A蛋白的抑制作用与其剂量及作用时间呈正相关.用IFN γ预处理的细胞对IFN α抗HCV的作用更敏感,且细胞内干扰素调节因子-1(IRF-1)、干扰素刺激基因因子3 γ(ISGF 3 γ)和2'5'-寡腺苷酸合成酶(2'5'-OAS)呈明显的诱导性表达.结论 IFN γ能有效抑制HCV RNA的复制,并与IFN α有协同抗HCV的作用.IRF-1、ISGF3γ和2'5'-OAS可能参与介导IFN γ抗HCV的作用.

  4. Identification of cytotoxic T lymphocyte epitopes in dengue virus serotype 1.

    Science.gov (United States)

    Duan, Zhiliang; Guo, Jianglong; Huang, Xi; Liu, Huifang; Chen, Xinyu; Jiang, Minghua; Wen, Jinsheng

    2015-07-01

    Dengue virus (DENV) has a serious and growing impact on global health and the exact role of DENV-specific CD8(+) T-cells in DENV infection is still uncertain. In the present study, SYFPEITHI algorithm was used to screen the amino acid sequence of Dengue virus serotype 1 (DENV-1) for potential epitopes, and seven putative HLA-A*1101-restricted and five putative HLA-A*2402-restricted epitopes conserved in hundreds of DENV-1 strains were synthesized. The binding affinity of these epitope candidates to corresponding HLA molecules was evaluated using competitive peptide-binding assay. The immunogenicity and specificity of peptides were further tested in HLA-A*1101 transgenic mice, HLA-A*2402 transgenic mice and peripheral blood mononuclear cells (PBMCs) of patients infected with DENV-1. Percentage inhibition (PI) values calculated in competitive peptide-binding assay showed that six peptides (E39-47 PTLDIELLK, NS5(505-513) GVEGEGLHK, NS2b(15-23) SILLSSLLK, NS5(561-569) ALLATSIFK, NS3(99-107) AVEPGKNPK, and NS4b(159-167) VVYDAKFEK) could bind to HLA-A*1101 molecule with high affinity and five peptides (NS3472-480 QYIYMGQPL, NS4a40-48 AYRHAMEEL, NS5(880-888) DYMTSMKRF, NS3(548-556) SYKVASEGF, and NS3(22-30) IYRILQRGL) have a high affinity for HLA-A*2402 molecule. Enzyme-linked immunospot (ELISPOT) results indicated that these high-affinity peptides were recognized by splenocytes of DENV-1-infected transgenic mice and high-affinity peptide-immunized transgenic mice displayed high levels of peptide-specific IFN-γ-secreting cells. In addition, both peptide-pulsed splenocytes and DENV-1-infected splenic monocytes were efficiently killed by these peptide-specific cytotoxic T lymphocytes. Finally, except NS2b(15-23), 10 high-affinity peptides were recognized by PBMCs of patients infected with DENV-1. These identified epitopes would contribute to the understanding of the function of DENV-specific CD8(+) T-cells.

  5. Drug-Drug Interactions between Sofosbuvir and Ombitasvir-Paritaprevir-Ritonavir with or without Dasabuvir.

    Science.gov (United States)

    King, Jennifer R; Dutta, Sandeep; Cohen, Daniel; Podsadecki, Thomas J; Ding, Bifeng; Awni, Walid M; Menon, Rajeev M

    2016-02-01

    The combination of ombitasvir (an NS5A inhibitor), paritaprevir (an NS3/4A inhibitor) coadministered with ritonavir (r), and dasabuvir (an NS5B nonnucleoside polymerase inhibitor), referred to as the 3D regimen, and the combination of ombitasvir-paritaprevir-r, referred to as the 2D regimen, have demonstrated high efficacy with and without ribavirin in hepatitis C virus (HCV)-infected subjects. These regimens have potential for coadministration with sofosbuvir (nucleoside NS5B inhibitor) in the treatment of HCV. This phase 1, drug-drug interaction, open-label, multiple-dose study enrolled 32 healthy subjects to receive the 3D or 2D regimen in combination with sofosbuvir. Doses of study drugs were as follows: ombitasvir-paritaprevir-r, 25/150/100 mg daily (QD); dasabuvir, 250 mg twice daily (BID); and sofosbuvir, 400 mg QD. Blood samples were collected on study days 7, 14, and 21 for evaluating drug interaction at steady state. The effect of the 3D and 2D regimens on the pharmacokinetics of sofosbuvir and its circulating metabolite GS-331007 and vice versa was assessed by a repeated-measures analysis. Exposures of the 3D and 2D regimens were similar (≤20% change) during coadministration with sofosbuvir and during administration alone. Sofosbuvir exposures were 61% to 112% higher with the 3D regimen and 64% to 93% higher with the 2D regimen than with sofosbuvir alone. GS-331007 total exposures were 27% and 32% higher with the 3D and 2D regimens, respectively, than with sofosbuvir alone. Increases in sofosbuvir and GS-331007 exposures likely resulted from breast cancer resistance protein (BCRP) and/or P glycoprotein (P-gp) transporter inhibition by paritaprevir and ritonavir. No subjects discontinued the study due to study drug-related adverse events. No dose adjustment is recommended for 3D, 2D, or sofosbuvir in clinical trials exploring the safety and efficacy of the combination. (This study has been registered at ClinicalTrials.gov under registration no. NCT

  6. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

    Science.gov (United States)

    Li, Lian-Feng; Yu, Jiahui; Li, Yongfeng; Wang, Jinghan; Li, Su; Zhang, Lingkai; Xia, Shui-Li; Yang, Qian; Wang, Xiao; Yu, Shaoxiong; Luo, Yuzi; Sun, Yuan; Zhu, Yan; Munir, Muhammad

    2016-01-01

    ABSTRACT Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV

  7. Hepatitis C virus infection and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Richard Fischer; Thomas Baumert; Hubert E Blum

    2007-01-01

    Apoptosis is central for the control and elimination of viral infections. In chronic hepatitis C virus (HCV) infection,enhanced hepatocyte apoptosis and upregulation of the death inducing ligands CD95/Fas, TRAIL and TNFα occur.Nevertheless, HCV infection persists in the majority of patients. The impact of apoptosis in chronic HCV infection is not well understood. It may be harmful by triggering liver fibrosis, or essential in interferon (IFN)induced HCV elimination. For virtually all HCV proteins,pro- and anti-apoptotic effects have been described,especially for the core and NS5A protein. To date, it is not known which HCV protein affects apoptosis in vivo and whether the infectious virions act pro- or antiapoptotic. With the availability of an infectious tissue culture system, we now can address pathophysiologically relevant issues. This review focuses on the effect of HCV infection and different HCV proteins on apoptosis and of the corresponding signaling cascades.

  8. Impact of the Future Merchant Fleet on Coast Guard Operating and Support Programs Over the Next 25 Years.

    Science.gov (United States)

    1981-04-01

    ISO Golo 4f0 0 0 s4nf~-h~SS5 O2 Ŕ’S Ca....Sf--’c.,Cs~.a’s M Q Q’ r d . ’ NS 5 -a-4sSS h 9S rx ki u ’a- -. u. -.4S CA~ "- ’h SN s-1 13 - -5S 7 7 S...38 15000 20500 54 13500 18500 S5 8500 11500 6 26500 3550O o 15500 21000 59 26000 35000 60 23000 31000 s0 10000 13500 1 O1500 2000 82 4500 6000 83 11000...15000 84 15500 21000 01 23500 29500 ,9 3000 4000 92 19000 26000 03 1950 26500 61 300 300 Source: Reference A-19, Exhibit VIII-7. L-5 TABLE L-2 US

  9. Tetrazole and triazole as bioisosteres of carboxylic acid: discovery of diketo tetrazoles and diketo triazoles as anti-HCV agents.

    Science.gov (United States)

    Song, Wu-Hui; Liu, Ming-Ming; Zhong, Dong-Wei; Zhu, Ye-lin; Bosscher, Mike; Zhou, Lu; Ye, De-Yong; Yuan, Zheng-Hong

    2013-08-15

    A series of diketo tetrazoles and diketo triazoles were designed and synthesized as bioisosteres of α,γ-diketo acid, the active site inhibitor of HCV (Hepatitis C virus) polymerase NS5B. Among the synthesized compounds, 4-(4-fluorobenzyloxy)phenyl diketo triazole (30) exhibited anti-HCV activity with an EC50 value of 3.9 μM and an SI value more than 128. The reduction of viral protein and mRNA levels were also validated, supporting the anti-HCV activity of compound 30. These results provide convincing evidence that the diketo tetrazoles and diketo triazoles can be developed as bioisosteres of α,γ-diketo acid to exhibit potent inhibitory activity against HCV.

  10. Don't Panic! Closed String Tachyons in ALE Spacetimes

    CERN Document Server

    Adams, A; Silverstein, E

    2001-01-01

    We consider closed string tachyons localized at the fixed points of noncompact nonsupersymmetric orbifolds. We argue that tachyon condensation drives these orbifolds to flat space or supersymmetric ALE spaces. The decay proceeds via an expanding shell of dilaton gradients and curvature which interpolates between two regions of distinct angular geometry. The string coupling remains weak throughout. For small tachyon VEVs, evidence comes from quiver theories on D-branes probes, in which deformations by twisted couplings smoothly connect non-supersymmetric orbifolds to supersymmetric orbifolds of reduced order. For large tachyon VEVs, evidence comes from worldsheet RG flow and spacetime gravity. For $\\IC^2/\\IZ_n$, we exhibit infinite sequences of transitions producing SUSY ALE spaces via twisted closed string condensation from non-supersymmetric ALE spaces. In a $T$-dual description this provides a mechanism for creating NS5-branes via {\\it closed} string tachyon condensation similar to the creation of D-branes ...

  11. IIB Soliton Spectra with All Fluxes Activated

    CERN Document Server

    Evslin, J

    2003-01-01

    Building upon an earlier proposal for the classification of fluxes, a sequence is proposed which generalizes the AHSS by computing type IIB string theory's group of conserved RR and also NS charges, which is conjectured to be a K-theory of dual pairs. As a test, the formalism of Maldacena, Moore and Seiberg (hep-th/0108100) is applied to classify D-branes, NS5-branes, F-strings and their dielectric counterparts in IIB compactified on a 3-sphere with both NS and RR background fluxes. The soliton spectra on the 3-sphere are then compared with the output of the sequence, as is the baryon spectrum in Witten's non-spin^c example, AdS^5xRP^5. The group of conserved charges is seen to change during Brown-Teitelboim-like phase transitions which change the effective cosmological constant.

  12. Therapie der chronischen Hepatitis C Genotyp 1 – Update 2015

    Directory of Open Access Journals (Sweden)

    Gschwantler M

    2015-01-01

    Full Text Available Die Therapie der chronischen Hepatitis C hat sich während der vergangenen zwei Jahre fundamental verändert. Durch eine Kombination von zwei oder drei neuen DAAs („direct acting antiviral agents“ kann bei fast allen Patienten eine Elimination des Virus praktisch ohne Nebenwirkungen erzielt werden. Auch bei den meisten Patienten mit dekompensierter Leberzirrhose konnte nach virologischer Heilung innerhalb weniger Wochen eine Verbesserung der Leberfunktion beobachtet werden. Derzeit stehen in Österreich folgende neue DAAs für die Therapie zur Verfügung: Die Proteasehemmer Simeprevir und Paritaprevir, die NS5A-Inhibitoren Daclatasvir, Ledipasvir und Ombitasvir, sowie die Polymerasehemmer Sofosbuvir und Dasabuvir. Die effektivsten Therapieregime beim Genotyp 1 sind die Kombinationen Sofosbuvir/Simeprevir, Sofosbuvir/Daclatasvir, Sofosbuvir/Ledipasvir und Paritaprevir/Ritonavir/Ombitasvir/Dasabuvir.

  13. Relapse of HCV Genotype 1b Infection After Sofosbuvir/Ledipasvir Treatment Presenting as De Novo Cryoglobulinemic Vasculitis

    Science.gov (United States)

    Moreno, Alan D.; Joseph, Nora; Kim, George; Fimmel, Claus J.

    2017-01-01

    Relapse of hepatitis C virus (HCV) genotype 1 infection after combination therapy with sofosbuvir and ledipasvir is unusual. We report a treatment-naïve, non-cirrhotic patient in whom the relapse of genotype 1b HCV infection was accompanied by de novo cryoglobulinemic vasculitis and glomerulonephritis, requiring hemodialysis for acute renal failure. Sequence analysis revealed several resistance-associated variants in the HCV NS5a gene but not in NS3/4A. The patient’s vasculitis was successfully treated with immunosuppression and plasmapheresis, followed by retreatment of HCV with a combination of sofosbuvir, simeprevir, and ribavirin. The patient achieved sustained virological response, recovered his renal function, and remains in remission from cryoglobulinemia. PMID:28184378

  14. 2d (0,2) Quiver Gauge Theories and D-Branes

    CERN Document Server

    Franco, Sebastian; Lee, Sangmin; Seong, Rak-Kyeong; Yokoyama, Daisuke

    2015-01-01

    We initiate a systematic study of 2d (0,2) quiver gauge theories on the worldvolume of D1-branes probing singular toric Calabi-Yau 4-folds. We present an algorithm for efficiently calculating the classical mesonic moduli spaces of these theories, which correspond to the probed geometries. We also introduce a systematic procedure for constructing the gauge theories for arbitrary toric singularities by means of partial resolution, which translates to higgsing in the field theory. Finally, we introduce Brane Brick Models, a novel class of brane configurations that consist of D4-branes suspended from an NS5-brane wrapping a holomorphic surface, tessellating a 3-torus. Brane Brick Models are the 2d analogues of Brane Tilings and allow a direct connection between geometry and gauge theory.

  15. Thermodynamic Geometry and Extremal Black Holes in String Theory

    CERN Document Server

    Sarkar, Tapobrata; Tiwari, Bhupendra Nath

    2008-01-01

    We study a generalisation of thermodynamic geometry to degenerate quantum ground states at zero temperatures exemplified by charged extremal black holes in type II string theories. Several examples of extremal charged black holes with non degenerate thermodynamic geometries and finite but non zero state space scalar curvatures are established. These include black holes described by D1-D5-P and D2-D6-NS5-P brane systems and also two charged small black holes in Type II string theories. We also explore the modifications to the state space geometry and the scalar curvature due to the higher derivative contributions and string loop corrections as well as an exact entropy expression from quantum information theory. Our construction describes state space geometries arising out of a possible limiting thermodynamic characterisation of degenerate quantum ground states at zero temperatures.

  16. Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

    DEFF Research Database (Denmark)

    Russell, Rodney S; Meunier, Jean-Christophe; Takikawa, Shingo

    2008-01-01

    mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7......The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five......, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast...

  17. Defect (p,q) Five-branes

    CERN Document Server

    Kimura, Tetsuji

    2015-01-01

    We study the local description of composite five-branes of codimension two. The formulation is constructed by virtue of the $SL(2,{\\mathbb Z}) \\times SL(2,{\\mathbb Z)}$ monodromy associated with two-torus. Applying the conjugate monodromy transformations to the complex structures of the two-torus, we obtain the field configuration of the defect $(p,q)$ five-branes. This is the composite state of $p$ defect NS5-branes and $q$ exotic $5^2_2$-branes. We also obtain a new hyper-K\\"{a}hler geometry. This is an ALG space, a generalization of an ALF space which asymptotically has a tri-holomorphic two-torus action. This geometry appears in the conjugate configuration of the single defect KK5-brane.

  18. C-6 aryl substituted 4-quinolone-3-carboxylic acids as inhibitors of hepatitis C virus.

    Science.gov (United States)

    Chen, Yue-Lei; Zacharias, Jeana; Vince, Robert; Geraghty, Robert J; Wang, Zhengqiang

    2012-08-01

    Quinolone-3-carboxylic acid represents a highly privileged chemotype in medicinal chemistry and has been extensively explored as antibiotics and antivirals targeting human immunodeficiency virus (HIV) integrase (IN). Herein we describe the synthesis and anti-hepatitis C virus (HCV) profile of a series of C-6 aryl substituted 4-quinlone-3-carboxylic acid analogues. Significant inhibition was observed with a few analogues at low micromolar range against HCV replicon in cell culture and a reduction in replicon RNA was confirmed through an RT-qPCR assay. Interestingly, evaluation of analogues as inhibitors of NS5B in a biochemical assay yielded only modest inhibitory activities, suggesting that a different mechanism of action could operate in cell culture.

  19. Remarks on brane and antibrane dynamics

    CERN Document Server

    Michel, Ben; Polchinski, Joseph; Puhm, Andrea; Saad, Philip

    2014-01-01

    We develop the point of view that brane actions should be understood in the context of effective field theory, and that this is the correct way to treat classical as well as loop divergences. We illustrate this idea in a simple model. We then consider the implications for the dynamics of antibranes in flux backgrounds, focusing on the simplest case of a single antibrane. We argue that there is no tachyonic instability at zero temperature, but there is a nonperturbative process in which an antibrane annihilates with its screening cloud. This is distinct from the NS5-brane instanton decay. Constraints on models of metastable supersymmetry breaking by antibranes may be tightened, but there is no problem of principle with this mechanism.

  20. [Detection of TBEV RNA in ticks as a tool for valuation of endemic area wide and sensitivity of TBE surveillance].

    Science.gov (United States)

    Makówka, Agata; Gut, Włodzimierz; Rogalska, Justyna; Michalik, Jerzy; Wodecka, Beata; Rymaszewska, Anna; Stefanoff, Paweł

    2009-01-01

    In this study we present the nested RT-PCR strategy designed for detection of TBEV RNA in ticks Ixodes ricinus. The presented nested RT-PCR method using 2 different primer pairs specific primers for NS5 gene provides specific TBEV cDNA detectable by electroforesis in agarose gel. Of the 177 polls of ticks investigated, TBEV RNA was detected in 14, which accounts for 7.9% of all pools. We confront the PCR results of tested ticks to routine surveillance data. The obtained results showed that the TBEV RNA is detectable in ticks collected in areas in Poland, which are defined as an non-endemic. The nested RT-PCR method can be used as a tool of epidemiological surveillance as well as for screening of occurrence of circulating TBEV.

  1. An autochthonous case of hepatitis C virus genotype 5a in Brazil: phylogenetic analysis

    DEFF Research Database (Denmark)

    Ribeiro, L.C.; Souto, F.J.D.; do Espirito-Santo, M.P.;

    2009-01-01

    Genotype 5 of hepatitis C virus (HCV) has been rarely identified in South America. A female of African descent who never left Brazil was found to be infected by this genotype in Mato Grosso state, Central Brazil. The patient denied drug injections and revealed that she had received blood...... transfusions several years before. One of her blood donors was identified and tested negative for anti-HCV and HCV RNA, as were her husband and offspring. Phylogenetic analysis of the E1 and NS5B regions confirmed that this HCV strain belonged to genotype 5a. However, the E1 region analysis indicates that our...... strain is not closely related to any sequences of genotype 5a from other geographical areas, diverging from the African and European subclades known so far. These data suggest that genotype 5a HCV might have been circulating at a low level in Brazil longer than previously supposed....

  2. Anti-HCV RNA Aptamers Targeting the Genomic cis-Acting Replication Element

    Directory of Open Access Journals (Sweden)

    Alfredo Berzal-Herranz

    2011-12-01

    Full Text Available Hepatitis C virus (HCV replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE, located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80% of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.

  3. Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient.

    Science.gov (United States)

    Cao, Huang; Zhu, Wandi; Han, Qingxia; Pei, Rongjuan; Chen, Xinwen

    2014-02-01

    Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.

  4. T^{\\sigma}_{\\rho}(G) Theories and Their Hilbert Series

    CERN Document Server

    Cremonesi, Stefano; Mekareeya, Noppadol; Zaffaroni, Alberto

    2015-01-01

    We give an explicit formula for the Higgs and Coulomb branch Hilbert series for the class of 3d N=4 superconformal gauge theories T^{\\sigma}_{\\rho}(G) corresponding to a set of D3 branes ending on NS5 and D5-branes, with or without O3 planes. Here G is a classical group, \\sigma is a partition of G and \\rho a partition of the dual group G^\\vee. In deriving such a formula we make use of the recently discovered formula for the Hilbert series of the quantum Coulomb branch of N=4 superconformal theories. The result can be expressed in terms of a generalization of a class of symmetric functions, the Hall-Littlewood polynomials, and can be interpreted in mathematical language in terms of localization. We mainly consider the case G=SU(N) but some interesting results are also given for orthogonal and symplectic groups.

  5. Emergence of occult minority genotype 2b hepatitis C infection in an HIV-1-co-infected patient treated for genotype 5a HCV infection with 48 weeks of pegylated-interferon-alpha 2b and ribavirin.

    Science.gov (United States)

    Buckton, A J; Kulasegaram, R; Ngui, S L; Fisher, M; James, R; Rangarajan, S; Teo, C G

    2007-09-01

    An HIV-1/hepatitis C virus (HCV) co-infected patient with haemophilia received a 48-week course of pegylated interferon-alpha-2b and ribavirin therapy for genotype 5a HCV infection. Virological response was achieved at week 24. At the end of treatment, HCV RNA in serum was detected and identified to belong to genotype 2b, rather than genotype 5a. A sensitive method for identifying minority HCV genotypes in pre-treatment serum showed genotype 2b HCV carriage prior to treatment. Sequencing the interferon sensitivity-determining region of the HCV NS5A gene obtained from pre-, intra- and post-treatment sera revealed emergence of quasispecies bearing R-->K and M-->A/T mutations at codons 2222 and 2223, respectively. Occult presence of minority HCV subpopulations and their acquisition of mutations following therapy can result in poor treatment outcome.

  6. Distribution of hepatitis C virus genotypes in volunteer blood donors from Chengdu, China.

    Science.gov (United States)

    Gong, Tianxiang; Zhao, Xin; Luo, Yijia; Hong, Ying; Li, Shuping; Fu, Xuemei

    2016-07-01

    Hepatitis C virus (HCV) is a significant pathogen of global concern. The virus is usually spread through blood contact, such as transfusion, hemodialysis and injection of illegal drugs. HCV genotypes have a geographic distribution in different areas. In this paper, we focus on the distribution of HCV genotypes from volunteer blood donors in Chengdu. The prevalence of genotypes was analyzed using phylogenetic analysis. Phylogenetic trees were constructed based on the HCV core and NS5B regions from 313 sequences. HCV sequences were classified into six subtypes, and HCV genotypes were determined with the following results: 1b in 283, 2a in 14, 3b in seven, 3a in three, 6a in five and 6u in one. Subtype 1b was the most common and accounted for approximately 90.41 % (283/313), and a virus of subtype 6u was isolated for the first time from the Chengdu area. Genotypes 4 and 5 were not detected.

  7. Molecular design, synthesis and cell based HCV replicon assay of novel benzoxazole derivatives.

    Science.gov (United States)

    Ismail, M A H; Adel, M; Ismail, N S M; Abouzid, K A M

    2013-03-01

    Hepatitis C virus inhibitors based on benzoxazole scaffold were designed based on molecular modeling simulation study including docking into the NS5B polymerase active site. Several compounds showed significant high simulation docking scores relative to the assigned benzimidazole lead compound. The designed compounds were synthesized, structurally elucidated and their antiviral activity was evaluated through cell-based replicon in cultured Huh 5-2 cells. A number of the synthesized compounds showed significant inhibitory activity ranging from (52.2% inhibition up to 98% at<50 µg/mL). N-Benzyl-2-phenylbenzo[1,3]oxazole-5-carboxamide (8b) and N-Phenethyl-2-phenylbenzo[1,3] oxazole-5-carboxamide (8c) demonstrated genuine HCV inhibitory activity with EC50 values of 41.6 and 24.5 µg/mL respectively.

  8. The IR Obstruction to UV Completion for Dante's Inferno Model with Higher-Dimensional Gauge Theory Origin

    CERN Document Server

    Furuuchi, Kazuyuki

    2015-01-01

    We continue our investigation of large field inflation models obtained from higher-dimensional gauge theories, initiated in our previous study \\cite{Furuuchi:2014cwa}. We focus on Dante's Inferno model which was the most preferred model in our previous analysis. We point out the relevance of the IR obstruction to UV completion, which constrains the form of the potential of the massive vector field, under the current observational upper bound on the tensor to scalar ratio. We also show that in simple examples of the potential arising from DBI action of D5- and NS5- brane that inflation occurs in the field range which is within the convergence radius of the Taylor expansion. This is in contrast to the well known examples of axion monodromy inflation. The difference arises from the very essence of Dante's Inferno model that the effective inflaton potential is stretched in the inflaton field direction compared with the potential for the original field.

  9. Regulatory mechanisms of viral hepatitis B and C

    Indian Academy of Sciences (India)

    G Waris; A Siddiqui

    2003-04-01

    Of all the hepatitis viruses, only the hepatitis B virus (HBV) and hepatitis C virus (HCV) cause chronic hepatitis, which can progress to cirrhosis and hepatocellular carcinoma. In this review, we discuss how these two biologically diverse viruses use common pathways to induce oxidative stress and activation of key transcription factors, known to be involved in inflammatory processes in cells. Activation of NF-B and STAT-3 most likely contribute to the progression of viral infections to chronic hepatitis and liver oncogenesis associated with HBV and HCV infections. In this review, we focus on the mechanisms of action of HBx and HCV NS5A proteins in inducing intracellular events associated with the viral infections.

  10. The squares of the dirac and spin-dirac operators on a riemann-cartan space(time)

    Science.gov (United States)

    Notte-Cuello, E. A.; Rodrigues, W. A.; Souza, Q. A. G.

    2007-08-01

    In this paper we introduce the Dirac and spin-Dirac operators associated to a connection on Riemann-Cartan space(time) and standard Dirac and spin-Dirac operators associated with a Levi-Civita connection on a Riemannian (Lorentzian) space(time) and calculate the squares of these operators, which play an important role in several topics of modern mathematics, in particular in the study of the geometry of moduli spaces of a class of black holes, the geometry of NS-5 brane solutions of type II supergravity theories and BPS solitons in some string theories. We obtain a generalized Lichnerowicz formula, decompositions of the Dirac and spin-Dirac operators and their squares in terms of the standard Dirac and spin-Dirac operators and using the fact that spinor fields (sections of a spin-Clifford bundle) have representatives in the Clifford bundle we present also a noticeable relation involving the spin-Dirac and the Dirac operators.

  11. The hidden seasonality of the rare biosphere in coastal marine bacterioplankton.

    Science.gov (United States)

    Alonso-Sáez, Laura; Díaz-Pérez, Laura; Morán, Xosé Anxelu G

    2015-10-01

    Rare microbial taxa are increasingly recognized to play key ecological roles, but knowledge of their spatio-temporal dynamics is lacking. In a time-series study in coastal waters, we detected 83 bacterial lineages with significant seasonality, including environmentally relevant taxa where little ecological information was available. For example, Verrucomicrobia had recurrent maxima in summer, while the Flavobacteria NS4, NS5 and NS2b clades had contrasting seasonal niches. Among the seasonal taxa, only 4 were abundant and persistent, 20 cycled between rare and abundant and, remarkably, most of them (59) were always rare (contributing biosphere, hitherto mainly characterized by dormancy and episodes of 'boom and bust', as envisioned by the seed-bank hypothesis. The predictable patterns of seasonal reoccurrence are relevant for understanding the ecology of rare bacteria, which may include key players for the functioning of marine ecosystems.

  12. Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease

    Science.gov (United States)

    Martínez-González, José Ángel; Rodríguez, Alex; Puyuelo, María Pilar; González, Miguel; Martínez, Rodrigo

    2015-01-01

    The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second-order Møller-Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different substrates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a concerted mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated.

  13. Sofosbuvir as backbone of interferon free treatments.

    Science.gov (United States)

    Bourlière, Marc; Oules, Valèrie; Ansaldi, Christelle; Adhoute, Xavier; Castellani, Paul

    2014-12-15

    Sofosbuvir is the first-in-class NS5B nucleotide analogues to be launched for hepatitis C virus (HCV) treatment. Its viral potency, pangenotypic activity and high barrier to resistance make it the ideal candidate to become a backbone for several IFN-free regimens. Recent data demonstrated that sofosbuvir either with ribavirin alone or in combination with other direct-acting antivirals (DAAs) as daclatasvir, ledipasvir or simeprevir are able to cure HCV in at least 90% or over of patients. Treatment experienced genotype 3 population may remain the most difficult to treat population, but ongoing DAA combination studies will help to fill this gap. Safety profile of sofosbuvir or combination with other DAAs is good. Resistance to sofosbuvir did not appear as a significant issue. The rationale for using this class of drug and the available clinical data are reviewed.

  14. Sofosbuvir-based therapy cures hepatitis C virus infection after prior treatment failures in a patient with concurrent lymphoma.

    Science.gov (United States)

    Romagnoli, Dante; Marrazzo, Alessandra; Ballestri, Stefano; Lonardo, Amedeo; Bertolotti, Marco

    2015-08-01

    We report on the first well-tolerated and successful use of sofosbuvir-based therapy in a patient in whom chronic infection with hepatitis C had preceded the development of B-cell non-Hodgkin's lymphoma. The patient had previously failed numerous attempts to clear the hepatitis C virus with traditional antiviral schedules. We demonstrate that sofosbuvir-based therapy resulted in cure of hepatitis C in a patient who had relapsed during combination therapy with an NS5A inhibitor, an NS3 protease inhibitor and ribavirin, as well as treatment failures to multiple courses of interferon-based therapy. This report also suggests that eradication of hepatitis C virus may result in the short-term prevention of B-cell non-Hodgkin's lymphoma relapse. The findings from our case require further validation in future cohorts of patients.

  15. A silence black hole: Hawking radiation at the Hagedorn temperature

    CERN Document Server

    Lorente-Espin, O

    2008-01-01

    We compute semi-classically the Hawking emission for different types of black hole in type II string theory. In particular we analyze the thermal transition between NS5 branes and Little String Theory, finding compelling evidence for information recovering. We find that once the near horizon limit is taken the emission of a full family of models is exactly thermal even if back-reaction is taken into account. Consequently these theories are non-unitary and can not convey any information about the black hole internal states. It is argue that this behaviour matches the string theory expectations. We suggest a plausible reason for the vanishing of the jet-quenching parameter in such theories.

  16. Genetic history of hepatitis C virus in Pakistan.

    Science.gov (United States)

    ur Rehman, Irshad; Vaughan, Gilberto; Purdy, Michael A; Xia, Guo-liang; Forbi, Joseph C; Rossi, Livia Maria Gonçalves; Butt, Sadia; Idrees, Muhammad; Khudyakov, Yury E

    2014-10-01

    Hepatitis C virus (HCV) genotype 3a accounts for ∼80% of HCV infections in Pakistan, where ∼10 million people are HCV-infected. Here, we report analysis of the genetic heterogeneity of HCV NS3 and NS5b subgenomic regions from genotype 3a variants obtained from Pakistan. Phylogenetic analyses showed that Pakistani genotype 3a variants were as genetically diverse as global variants, with extensive intermixing. Bayesian estimates showed that the most recent ancestor for genotype 3a in Pakistan was last extant in ∼1896-1914 C.E. (range: 1851-1932). This genotype experienced a population expansion starting from ∼1905 to ∼1970 after which the effective population leveled. Death/birth models suggest that HCV 3a has reached saturating diversity with decreasing turnover rate and positive extinction. Taken together, these observations are consistent with a long and complex history of HCV 3a infection in Pakistan.

  17. Sofosbuvir for the treatment of chronic hepatitis C virus infection.

    Science.gov (United States)

    Temesgen, Z; Talwani, R; Rizza, S A

    2014-06-01

    Sofosbuvir is a nucleotide analogue selective inhibitor of the RNA-directed RNA polymerase (NS5B) enzyme of the hepatitis C virus (HCV) genome. It has shown potent antiviral activity across all HCV genotypes and in a variety of patient populations, including treatment-naive patients; treatment-experienced patients who had failed previous standard therapy; patients with decompensated liver disease, including cirrhosis; and HIV co-infected patients. It is administered as a single, once-daily 400-mg tablet, has no food restrictions, has low potential for drug interactions, and requires no dose adjustment in mild to moderate kidney or liver impairment. When sofosbuvir is combined with pegylated interferon and/or ribavirin, its clinical and laboratory safety profile is similar to that which is expected from pegylated interferon or ribavirin alone. Rates of treatment discontinuation and dose reduction with sofosbuvir-containing regimens were lower than those commonly observed with pegylated interferon and ribavirin.

  18. Sofosbuvir for treatment of chronic hepatitis C.

    Science.gov (United States)

    Kattakuzhy, Sarah; Levy, Rachel; Kottilil, Shyam

    2015-04-01

    Chronic hepatitis C is a leading cause of liver-related morbidity and mortality worldwide. If untreated, chronic hepatitis C can progress to advanced liver fibrosis, cirrhosis, liver failure, hepatocellular carcinoma and death. Until recently, treatment of hepatitis C predominantly constituted an immunomodulatory agent, peg-interferon-alfa and ribavirin. In 2011, the first class of directly acting antiviral agents, HCV NS3/4A serine protease inhibitors, was added to peg-interferon-alfa and ribavirin with increased efficacy. In the past year, an NS5B inhibitor, sofosbuvir, has emerged as a potent agent with pangenotypic efficacy, resulting in the first interferon-free regimen for the treatment of hepatitis C. This review summarizes the data that resulted in regulatory approval of sofosbuvir and highlights the future of hepatitis C therapy with sofosbuvir as the backbone of a highly effective antiviral regimen.

  19. Monitoring the antiviral effect of alpha interferon on individual cells.

    Science.gov (United States)

    Kim, Chon Saeng; Jung, Jong Ha; Wakita, Takaji; Yoon, Seung Kew; Jang, Sung Key

    2007-08-01

    An infectious hepatitis C virus (HCV) cDNA clone (JFH1) was generated recently. However, quantitative analysis of HCV infection and observation of infected cells have proved to be difficult because the yield of HCV in cell cultures is fairly low. We generated infectious HCV clones containing the convenient reporters green fluorescent protein (GFP) and Renilla luciferase in the NS5a-coding sequence. The new viruses responded to antiviral agents in a dose-dependent manner. Responses of individual cells containing HCV to alpha interferon (IFN-alpha) were monitored using GFP-tagged HCV and time-lapse confocal microscopy. Marked variations in the response to IFN-alpha were observed among HCV-containing cells.

  20. Brane polarization is no cure for tachyons

    CERN Document Server

    Bena, Iosif

    2015-01-01

    Anti-M2 and anti-D3 branes placed in regions with charges dissolved in fluxes have a tachyon in their near-horizon region, which causes these branes to repel each other. If the branes are on the Coulomb branch this tachyon gives rise to a runaway behavior, but when the branes are polarized into five-branes this tachyon only appears to lower the energy of the polarized branes, without affecting its stability. We analyze brane polarization in the presence of a brane-brane-repelling tachyon and show that when the branes are polarized along the direction of the tachyon the polarized shell is unstable. This implies that tachyons cannot be cured by brane polarization and indicates that, at least in a certain regime of parameters, anti-D3 branes polarized into NS5 branes at the bottom of the Klebanov-Strassler solution have an instability.

  1. $SL(2, Z)$ invariant rotating $(m,n)$ strings in $AdS_3\\times S^3$ with mixed flux

    CERN Document Server

    Barik, Sorna Prava; Kluson, Josef; Panigrahi, Kamal L

    2016-01-01

    We study rigidly rotating and pulsating $(m,n)$ strings in $AdS_3 \\times S^3$ with mixed three form flux. The $AdS_3 \\times S^3$ background with mixed three form flux is obtained in the near horizon limit of $SL(2,Z)$-transformed solution, corresponding to the bound state of NS5-branes and fundamental strings. We study the probe $(m,n)-$string in this background by solving the manifest $SL(2,Z)-$covariant form of the action. We find out the dyonic giant magnon and single spike solutions corresponding to the equations of motion of a probe string in this background and find out various relationships among the conserved charges. We further study a class of pulsating $(m,n)$ string in $AdS_3$ with mixed three form flux.

  2. Holography, brane intersections and six-dimensional SCFTs

    Science.gov (United States)

    Bobev, Nikolay; Dibitetto, Giuseppe; Gautason, Friðrik Freyr; Truijen, Brecht

    2017-02-01

    We study supersymmetric intersections of NS5-, D6- and D8-branes in type IIA string theory. We focus on the supergravity description of this system and identify a "near horizon" limit in which we recover the recently classified supersymmetric seven-dimensional AdS solutions of massive type IIA supergravity. Using a consistent truncation to seven-dimensional gauged supergravity we construct a universal supersymmetric deformation of these AdS vacua. In the holographic dual six-dimensional (1,0) superconformal field theory this deformation describes a universal RG flow on the tensor branch of the vacuum moduli space triggered by a vacuum expectation value for a protected scalar operator of dimension four.

  3. Rational sphere valued supercocycles in M-theory and type IIA string theory

    Science.gov (United States)

    Fiorenza, Domenico; Sati, Hisham; Schreiber, Urs

    2017-04-01

    We show that supercocycles on super L∞-algebras capture, at the rational level, the twisted cohomological charge structure of the fields of M-theory and of type IIA string theory. We show that rational 4-sphere-valued supercocycles for M-branes in M-theory descend to supercocycles in type IIA string theory with coefficients in the free loop space of the 4-sphere, to yield the Ramond-Ramond fields in the rational image of twisted K-theory, with the twist given by the B-field. In particular, we derive the M2/M5 ↔ F1/Dp/NS5 correspondence via dimensional reduction of sphere-valued super-L∞-cocycles.

  4. Exploring String Theory Backgrounds

    CERN Document Server

    Williams, B P

    2004-01-01

    This thesis examines phenomenological and theoretical questions by exploring string theoretic backgrounds. Part I focuses on cosmology. First we propose that the induced metric along a brane moving through a curved bulk may be interpreted as the cosmology of the brane universe, providing a resolution to the apparent cosmological singularity on the brane. We then look at various decay channels of the certain meta-stable de Sitter vacua and show that there exist NS5-brane meditated decays which are much faster than decays to decompactification. Part II discusses a new class of nongeometric vacua in string theory. These backgrounds may be described locally as T2 fibrations. By enlarging the monodromy group of the fiber to include perturbative stringy duality symmetries we are able to explicitly construct nongeometric backgrounds.

  5. Probing the Hydrodynamic Limit of (Super)gravity

    CERN Document Server

    Di Dato, Adriana; Pedersen, Andreas Vigand

    2015-01-01

    We study the long-wavelength effective description of two general classes of charged dilatonic (asymptotically flat) black p-branes including D/NS/M-branes in ten and eleven dimensional supergravity. In particular, we consider gravitational brane solutions in a hydrodynamic derivative expansion (to first order) for arbitrary dilaton coupling and for general brane and co-dimension and determine their effective electro-fluid-dynamic descriptions by exacting the characterizing transport coefficients. We also investigate the stability properties of the corresponding hydrodynamic systems by analyzing their response to small long-wavelength perturbations. For branes carrying unsmeared charge, we find that in a certain regime of parameter space there exists a branch of stable charged configurations. This is in accordance with the expectation that D/NS/M-branes have stable configurations, except for the D5, D6, and NS5. In contrast, we find that Maxwell charged brane configurations are Gregory-Laflamme unstable indep...

  6. A new 6d fixed point from holography

    Science.gov (United States)

    Apruzzi, Fabio; Dibitetto, Giuseppe; Tizzano, Luigi

    2016-11-01

    We propose a stringy construction giving rise to a class of interacting and non-supersymmetric CFT's in six dimensions. Such theories may be obtained as an IR conformal fixed point of an RG flow ending up in a (1, 0) theory in the UV. We provide the due holographic evidence in the context of massive type IIA on AdS7 × M 3, where M 3 is topologically an S 3. In particular, in this paper we present a 10d flow solution which may be interpreted as a non-BPS bound state of NS5, D6 and overline{D6} branes. Moreover, by adopting its 7d effective description, we are able to holographically compute the free energy and the operator spectrum in the novel IR conformal fixed point.

  7. Molecular characterization of two Rocio flavivirus strains isolated during the encephalitis epidemic in São Paulo State, Brazil and the development of a one-step RT-PCR assay for diagnosis.

    Science.gov (United States)

    Coimbra, Terezinha Lisieux Moraes; Santos, Raimundo N; Petrella, Selma; Nagasse-Sugahara, Teresa Keico; Castrignano, Silvana Beres; Santos, Cecília L Simões

    2008-01-01

    Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007).

  8. Empirical fitness models for hepatitis C virus immunogen design

    Science.gov (United States)

    Hart, Gregory R.; Ferguson, Andrew L.

    2015-12-01

    Hepatitis C virus (HCV) afflicts 170 million people worldwide, 2%-3% of the global population, and kills 350 000 each year. Prophylactic vaccination offers the most realistic and cost effective hope of controlling this epidemic in the developing world where expensive drug therapies are not available. Despite 20 years of research, the high mutability of the virus and lack of knowledge of what constitutes effective immune responses have impeded development of an effective vaccine. Coupling data mining of sequence databases with spin glass models from statistical physics, we have developed a computational approach to translate clinical sequence databases into empirical fitness landscapes quantifying the replicative capacity of the virus as a function of its amino acid sequence. These landscapes explicitly connect viral genotype to phenotypic fitness, and reveal vulnerable immunological targets within the viral proteome that can be exploited to rationally design vaccine immunogens. We have recovered the empirical fitness landscape for the HCV RNA-dependent RNA polymerase (protein NS5B) responsible for viral genome replication, and validated the predictions of our model by demonstrating excellent accord with experimental measurements and clinical observations. We have used our landscapes to perform exhaustive in silico screening of 16.8 million T-cell immunogen candidates to identify 86 optimal formulations. By reducing the search space of immunogen candidates by over five orders of magnitude, our approach can offer valuable savings in time, expense, and labor for experimental vaccine development and accelerate the search for a HCV vaccine. Abbreviations: HCV—hepatitis C virus, HLA—human leukocyte antigen, CTL—cytotoxic T lymphocyte, NS5B—nonstructural protein 5B, MSA—multiple sequence alignment, PEG-IFN—pegylated interferon.

  9. Hepatitis C virus genotype 4 in Southern and Central Spain does not originate from recent foreign migration waves.

    Science.gov (United States)

    Di Lello, Federico A; Neukam, Karin; Parra-Sanchez, Manuel; Plaza, Zulema; Soriano, Vicente; Cifuentes, Celia; Mira, José A; Poveda, Eva; Pineda, Juan A

    2013-10-01

    Hepatitis C virus genotype 4 (HCV-4) is highly prevalent in Spain, but the information on the molecular characterization of HCV-4 in this region is scarce. Due to this, the molecular characteristics and the evolution of HCV-4 infection in Seville were analyzed (Southern Spain) and compared them with samples from Madrid. HCV genotype was determined by LIPA 2.0 assay and confirmed by sequence analysis of NS5B. Phylogenetic tree was estimated by MEGA 5.10. Bayesian coalescent-based methods were used to estimate the substitution rate and the age of the most recent common ancestor (MRCA). In the phylogenetic analysis of 50 NS5B HCV-4 from Seville and 11 from Madrid, 2 clusters were distinguished: The first cluster (HCV-4a) included 48% of the sequences from Seville and 9% of sequences from Madrid. The second cluster included the remaining sequences belonging to HCV-4d. The mean estimated substitution rate was 2.39 × 10(-3) for HCV-4a and 1.81 × 10(-3) for HCV-4d for Seville and 2.32 × 10(-3) for HCV-4d from Madrid. The date for MRCA was estimated to be around 1981-1984 for HCV-4 from Seville. The dates for MRCA were dated before the recent flow of immigration in Spain. Therefore, the results presented in this study argues against the possibility of a foreign introduction of the HCV-4 from other regions with high prevalence, at least during the last two, decades in which there was a great flow of immigrants. Additionally, an unusual high prevalence of subtype 4a was observed in Seville.

  10. Association of filamin A and vimentin with hepatitis C virus proteins in infected human hepatocytes.

    Science.gov (United States)

    Ghosh, S; Ahrens, W A; Phatak, S U; Hwang, S; Schrum, L W; Bonkovsky, H L

    2011-10-01

    Chronic hepatitis C (CHC) infection caused by hepatitis C virus (HCV) is a major cause of liver disease and remains a major therapeutic challenge. A variety of host proteins interact with HCV proteins. The definitive role of cytoskeletal (CS) proteins in HCV infection remains to be determined. In this study, our aim was to determine the expression profile of differentially regulated and expressed selected CS proteins and their association with HCV proteins in infected hepatocytes as possible therapeutic targets. Using proteomics, qRT-PCR, Western blot and immunofluorescence techniques, we revealed that filamin A (fila) and vimentin (vim) were prominently increased proteins in HCV-expressing human hepatoma cells compared with parental cells and in liver biopsies from patients with CHC vs controls. HCV nonstructural (NS) 3 and NS5A proteins were associated with fila, while core protein partially with fila and vim. Immunoprecipitation showed interactions among fila and NS3 and NS5A proteins. Cells treated with interferon-α showed a dose- and time-dependent decrease in CS and HCV proteins. NS proteins clustered at the perinuclear region following cytochalasin b treatment, whereas disperse cytoplasmic and perinuclear distribution was observed in the no-treatment group. This study demonstrates and signifies that changes occur in the expression of CS proteins in HCV-infected hepatocytes and, for the first time, shows the up-regulation and interaction of fila with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection.

  11. Very-low-density lipoprotein (VLDL)-producing and hepatitis C virus-replicating HepG2 cells secrete no more lipoviroparticles than VLDL-deficient Huh7.5 cells.

    Science.gov (United States)

    Jammart, Baptiste; Michelet, Maud; Pécheur, Eve-Isabelle; Parent, Romain; Bartosch, Birke; Zoulim, Fabien; Durantel, David

    2013-05-01

    In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.

  12. Geno2pheno[HCV] - A Web-based Interpretation System to Support Hepatitis C Treatment Decisions in the Era of Direct-Acting Antiviral Agents.

    Directory of Open Access Journals (Sweden)

    Prabhav Kalaghatgi

    Full Text Available The face of hepatitis C virus (HCV therapy is changing dramatically. Direct-acting antiviral agents (DAAs specifically targeting HCV proteins have been developed and entered clinical practice in 2011. However, despite high sustained viral response (SVR rates of more than 90%, a fraction of patients do not eliminate the virus and in these cases treatment failure has been associated with the selection of drug resistance mutations (RAMs. RAMs may be prevalent prior to the start of treatment, or can be selected under therapy, and furthermore they can persist after cessation of treatment. Additionally, certain DAAs have been approved only for distinct HCV genotypes and may even have subtype specificity. Thus, sequence analysis before start of therapy is instrumental for managing DAA-based treatment strategies. We have created the interpretation system geno2pheno[HCV] (g2p[HCV] to analyse HCV sequence data with respect to viral subtype and to predict drug resistance. Extensive reviewing and weighting of literature related to HCV drug resistance was performed to create a comprehensive list of drug resistance rules for inhibitors of the HCV protease in non-structural protein 3 (NS3-protease: Boceprevir, Paritaprevir, Simeprevir, Asunaprevir, Grazoprevir and Telaprevir, the NS5A replicase factor (Daclatasvir, Ledipasvir, Elbasvir and Ombitasvir, and the NS5B RNA-dependent RNA polymerase (Dasabuvir and Sofosbuvir. Upon submission of up to eight sequences, g2p[HCV] aligns the input sequences, identifies the genomic region(s, predicts the HCV geno- and subtypes, and generates for each DAA a drug resistance prediction report. g2p[HCV] offers easy-to-use and fast subtype and resistance analysis of HCV sequences, is continuously updated and freely accessible under http://hcv.geno2pheno.org/index.php. The system was partially validated with respect to the NS3-protease inhibitors Boceprevir, Telaprevir and Simeprevir by using data generated with recombinant

  13. HCV genotyping from NGS short reads and its application in genotype detection from HCV mixed infected plasma.

    Science.gov (United States)

    Qiu, Ping; Stevens, Richard; Wei, Bo; Lahser, Fred; Howe, Anita Y M; Klappenbach, Joel A; Marton, Matthew J

    2015-01-01

    Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection.

  14. Direct-acting antiviral treatment in adults infected with hepatitis C virus: Reactivation of hepatitis B virus coinfection as a further challenge.

    Science.gov (United States)

    De Monte, Anne; Courjon, Johan; Anty, Rodolphe; Cua, Eric; Naqvi, Alissa; Mondain, Véronique; Cottalorda, Jacqueline; Ollier, Laurence; Giordanengo, Valérie

    2016-05-01

    Use of direct-acting antiviral drugs (DAAs) greatly improves management of adults infected with hepatitis C virus (HCV) whether patients are treatment-naive or unsuccessfully pre-treated. Several inhibitors of viral nonstructural proteins (NS3/4A protease, NS5A and NS5B polymerase) allow a rapid HCV clearance and increase rates of sustained virological response. Both the EASL and AASLD guidelines have recently published up-to-date recommendations for their use, addressing each HCV genotype and particular situations. However, management of patients coinfected with hepatitis B virus (HBV) has been developed by these guidelines with reference to cases of HBV reactivation reported during previous anti-HCV regimens containing interferon known active against both HBV and HCV. In the setting of the interferon-free HCV therapies with DAAs only, the possibility of HBV reactivation during treatment of hepatitis C is raised due to viral interferences in HCV/HBV coinfected persons. Herein, we report a case of early HBV reactivation during DAAs-based anti-HCV treatment (ledipasvir/sofosbuvir) in a patient having a resolved HBV infection and chronically infected with HCV genotype 4 and HIV. Moreover, we review similar recent cases of HBV reactivation in patients infected with HBV and HCV genotype 1 during treatment of hepatitis C by regimen incorporating other combination of DAAs (sofosbuvir/simeprevir or daclatasvir/asunaprevir). Due to the potential risk of early HBV reactivation in HCV/HBV-coinfected patients during interferon-free DAAs-based HCV therapies, altogether these cases highlight the necessity to closely monitor HBV coinfection, regardless its stage (chronic, occult, resolved), whatever HCV genotype or class of DAAs used.

  15. Anti-HCV Activity from Semi-purified Methanolic Root Extracts of Valeriana wallichii.

    Science.gov (United States)

    Ganta, Krishna Kumar; Mandal, Anirban; Debnath, Sukalyani; Hazra, Banasri; Chaubey, Binay

    2017-03-01

    Hepatitis C virus (HCV) is a serious global health problem affecting approximately 130-150 million individuals. Presently available direct-acting anti-HCV drugs have higher barriers to resistance and also improved success rate; however, cost concerns limit their utilization, especially in developing countries like India. Therefore, development of additional agents to combat HCV infection is needed. In the present study, we have evaluated anti-HCV potential of water, chloroform, and methanol extracts from roots of Valeriana wallichii, a traditional Indian medicinal plant. Huh-7.5 cells infected with J6/JFH chimeric HCV strain were treated with water, chloroform, and methanol extracts at different concentrations. Semi-quantitative reverse transcription polymerase chain reaction result demonstrated that methanolic extract showed reduction in HCV replication. The methanolic extract was fractionated by thin layer chromatography, and the purified fractions (F1, F2, F3, and F4) were checked for anti-HCV activity. Significant viral inhibition was noted only in F4 fraction. Further, intrinsic fluorescence assay of purified HCV RNA-dependent RNA polymerase NS5B in the presence of F4 resulted in sharp quenching of intrinsic fluorescence with increasing amount of plant extract. Our results indicated that methanolic extract of V. wallichii and its fraction (F4) inhibited HCV by binding with HCV NS5B protein. The findings would be further investigated to identify the active principle/lead molecule towards development of complementary and alternative therapeutics against HCV. Copyright © 2017 John Wiley & Sons, Ltd.

  16. Establishment of RT-PCR for detecting duck hemorrhagic ovaritis causing abrupt%鸭出血性卵巢炎病毒RT-PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    万春和; 施少华; 程龙飞; 陈红梅; 傅光华; 彭春香; 林芳; 林建生; 黄瑜

    2011-01-01

    参照(GenBank上登录的黄病毒科黄病毒属Ns5基因片段序列设计引物,建立能检测引起鸭出血性卵巢炎病毒的RT-PCR方法.该办法能从鸭出血性卵巢炎病毒扩增出1条约470 bp的特异性片段,从H9亚型禽流感病毒、鸭呼肠孤病毒、鸭瘟病毒、鸭肝炎病毒、鸭源禽Ⅰ型副粘病毒均不能扩增出目的片段;敏感性试验显示建立的RT-PCR方法最低可检出20 pg病毒核酸.以上结果表明建立的RT-PCR方法具有较好的特异性、敏感性和准确性,可用于该病的临床诊断和流行病学调查.%A pair of primers based on NS5 gene sequences of different strains of genus Flavivirus was designed for amplification of 470 bp fragments. No positive target fragment was available from H9 subtype influenza virus,Muscovy duck reovirus, duck plague, duck hepatitis virus (DHV-I ) or duck-origin avian paramyxovirus type Ⅰ.The detection limit reached 20 pg under the optimized conditions. The results showed that the RT-PCR was sensitive, specific and applicable for rapid laboratory diagnosis and epidemiologic surveillance.

  17. Construction of a subgenomic replicon of classical swine fever virus and its stable cell lines%猪瘟病毒亚基因复制子及其细胞系的构建

    Institute of Scientific and Technical Information of China (English)

    张莉; 谢金鑫; 蒋大良; 余兴龙; 贾俊杰; 郭焕成; 龚文杰; 涂长春

    2016-01-01

    为探索猪瘟病毒(CSFV)的复制机理,本实验在前期CSFV流行株GD53全基因组测序的基础上,利用新霉素抗性基因(Neor)替换病毒结构蛋白基因以构建CSFV GD53株的亚基因组复制子(GD53-SGR).首先构建含有T7-5'UTR-Npro-Neor-EMCV/IRES-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3'UTR的重组质粒pcDNA-GD53-SGR,将其利用T7 RNA聚合酶经体外转录制备的RNA转录本转染猪睾丸细胞系(ST)后,利用G418筛选含有GD53-SGR的ST细胞,构建ST-GD53-SGR细胞系.酶切鉴定及测序结果表明,重组质粒pcDNA-GD53-SGR构建正确,利用T7 RNA聚合酶体外转录后获得了与预期大小一致的GD53-SGR RNA.经RT-PCR和间接免疫荧光鉴定结果表明,获得了含有GD53-SGR的ST细胞系.本研究构建的CSFV ST-GD53-SGR细胞系有助于进一步了解CSFV非结构蛋白的功能,同时为CSFV的复制机制研究及抗病毒药物的研发奠定基础.

  18. Inhibition of hepatitis C virus replicon RNA synthesis by PSI-352938, a cyclic phosphate prodrug of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine.

    Science.gov (United States)

    Lam, Angela M; Espiritu, Christine; Murakami, Eisuke; Zennou, Veronique; Bansal, Shalini; Micolochick Steuer, Holly M; Niu, Congrong; Keilman, Meg; Bao, Haiying; Bourne, Nigel; Veselenak, Ronald L; Reddy, P Ganapati; Chang, Wonsuk; Du, Jinfa; Nagarathnam, Dhanapalan; Sofia, Michael J; Otto, Michael J; Furman, Phillip A

    2011-06-01

    PSI-352938 is a novel cyclic phosphate prodrug of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine 5'-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 μM, respectively. The active 5'-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. In contrast, PSI-352938 did not inhibit the replication of hepatitis B virus or human immunodeficiency virus in vitro. PSI-352666 did not significantly affect the activity of human DNA and RNA polymerases. PSI-352938 and its cyclic phosphate metabolites did not affect the cyclic GMP-mediated activation of protein kinase G. Clearance studies using replicon cells demonstrated that PSI-352938 cleared cells of HCV replicon RNA and prevented replicon rebound. An additive to synergistic effect was observed when PSI-352938 was combined with other classes of HCV inhibitors, including alpha interferon, ribavirin, NS3/4A inhibitors, an NS5A inhibitor, and nucleoside/nucleotide and nonnucleoside inhibitors. Cross-resistance studies showed that PSI-352938 remained fully active against replicons containing the S282T or the S96T/N142T amino acid alteration. Replicons that contain mutations conferring resistance to various classes of nonnucleoside inhibitors also remained sensitive to inhibition by PSI-352938. PSI-352938 is currently being evaluated in a phase I clinical study in genotype 1-infected individuals.

  19. Hepatitis C virus RNA elimination and development of resistance in replicon cells treated with BMS-790052.

    Science.gov (United States)

    Wang, Chunfu; Huang, Haichang; Valera, Lourdes; Sun, Jin-Hua; O'Boyle, Donald R; Nower, Peter T; Jia, Lingling; Qiu, Dike; Huang, Xin; Altaf, Aneela; Gao, Min; Fridell, Robert A

    2012-03-01

    BMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time- and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-α) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-α and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from the in vitro anti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor.

  20. A rapid immunization strategy with a live attenuated tetravalent dengue vaccine elicits protective neutralizing antibody responses in non-human primates

    Directory of Open Access Journals (Sweden)

    Yuping eAmbuel

    2014-06-01

    Full Text Available Dengue viruses (DENVs cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine (TDV that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2 and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3 and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0 at two different anatomical locations with a needle-free disposable syringe jet injection (DSJI delivery devices (PharmaJet in non-human primates (NHP. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (two months later vaccination schedule. In addition, the vaccine induced CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3 and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post- challenge. RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas.

  1. Baseline quasispecies selection and novel mutations contribute to emerging resistance-associated substitutions in hepatitis C virus after direct-acting antiviral treatment

    Science.gov (United States)

    Kai, Yugo; Hikita, Hayato; Morishita, Naoki; Murai, Kazuhiro; Nakabori, Tasuku; Iio, Sadaharu; Hagiwara, Hideki; Imai, Yasuharu; Tamura, Shinji; Tsutsui, Syusaku; Naito, Masafumi; Nishiuchi, Meiko; Kondo, Yasuteru; Kato, Takanobu; Suemizu, Hiroshi; Yamada, Ryoko; Oze, Tsugiko; Yakushijin, Takayuki; Hiramatsu, Naoki; Sakamori, Ryotaro; Tatsumi, Tomohide; Takehara, Tetsuo

    2017-01-01

    Resistance-associated substitutions (RASs) in hepatitis C virus (HCV) appear upon failure of treatment with direct-acting antivirals (DAAs). However, their origin has not been clarified in detail. Among 11 HCV genotype 1b patients who experienced virologic failure with asunaprevir (ASV)/daclatasvir (DCV), 10 had major NS5A L31M/V-Y93H variants after treatment. L31M/V-Y93H variants were detected as a minor clone before therapy in 6 patients and were the most closely related to the post-treatment variants by phylogenetic tree analysis in 4 patients. Next, to consider the involvement of a trace amount of pre-existing variants below the detection limit, we analysed human hepatocyte chimeric mice infected with DAA-naïve patient serum. L31V-Y93H variants emerged after treatment with ledipasvir (LDV)/GS-558093 (nucleotide NS5B inhibitor) and decreased under the detection limit, but these variants were dissimilar to the L31V-Y93H variants reappearing after ASV/DCV re-treatment. Finally, to develop an infection derived from a single HCV clone, we intrahepatically injected full-genome HCV RNA (engineered based on the wild-type genotype 1b sequence) into chimeric mice. A new Y93H mutation actually occurred in this model after LDV monotherapy failure. In conclusion, post-treatment RASs appear by 2 mechanisms: the selection of pre-existing substitutions among quasispecies and the generation of novel mutations during therapy. PMID:28134353

  2. Interaction of Hepatitis C virus proteins with pattern recognition receptors

    Directory of Open Access Journals (Sweden)

    Imran Muhammad

    2012-06-01

    Full Text Available Abstract Hepatitis C virus (HCV is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I as the receptors for intracellular viral double stranded RNA (dsRNA, and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C, non structural 3/4 A (NS3/4A and non structural 5A (NS5A have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

  3. Geno2pheno[HCV] – A Web-based Interpretation System to Support Hepatitis C Treatment Decisions in the Era of Direct-Acting Antiviral Agents

    Science.gov (United States)

    Kalaghatgi, Prabhav; Sikorski, Anna Maria; Knops, Elena; Rupp, Daniel; Sierra, Saleta; Heger, Eva; Neumann-Fraune, Maria; Beggel, Bastian; Walker, Andreas; Timm, Jörg; Walter, Hauke; Obermeier, Martin; Kaiser, Rolf; Bartenschlager, Ralf; Lengauer, Thomas

    2016-01-01

    The face of hepatitis C virus (HCV) therapy is changing dramatically. Direct-acting antiviral agents (DAAs) specifically targeting HCV proteins have been developed and entered clinical practice in 2011. However, despite high sustained viral response (SVR) rates of more than 90%, a fraction of patients do not eliminate the virus and in these cases treatment failure has been associated with the selection of drug resistance mutations (RAMs). RAMs may be prevalent prior to the start of treatment, or can be selected under therapy, and furthermore they can persist after cessation of treatment. Additionally, certain DAAs have been approved only for distinct HCV genotypes and may even have subtype specificity. Thus, sequence analysis before start of therapy is instrumental for managing DAA-based treatment strategies. We have created the interpretation system geno2pheno[HCV] (g2p[HCV]) to analyse HCV sequence data with respect to viral subtype and to predict drug resistance. Extensive reviewing and weighting of literature related to HCV drug resistance was performed to create a comprehensive list of drug resistance rules for inhibitors of the HCV protease in non-structural protein 3 (NS3-protease: Boceprevir, Paritaprevir, Simeprevir, Asunaprevir, Grazoprevir and Telaprevir), the NS5A replicase factor (Daclatasvir, Ledipasvir, Elbasvir and Ombitasvir), and the NS5B RNA-dependent RNA polymerase (Dasabuvir and Sofosbuvir). Upon submission of up to eight sequences, g2p[HCV] aligns the input sequences, identifies the genomic region(s), predicts the HCV geno- and subtypes, and generates for each DAA a drug resistance prediction report. g2p[HCV] offers easy-to-use and fast subtype and resistance analysis of HCV sequences, is continuously updated and freely accessible under http://hcv.geno2pheno.org/index.php. The system was partially validated with respect to the NS3-protease inhibitors Boceprevir, Telaprevir and Simeprevir by using data generated with recombinant, phenotypic

  4. Barrier-Independent, Fitness-Associated Differences in Sofosbuvir Efficacy against Hepatitis C Virus

    Science.gov (United States)

    Gallego, Isabel; Sheldon, Julie; Moreno, Elena; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Domingo, Esteban

    2016-01-01

    Sofosbuvir displays a high phenotypic barrier to resistance, and it is a component of several combination therapies for hepatitis C virus (HCV) infections. HCV fitness can be a determinant of decreased sensitivity to direct-acting antiviral agents such as telaprevir or daclatasvir, but fitness-dependent decreased drug sensitivity has not been established for drugs with a high phenotypic barrier to resistance. Low- and high-fitness HCV populations and biological clones derived from them were used to infect Huh-7.5 hepatoma cells. Sofosbuvir efficacy was analyzed by measuring virus progeny production during several passages and by selection of possible sofosbuvir resistance mutations determined by sequencing the NS5B-coding region of the resulting populations. Sofosbuvir exhibited reduced efficacy against high-fitness HCV populations, without the acquisition of sofosbuvir-specific resistance mutations. A reduced sofosbuvir efficacy, similar to that observed with the parental populations, was seen for high-fitness individual biological clones. In independently derived high-fitness HCV populations or clones passaged in the presence of sofosbuvir, M289L was selected as the only substitution in the viral polymerase NS5B. In no case was the sofosbuvir-specific resistance substitution S282T observed. High HCV fitness can lead to decreased sensitivity to sofosbuvir, without the acquisition of specific sofosbuvir resistance mutations. Thus, fitness-dependent drug sensitivity can operate with HCV inhibitors that display a high barrier to resistance. This mechanism may underlie treatment failures not associated with selection of sofosbuvir-specific resistance mutations, linked to in vivo fitness of pretreatment viral populations. PMID:27067341

  5. HCV Diversity among Chinese and Burmese IDUs in Dehong, Yunnan, China

    Science.gov (United States)

    Chen, Xin; Duo, Lin; Li, Peilu; Zheng, Yong-Tang; Zhang, Chiyu

    2016-01-01

    HCV transmission is closely associated with drug-trafficking routes in China. Dehong, a prefecture of Yunnan, is the important trade transfer station linking Southeast Asia and China, as well as the drug-trafficking channel linking “Golden triangle” and other regions of China and surrounding countries. In this study, we investigated the HCV genotype diversity among IDUs in Dehong based on 259 HCV positive samples from 118 Chinese and 141 Burmese IDUs. HCV genotypes were determined based on the phylogenies of C/E2 and NS5B genomic sequences. Six HCV subtypes, including 1a, 1b, 3a, 3b, 6n and 6u, were detected. Interestingly, 4 HCV sequences from Burmese IDUs did not cluster with any known HCV subtypes, but formed a well-supported independent clade in the phylogenetic trees of both C/E2 and NS5B, suggesting a potential new HCV subtype circulating in Dehong. Subtype 3b was the predominant subtype, followed by subtypes 6n and 6u. Comparison showed that Dehong had a unique pattern of HCV subtype distribution, obviously different from other regions of China. In particular, HCV subtypes 6u and the potential new HCV subtype had a relatively high prevalence in Dehong, but were rarely detected in other regions of China. There was no significant difference in HCV subtype distribution between Burmese and Chinese IDUs. Few HCV sequences from Burmese and Chinese IDUs clustered together to form transmission clusters. Furthermore, about half of HCV sequences from Burmese IDUs formed small transmission clusters, significantly higher than that from Chinese IDUs (p<0.01). These suggest that the Chinese and Burmese IDUs were relatively isolated from each other in injection drug use behavior and the Burmese IDUs might prefer to inject drugs themselves together. The unique genotype distribution and complex diversity of genotype 6 among IDUs may be associated with the special geographical position of Dehong. PMID:27657722

  6. Identification of class I HLA T cell control epitopes for West Nile virus.

    Directory of Open Access Journals (Sweden)

    Saghar Kaabinejadian

    Full Text Available The recent West Nile virus (WNV outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1 the number of viral ligands presented by the HLA of infected cells, and 2 the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.

  7. Treatment of Chronic Hepatitis C in the Aged - Does It Impact Life Expectancy? A Decision Analysis.

    Directory of Open Access Journals (Sweden)

    Yaakov Maor

    Full Text Available Recent studies have demonstrated that the efficacy of interferon-free direct-acting antiviral agents (DAAs in patients over 70 is similar to that of younger age groups. Evidence continues to mount that life expectancy (LE increases with successful treatment of hepatitis C (HCV patients with advanced fibrosis. The evidence in older people is more limited. Our aim was to estimate the life year (LY and quality-adjusted life year (QALY gained by treatment of naïve patients with HCV as a function of patient's age and fibrosis stage.We constructed a Markov model of HCV progression toward advanced liver disease. The primary outcome was LY and QALY saved. The model and the sustained virological response of HCV infected subjects treated with a fixed-dose combination of the NS5B polymerase inhibitor Sofosbuvir and the NS5A replication complex inhibitor Ledipasvir were based on the published literature and expert opinion.Generally, both the number of LY gained and QALY gained gradually decreased with advancing age but the rate of decline was slower with more advanced fibrosis stage. For patients with fibrosis stage F1, F2 and F3, LY gained dropped below six months if treated by the age of 55, 65 or 70 years, respectively, while for a patient with fibrosis stage F4, the gain was one LY if treated by the age of 75. The QALY gained for treated over untreated elderly were reasonably high even for those treated at early fibrosis stage.There is a significant life expectancy benefit to HCV treatment in patients up to age 75 with advanced-stage fibrosis.

  8. Daclatasvir–sofosbuvir combination therapy with or without ribavirin for hepatitis C virus infection: from the clinical trials to real life

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    Pol S

    2016-03-01

    Full Text Available Stanislas Pol, Marion Corouge, Anaïs Vallet-Pichard Université Paris Descartes, Liver Department, Assistance Publique Hôpitaux de Paris, Cochin Hospital, French Institute of Health and Medical Research UMS20, Institut Pasteur, Paris, France Abstract: The treatment of hepatitis C virus has changed dramatically with the rapid advent of numerous new antiviral agents, including direct-acting antivirals and agents with non-viral targets (cyclophilin inhibitors, interferon-lambda, vaccine therapy. Given the better safety profile and high antiviral potency of direct-acting antivirals, their combination in interferon-free oral regimens is becoming the standard of care for hepatitis C virus infection, tailored to individual patients according to the degree of disease progression (fibrosis, hepatitis C virus genotype and subtype, resistance profile, and prior therapeutic history. Results from clinical studies as well as preliminary real-life data regarding the combination of sofosbuvir (a nucleotide polymerase inhibitor and daclatasvir, a first-in-class NS5A replication complex inhibitor, demonstrate that it is one of the most promising antiviral therapies, with once-daily oral dosing, a low pill burden, good tolerability, and limited drug–drug interactions, in addition to high antiviral potency, with >90% sustained virologic response rates. This combination has high pangenotypic antiviral potency regardless of the severity and patient characteristics. The combination of sofosbuvir and an NS5A inhibitor with ribavirin for 12 weeks appears to be a very good further treatment option in both cirrhotic and treatment-experienced patients whatever the stage of fibrosis. Keywords: hepatitis C virus, direct-acting antivirals, sofosbuvir, daclatasvir

  9. Combination ledipasvir-sofosbuvir for the treatment of chronic hepatitis C virus infection: a review and clinical perspective

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    Nkuize M

    2016-06-01

    Full Text Available Marcel Nkuize,1 Thomas Sersté,1,2 Michel Buset,1 Jean-Pierre Mulkay11Department of Gastroenterology and Hepatology, Saint-Pierre University Hospital, 2Department of Gastroenterology, Pancreatology and Hepatology, Hôpital Academique Erasme, Université Libre de Bruxelles, Brussels, Belgium Abstract: Chronic hepatitis C treatment has continued to evolve, and interferon-free, oral treatment with direct-acting antiviral agents is the current standard of care. Recently, a new treatment, which is a combination of two direct-acting antiviral agents, ledipasvir 90 mg (anti-NS5A and sofosbuvir 400 mg (anti-NS5B, has been approved in the US and the European Union for the treatment of chronic hepatitis C viral infection. In Phase III trials among chronic hepatitis C virus genotype 1 monoinfected (treatment-naïve, treatment-experienced, and with advanced liver disease or posttransplant patients and HIV–hepatitis C virus coinfected patients, the ledipasvir-sofosbuvir fixed-dose combination is associated with a higher rate of sustained virologic response at 12 weeks after therapy has ceased. According to preliminary data, the ledipasvir-sofosbuvir combination also may be effective against hepatitis C genotype 4 virus infection. The ledipasvir-sofosbuvir combination taken orally is generally well-tolerated. Moreover, the combination treatment may suppress the effect of predictive factors of chronic hepatitis C that have historically been known to be associated with treatment failure. Thus, the fixed-dose single-tablet combination of ledipasvir-sofosbuvir offers a new era for the effective treatment of a variety of patients suffering from chronic hepatitis C virus infection.Keywords: ledipasvir, liver disease, ethnicity, DAA, HIV

  10. A Complex Network of Interactions between S282 and G283 of Hepatitis C Virus Nonstructural Protein 5B and the Template Strand Affects Susceptibility to Sofosbuvir and Ribavirin.

    Science.gov (United States)

    Kulkarni, Anupriya S; Damha, Masad J; Schinazi, Raymond F; Mo, Hongmei; Doehle, Brian; Sagan, Selena M; Götte, Matthias

    2016-04-01

    The hepatitis C virus (HCV) RNA-dependent RNA-polymerase NS5B is essentially required for viral replication and serves as a prominent drug target. Sofosbuvir is a prodrug of a nucleotide analog that interacts selectively with NS5B and has been approved for HCV treatment in combination with ribavirin. Although the emergence of resistance to sofosbuvir is rarely seen in the clinic, the S282T mutation was shown to decrease susceptibility to this drug. S282T was also shown to confer hypersusceptibility to ribavirin, which is of potential clinical benefit. Here we devised a biochemical approach to elucidate the underlying mechanisms. Recent crystallographic data revealed a hydrogen bond between S282 and the 2'-hydroxyl of the bound nucleotide, while the adjacent G283 forms a hydrogen bond with the 2'-hydroxyl of the residue of the template that base pairs with the nucleotide substrate. We show that DNA-like modifications of the template that disrupt hydrogen bonding with G283 cause enzyme pausing with natural nucleotides. However, the specifically introduced DNA residue of the template reestablishes binding and incorporation of sofosbuvir in the context of S282T. Moreover, the DNA-like modifications of the template prevent the incorporation of ribavirin in the context of the wild-type enzyme, whereas the S282T mutant enables the binding and incorporation of ribavirin under the same conditions. Together, these findings provide strong evidence to show that susceptibility to sofosbuvir and ribavirin depends crucially on a network of interdependent hydrogen bonds that involve the adjacent residues S282 and G283 and their interactions with the incoming nucleotide and complementary template residue, respectively.

  11. Ledipasvir/Sofosbuvir: a review of its use in chronic hepatitis C.

    Science.gov (United States)

    Keating, Gillian M

    2015-04-01

    The single-tablet regimen of the hepatitis C virus (HCV) NS5A inhibitor ledipasvir and the HCV NS5B polymerase inhibitor sofosbuvir (ledipasvir/sofosbuvir; Harvoni(®)) was recently approved in the US and the EU. The phase III ION trials included treatment-naive (ION-1 and -3) or treatment-experienced (ION-2) patients with chronic HCV genotype 1 infection (≈20 % of patients in ION-1 and -2 had cirrhosis, whereas no patient in ION-3 had cirrhosis). A sustained virological response 12 weeks' post-treatment (SVR12) was seen in 99 % of treatment-naive patients receiving ledipasvir/sofosbuvir for 12 weeks in ION-1, with no additional benefit conferred by the addition of ribavirin or extending the treatment duration to 24 weeks. Moreover, in ION-3, an 8-week regimen achieved an SVR12 rate of 94 % overall and 97 % in the subgroup of patients with a baseline HCV RNA level of sofosbuvir for 12 and 24 weeks in ION-2. Data also support the use of ledipasvir/sofosbuvir in chronic HCV genotype 4 infection, in HCV and HIV co-infection and, in combination with ribavirin, in patients with chronic HCV genotype 1 or 4 infection who have decompensated cirrhosis or are liver transplant recipients and in chronic HCV genotype 3 infection. Oral ledipasvir/sofosbuvir was generally well tolerated. In conclusion, ledipasvir/sofosbuvir is an important new single-tablet regimen that represents a significant advance in the treatment of chronic hepatitis C.

  12. Acute hepatitis C in a chronically HIV-infected patient: Evolution of different viral genomic regions

    Institute of Scientific and Technical Information of China (English)

    Diego Flichman; Veronica Kott; Silvia Sookoian; Rodolfo Campos

    2003-01-01

    AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV infected patient that seroconverted to anti HCV antibodies were sequenced, from the event of superinfection through a period of 17 months and in a late sample (42nd month). Hypervariable genomic regions of HIV (V3 loop of the gp120) and HCV (HVR-1 on the E2 glycoprotein gene) were studied. In order to analyze genomic regions involved in different biological functions and with the cellular immune response, HCV core and NS5A were also chosen to be sequenced. Amplification of the different regions was done by RT-PCR and directly sequenced. Confirmation of sequences was done on reamplified material. Nucleotide sequences of the different time points were aligned with CLUSTAL W 1.5, and the corresponding amino acid ones were deduced.RESULTS: Hypervariable genomic regions of both viruses (HVR1 and gp120 V3 loop) presented several nonsynonymous changes but, while in the gp120 V3 loop mutations were detected in the sample obtained right after HCV superinfection and maintained throughout, they occurred following a sequential and cumulative pattern in the HVR1. In the NS5A region of HCV, two amino acid changes were detected during the follow-up period, whereas the core region presented several amino acid replacements, once the HCV chronic infection had been established.CONCLUSION: During the HIV-HCV superinfection, each genomic region analyzed shows a different evolutionary pattem.Most of the nucleotide substitutions observed are nonsynonymous and clustered in previously described epitopes,thus suggesting an immune-driven evolutionary process.

  13. Next-Generation Sequencing Reveals Frequent Opportunities for Exposure to Hepatitis C Virus in Ghana.

    Science.gov (United States)

    Forbi, Joseph C; Layden, Jennifer E; Phillips, Richard O; Mora, Nallely; Xia, Guo-Liang; Campo, David S; Purdy, Michael A; Dimitrova, Zoya E; Owusu, Dorcas O; Punkova, Lili T; Skums, Pavel; Owusu-Ofori, Shirley; Sarfo, Fred Stephen; Vaughan, Gilberto; Roh, Hajung; Opare-Sem, Ohene K; Cooper, Richard S; Khudyakov, Yury E

    2015-01-01

    Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5'-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3-4 centuries, indicating a long epidemic history of HCV-2 in Ghana.

  14. Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals.

    Science.gov (United States)

    Seronello, Scott; Montanez, Jessica; Presleigh, Kristen; Barlow, Miriam; Park, Seung Bum; Choi, Jinah

    2011-01-01

    Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.

  15. Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

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    Seiichi Mawatari

    Full Text Available BACKGROUND: It has been hypothesized that persistent hepatitis C virus (HCV infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4, composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. METHODS: Human C4 was incubated with HCV nonstructural (NS 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. RESULTS: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. CONCLUSIONS: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

  16. Transmission dynamics of hepatitis C virus among intra venous drug users in the border state of Manipur, India.

    Science.gov (United States)

    Saha, Kallol; Firdaus, Rushna; Biswas, Aritra; Mukherjee, Anirban; Sarkar, Kamalesh; Chakrabarti, Sekhar; Sadhukhan, Provash Chandra

    2014-06-01

    Intra venous drug users (IVDUs) are at high risk for hepatitis C virus (HCV) infection owing to their high rate of drug abuses. The north-eastern part of India has a high prevalence of IVDUs with Manipur being the worst hit state. The aim of the study was to document the molecular epidemiology, the patterns of HCV transmission, genomic variation and recombination events within HCV genome among IVDUs of Manipur, India. 91 anti-HCV sero-reactive blood samples were collected from IVDUs in Manipur. The samples were processed for RNA extraction, nested RT-PCR, sequencing and quantitative viral RNA estimation. Phylogeographic analysis of the sequenced core and NS5B regions of HCV genome was performed to determine the probable transmission route and recombinant HCV strains. 83 out of 91 anti-HCV seropositive samples were RNA positive (91.20%) based on 5'UTR of HCV genome by nested RT-PCR. Of the RNA positive samples, 73 paired partial core and NS5B gene were sequenced. Three major genotype and eight subtypes were detected while no recombinant strains were found. Individuals with genotype 1 had the mean viral load (5.94 ± 0.705 log10IU/ml) followed by genotype 3 (4.91 ± 0.49 log10IU/ml) and 6 (3.96 ± 0.32 log10IU/ml). The viral load was statistically significant among the male individuals at 4.822 ± 1.36 log10IU/ml compared to 4.767 ± 0.49 log10IU/ml for females (t=3.249, pManipur reflects the transmission of these strains from the "Golden Triangle" drug trafficking regions. Sequence analysis confirmed the transmission routes of HCV, which is linked to China and Vietnam for the newly emergent genotype 6 in north-eastern India.

  17. Novel drugs in the management of difficult-to-treat hepatitis C genotypes

    Directory of Open Access Journals (Sweden)

    Cartwright EJ

    2013-08-01

    Full Text Available Emily J Cartwright, Lesley MillerDepartment of Medicine, Emory University School of Medicine, Atlanta, GA, USABackground: Hepatitis C virus (HCV infection is the most common chronic bloodborne infection in the United States, with approximately 3.2 million Americans being chronically infected. Rates of HCV-related end-stage liver disease and its associated morbidity and mortality have yet to peak, so there is a pressing need for more effective and tolerable HCV treatment. HCV genotypes 1, 4, 5, and 6 are considered difficult to treat, and the need for improved therapies is especially great for persons infected with these genotypes.Current strategies for HCV treatment: Current therapy for genotype 1 HCV infection includes triple therapy with pegylated interferon, ribavirin, and a NS3/4A protease inhibitor. Sustained virologic response (SVR rates with triple therapy range from 42% to 75%, a vast improvement over pegylated interferon and ribavirin therapy alone. However, response rates remain suboptimal, and triple therapy is associated with significant adverse effects and is only indicated for genotype 1 HCV infection.Novel drugs for HCV treatment: HCV drug development is proceeding at a rapid pace to meet this need. Novel direct acting antiviral agents in several classes, including new NS3/4A serine protease inhibitors, NS5A replication complex inhibitors, NS5B polymerase inhibitors, interferon lambda, and microRNAs, are in varying stages of development. These new therapeutic agents promise SVR rates of up to 100% with durations as short as 12 weeks and, often, fewer adverse effects.Conclusion: New drug development in HCV is proceeding at an unprecedented pace. Novel agents promise higher SVR rates, shorter duration of therapy, and fewer adverse effects than have been possible with HCV therapy to date.Keywords: hepatitis C, direct acting antivirals, genotype

  18. 丙型肝炎病毒HLA-A*1101和A*2402限制性CD8+T细胞表位的研究%Identification of hepatitis C virus-specific HLA-A*1101-and A*2402-restricted CD8+ T-cell epitopes

    Institute of Scientific and Technical Information of China (English)

    钟晓芝; 段志良; 郭江龙; 李强; 王思娜; 李静; 林荣; 文金生

    2014-01-01

    目的:鉴定出丙型肝炎病毒(HCV)特异性HLA-A*1101限制性和A*2402限制性CD8+T细胞表位.方法:采用T细胞表位预测软件SYFPEITHI预测HCV特异性CD8+T细胞表位,合成HLA-A*1101限制性、A*2402限制性候选表位;建立永生化的HLA-A*1101阳性、A*2402阳性B细胞株,采用竞争性肽结合实验检测候选表位与HLA-A*1101或A*2402分子的结合力;候选表位体外分别刺激HLA-A*1101阳性或A*2402阳性HCV感染者外周血单个核细胞(PBMCs)后,采用酶联免疫斑点(ELISPOT)和细胞内细胞因子染色(ICS)实验分别检测肽特异性分泌γ-干扰素(IFN-γ)的细胞的水平和(肝) 异性IFN-γ+CD8+T细胞的水平.结果:5条HLA-A*1101限制性候选表位中,NS3_609(ITLTHPITK)和NS2_165(VVFSDMETK)与HLA-A*1101分子具有高结合力.3条HLA-A*2402限制性候选表位中,NS3_373 (KCDELASKL)和NS5b_382 (YYLTRDPTI)与HLA-A*2402具有高结合力;在HLA-A*1101阳性HCV感染者PBMCs中存在NS3 609和NS2_165特异性分泌IFN-γ的CD8+T细胞,而在HLA-A*2402阳性HCV感染者PBMCs中存在NS3_373和NS5b 382特异性分泌IFN-γ的CD8+T细胞.结论:本研究证实NS3_609(ITLTHPITK)和NS2_165(WFSDMETK)为全新的HCV特异性HLA-A*1101限制性CD8+T细胞表位,NS3_373 (KCDELASKL)和NS5b_382 (YYLTRDPTI)为全新的HCV特异性HLA-A*2402限制性CD8+T细胞表位.

  19. 基因重组HCV抗原斑点酶免疫法检测血清中抗-HCV%Immunoblotting Assay Using Recombinant Antigens for Detecting Antibodies against HCV

    Institute of Scientific and Technical Information of China (English)

    王百锁; 胡兴菊; 向前; 邢爱华; 王敬军

    2001-01-01

    目的 建立检测抗-HCV的斑点酶免疫法。方法 将基因重组HCV核心、NS3/NS4、NS5抗原和马抗人IgG、人IgG以最佳浓度固定在硝酸纤维膜条带上,用辣根过氧化酶和改进的底物进行斑点酶免疫反应。结果 检测了14份经ORTHO第三代抗-HCVELISA检测的血清,符合率为100%;72份经UBI第三代抗-HCVELISA监测的血清,符合率95.83%;103份经国产厦门新创抗-HCVELISA监测的血清,符合率95.14%。54份自然人群血清,阳性率3.47%。89份阳性标本中,三种抗体同时阳性的占30.34%,两种抗体阳性占69.66%,没有检出单一抗体阳性标本。核心抗体检出率最高87.64%,NS3/NS4抗体次之76.40%,NS5抗体最低61.80%。结论 用基因重组抗原建立的斑点酶免疫法与国内外广泛应用的ELISA试剂有较高的符合率,对进一步研究HCV感染诊断的确认试剂有意义。%Objective To set up a configuration of immunoblo tting assay forcletecting serum antibodies against HCV.Methods The proteins of the said virus that Core and NS3/NS4 and NS5 expres sed in E.coli cells were immobilized separately on nitrocellulose filter as antigens.HRP and a modified preparation of chromogenic substrate were used in the following procedure.Results Among 189 samples of sera tested by this Blotting Assay,14 were tested also by using ORTHO 3rd Generation ELISA regent and the coincident rate was 100% between the two assays,72 were tested by UBI 3rd Generation ELISA and their coincident rate was 95.83%,103 were tested by Xia Meng Xin Chuang ELISA and the coincident rate was 95.14%,Among 89 posi tive sera determined by Blotting Assay,those showed positive for all three antig ens accounted for 30.34%,and those positive for two of three antigens accounted for 69.66%,positive for only one antigen was not found.Among the 3 antibodies of Core was most efficient that 78 out of 89 positive sera reacted to it,the next was NS3/NS4with that number of 68

  20. Influence of quasispecies on virological responses and disease severity in patients with chronic hepatitis C

    Institute of Scientific and Technical Information of China (English)

    Deepak Kumar; Abdul Malik; Mohammad Asim; Anita Chakravarti; Rakha H Das; Premashis Kar

    2008-01-01

    AIM:To elucidate the influence of quasispecies on virological response and disease severity in patients with chronic hepatitis C.METHODS:Forty seven patients with hepatitis C [32 with chronic active hepatitis (CAH),9 with cirrhosis,and 6 with hepatocellular carcinoma (HCC)] were screened for the presence of quasispecies by single stranded conformational polymorphism (SSCP) analysis in the hypervariable region (HVR) and non-structural 5B (NS5B) viral genes of hepatitis C virus.The 41 patients excluding those with HCC were on therapy and followed up for a year with the determination of virological response and disease severity.Virus isolated from twenty three randomly selected patients (11 non-responders and 12 showing a sustained virological response) was sequenced for the assessment of mutations.RESULTS:The occurrence of quasispecies was proportionately higher in patients with HCC and cirrhosis than in those with CAH,revealing a significant correlation between the molecular evolution of quasispecies and the severity of disease in patients with hepatitis C.The occurrence of complex quasispecies has a significant association (P<0.05) with the non-responders,and leads to persistence of infection.Significant differences (P<0.05) in viral load (log10 IU/mL) were observed among patients infected with complex quasispecies (CQS),those infected with simple quasispecies (SQS) and those with no quasispecies (NQS),after 12 wk (CQS-5.2±2.3,SQS-3.2±1.9,NQS-2.8±2.4) and 24 wk (CQS-3.9±2.2,SQS-3.0±2.2,NQS-2.1±2.3) in the HVR region.However,a statistically significant difference (P<0.05) was observed between the viral loads of patients infected with CQS and those infected with NQS in NSSB viral gene after 24 wk (CQS-3.9±2.2,SQS-3.0±2.2,and NQS-2.1±2.3) and 48 wk (CQS-3.1±2.7,SQS-2.3±2.4,NQS-2.0±2.3) of therapy.Disease severity was significantly associated with viral load during therapy.The strains isolated from non-responders showed close pairing on phylogeny

  1. Molecular characterization of complete genome of Japanese encephalitis virus HEN0701 strain isolated from swine%猪源乙型脑炎病毒HEN0701株全基因组的分子特征

    Institute of Scientific and Technical Information of China (English)

    郑浩; 孙春清; 袁世山

    2011-01-01

    Four overlapping cDNA fragments from Japanese encephalitis virus(JEV) HEN0701 strain were cloned into pCR Ⅱ-Blunt-TOPO vector, followed by determination of complete genomic cDNA sequence. The complete genome of HEN0701 strain was 10 965 nucleotides in length,including a long open reading frame from 97th to 10395th nucleotide in the genome. Comparing to 6 strains of genotype Ⅰ (G Ⅰ)JEV and 5 strains of G Ⅲ, HEN0701 had high nucleotide sequence identity with G Ⅰ strains, but low nucleotide sequence identity with G Ⅲ strains in both complete genome and each gene. However, HEN0701 had high amino acid sequence identity with both G Ⅰ and GⅢ strains in both full polyprotein and each viral protein. Phylogenetic trees based on the C,E,NS3 and NS5 gene sequences of HEN0701 and 11 JEV strains were constructed with the software MEGA 5. Four phylogenetic trees had similar topology,indicating that the four genes from JEV had the similar evolutionary trend.%将乙型脑炎病毒HEN0701株基因组RNA分4段进行RT-PCR扩增,并克隆入pCRⅡ-Blunt-TOPO载体中,测定全长cDNA序列.以MapDraw软件分析了乙型脑炎病毒HEN0701株的基因组结构,并将病毒基因组分成2个非编码区和10个基因,比较了HEN0701株和11株乙型脑炎病毒在全基因组和各基因的核苷酸及推导氨基酸的同源性.以MEGA5软件绘制HEN0701株和11株病毒C、E、NS3、NS5 基因的进化树.结果显示,HEN0701株全基因组为10965 nt,包含一长10299 nt的开放阅读框.在核苷酸水平,HEN0701株与GⅠ分离株的全基因组和各基因的同源性均高,而与GⅢ分离株则较低;而在氨基酸水平,HEN0701株与GⅠ分离株在多聚蛋白和各病毒蛋白的同源性和HEN0701株与GⅢ分离株的相近.进化分析显示,不同基因进化树的拓扑结构相近,12株病毒的C、E、NS3、NS5基因进化方向相同,GⅠ分离株和GⅢ分离株间C基因进化距离最近.

  2. Effect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate

    Directory of Open Access Journals (Sweden)

    Michael Ho

    2011-01-01

    Full Text Available Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP at alternative sites to produce Aβ and the APP intracellular domain (AICD. Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro  γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg2+ stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca2+ and Mg2+ stabilize γ-secretase and enhance its activity.

  3. Genotypic distribution of hepatitis C virus in Thailand and Southeast Asia.

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    Rujipat Wasitthankasem

    Full Text Available The majority of hepatitis C virus (HCV infection results in chronic infection, which can lead to liver cirrhosis and hepatocellular carcinoma. Global burden of hepatitis C virus (HCV is estimated at 150 million individuals, or 3% of the world's population. The distribution of the seven major genotypes of HCV varies with geographical regions. Since Asia has a high incidence of HCV, we assessed the distribution of HCV genotypes in Thailand and Southeast Asia. From 588 HCV-positive samples obtained throughout Thailand, we characterized the HCV 5' untranslated region, Core, and NS5B regions by nested PCR. Nucleotide sequences obtained from both the Core and NS5B of these isolates were subjected to phylogenetic analysis, and genotypes were assigned using published reference genotypes. Results were compared to the epidemiological data of HCV genotypes identified within Southeast Asian. Among the HCV subtypes characterized in the Thai samples, subtype 3a was the most predominant (36.4%, followed by 1a (19.9%, 1b (12.6%, 3b (9.7% and 2a (0.5%. While genotype 1 was prevalent throughout Thailand (27-36%, genotype 3 was more common in the south. Genotype 6 (20.9% constituted subtype 6f (7.8%, 6n (7.7%, 6i (3.4%, 6j and 6m (0.7% each, 6c (0.3%, 6v and 6xa (0.2% each and its prevalence was significantly lower in southern Thailand compared to the north and northeast (p = 0.027 and p = 0.030, respectively. Within Southeast Asia, high prevalence of genotype 6 occurred in northern countries such as Myanmar, Laos, and Vietnam, while genotype 3 was prevalent in Thailand and Malaysia. Island nations of Singapore, Indonesia and Philippines demonstrated prevalence of genotype 1. This study further provides regional HCV genotype information that may be useful in fostering sound public health policy and tracking future patterns of HCV spread.

  4. Metabolism of phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity.

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    Annalisa Bianco

    Full Text Available 4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV--by recruiting PI4KIIIα in the RNA replication complex--hijacks PI4P metabolism, ultimately resulting in a markedly altered

  5. Changing the face of hepatitis C management – the design and development of sofosbuvir

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    Noell BC

    2015-04-01

    Full Text Available Bennett C Noell,* Siddesh V Besur,* Andrew S deLemos Department of Medicine, Center for Liver Diseases and Transplantation, Carolinas Medical Center, Charlotte, NC, USA *These authors contributed equally to this work Abstract: The availability of direct-acting antiviral (DAA therapy has launched a new era in the management of chronic hepatitis C. Sofosbuvir, a uridine nucleotide analog that inhibits the hepatitis C RNA-dependent RNA polymerase, is the backbone of chronic hepatitis C therapy. Acting at the catalytic site of the polymerase, sofosbuvir is highly potent in suppressing viral replication and has a high genetic barrier to resistance. Sofosbuvir is effective across all hepatitis C genotypes, and is a mainstay of interferon-free combination therapy. In Phase II and III studies, genotype 1 patients who took sofosbuvir in combination with another DAA such as the NS3-4A protease inhibitor, simeprevir, or the NS5A replication complex inhibitors, ledipasvir or daclatasvir, achieved a sustained virologic response rate of over 90%. Harvoni®, a combination tablet of sofosbuvir and ledipasvir, dosed once daily is recommended for 24 weeks for treatment-experienced genotype 1 patients with cirrhosis, but 12 weeks of therapy is sufficient for all other populations. While genotype 2 (12 weeks or 16 weeks and treatment-naïve genotype 3 patients (24 weeks have excellent response rates with sofosbuvir and ribavirin, treatment-experienced cirrhotic genotype 3 patients may need the addition of another DAA such as daclatasvir. Sofosbuvir is efficacious in special populations such as HIV–hepatitis C virus-coinfected patients and liver transplant recipients and has already made a profound impact in these groups. Since it is renally eliminated, patients with advanced kidney disease or on dialysis must await dosing recommendations. Sofosbuvir-based regimens appear to be well tolerated with headache and fatigue being the most common side effects. The

  6. Therapie der chronischen Hepatitis C: Der Beginn einer neuen Ära

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    Stättermayer AF

    2014-01-01

    Full Text Available Im letzten Jahrzehnt stellte die Kombination aus pegyliertem Interferon-α (PegINF-α und Ribavirin (RBV die Standardtherapie bei Patienten mit chronischer Hepatitis C dar. Mit der Zulassung der Protease-Inhibitoren Telaprevir und Boceprevir stehen seit 2011 erstmals direkt antiviral wirksame Substanzen („direct-acting antiviral agents“ [DAA] für die Therapie des HCV-Genotyps 1 zur Verfügung. Zwar konnte hiermit der Therapieerfolg deutlich erhöht werden, gleichzeitig führte die neue Triple-Therapie allerdings auch zu vermehrten Nebenwirkungen und höheren Therapiekosten. Zusätzliche Probleme wie Virusresistenz, Medikamenteninteraktionen und eine hohe Anzahl an täglich einzunehmenden Tabletten erschweren die antivirale Therapie mit Protease-Inhibitoren der ersten Generation. Insbesondere Patienten mit fortgeschrittener Lebererkrankung, welche den dringendsten Bedarf für eine antivirale Therapie aufweisen, haben ein hohes Risiko, schwere Nebenwirkungen unter der Triple-Therapie zu entwickeln. Die Entwicklungen in der Therapie der Hepatitis C sind rasant. So stehen gegenwärtig mit der Zulassung von Sofosbuvir (Sovaldi®, NS5BInhibitor, Simeprevir (Olysio®, Protease-Inhibitor und Daclatasvir (Daklinza®, NS5A-Inhibitor in Österreich 3 weitere direkt antiviral wirkende Substanzen zur Verfügung, welche in der Kombination bei Genotyp 1 (GT1 und Genotyp 4 (GT4 somit erstmalig eine Interferon-freie Therapie erlauben. Für die HCV-Genotypen 2 und 3 (GT2, GT3 ist die Interferon-freie Therapie in Form von Sofosbuvir/Ribavirin ebenfalls bereits Realität. Eine Reihe zusätzlicher Substanzen wird in Kürze folgen. Eine Vielzahl von klinischen Studien erbrachte diesbezüglich vielversprechende Ergebnisse. Diese zusätzlichen wirksamen, nebenwirkungsarmen, hocheffektiven und kürzeren Therapien für alle Patienten mit chronischer HCV-Infektion werden in absehbarer Zeit zur Verfügung stehen.

  7. Permissivity of primary human hepatocytes and different hepatoma cell lines to cell culture adapted hepatitis C virus.

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    Francois Helle

    Full Text Available Significant progress has been made in Hepatitis C virus (HCV culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells' permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.

  8. Mirror symmetry in the presence of branes

    Energy Technology Data Exchange (ETDEWEB)

    Mertens, Adrian

    2011-10-11

    over the plain. We study complex structure monodromies of the fibers and find evidence that they are mirror to the Calabi-Yau manifold with hypersurface that defines the combined deformation space, provided an NS5 brane is wrapped on the hypersurface. This gives a simple rule how to construct mirrors to Calabi-Yau manifolds with NS5 branes wrapped on hypersurfaces. (orig.)

  9. Hepatitis C virus core protein induces apoptosis-like caspase independent cell death

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    Gregor Michael

    2009-12-01

    Full Text Available Abstract Background Hepatitis C virus (HCV associated liver diseases may be related to apoptotic processes. Thus, we investigated the role of different HCV proteins in apoptosis induction as well as their potency to interact with different apoptosis inducing agents. Methods and Results The use of a tightly adjustable tetracycline (Tet-dependent HCV protein expression cell system with the founder osteosarcoma cell line U-2 OS allowed switch-off and on of the endogenous production of HCV proteins. Analyzed were cell lines expressing the HCV polyprotein, the core protein, protein complexes of the core, envelope proteins E1, E2 and p7, and non-structural proteins NS3 and NS4A, NS4B or NS5A and NS5B. Apoptosis was measured mainly by the detection of hypodiploid apoptotic nuclei in the absence or presence of mitomycin C, etoposide, TRAIL and an agonistic anti-CD95 antibody. To further characterize cell death induction, a variety of different methods like fluorescence microscopy, TUNEL (terminal deoxynucleotidyl transferase (TdT-catalyzed deoxyuridinephosphate (dUTP-nick end labeling assay, Annexin V staining, Western blot and caspase activation assays were included into our analysis. Two cell lines expressing the core protein but not the total polyprotein exerted a strong apoptotic effect, while the other cell lines did not induce any or only a slight effect by measuring the hypodiploid nuclei. Cell death induction was caspase-independent since it could not be blocked by zVAD-fmk. Moreover, caspase activity was absent in Western blot analysis and fluorometric assays while typical apoptosis-associated morphological features like the membrane blebbing and nuclei condensation and fragmentation could be clearly observed by microscopy. None of the HCV proteins influenced the apoptotic effect mediated via the mitochondrial apoptosis pathway while only the core protein enhanced death-receptor-mediated apoptosis. Conclusion Our data showed a caspase

  10. New treatment for hepatitis C in chronic kidney disease, dialysis, and transplant.

    Science.gov (United States)

    Fabrizi, Fabrizio; Martin, Paul; Messa, Piergiorgio

    2016-05-01

    The evidence that chronic hepatitis C plays a detrimental role in survival among patients on maintenance dialysis or renal transplant recipients promotes the antiviral treatment of hepatitis C virus (HCV) among chronic kidney disease patients. Also, it seems that HCV infection is associated with an increased risk of developing chronic kidney disease in the adult general population. Interferon-based regimens have provided limited efficacy and safety among chronic kidney disease patients, whereas the advent of the new direct-acting antivirals for the treatment of hepatitis C (launched over the past 5 years) has given the opportunity to reach sustained virologic response rates of 90% for many patient groups. Unfortunately, poor information exists regarding the antiviral treatment of hepatitis C in the chronic kidney disease population. The first published data on the treatment of hepatitis C among patients with chronic kidney disease (stage 4-5) and HCV genotype 1 regard the grazoprevir (NS3/4A protease inhibitor) and elbasvir (NS5A inhibitor) combination; excellent efficacy (sustained viral response, 94.3%; 115/122) and safety have been achieved. Preliminary evidence on the combined treatment of sofosbuvir (NS5B inhibitor) and simeprevir (NS3/4A inhibitor) has given a viral response of 89%, but the size of the study group (n = 38 patients with end-stage renal disease) was small. Some phase 2 and 3 clinical trials based on other antiviral combinations (3D regimen, sofosbuvir/ledipasvir, or other sofosbuvir-containing approaches) are ongoing. Thus, the antiviral regimens based on direct-acting antivirals promise to play a pivotal role in the eradication of hepatitis C among kidney disease patients. Direct-acting antivirals are very expensive; in an era of cost containment this is a crucial point either in developed and developing countries. Adverse drug reactions resulting from concomitantly administered medications are another ongoing concern for patients undergoing

  11. The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control.

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    Bukh, Jens

    2016-10-01

    The discovery of hepatitis C virus (HCV) in 1989 permitted basic research to unravel critical components of a complex life cycle for this important human pathogen. HCV is a highly divergent group of viruses classified in 7 major genotypes and a great number of subtypes, and circulating in infected individuals as a continuously evolving quasispecies destined to escape host immune responses and applied antivirals. Despite the inability to culture patient viruses directly in the laboratory, efforts to define the infectious genome of HCV resulted in development of experimental recombinant in vivo and in vitro systems, including replicons and infectious cultures in human hepatoma cell lines. And HCV has become a model virus defining new paradigms in virology, immunology and biology. For example, HCV research discovered that a virus could be completely dependent on microRNA for its replication since microRNA-122 is critical for the HCV life cycle. A number of other host molecules critical for HCV entry and replication have been identified. Thus, basic HCV research revealed important molecules for development of host targeting agents (HTA). The identification and characterization of HCV encoded proteins and their functional units contributed to the development of highly effective direct acting antivirals (DAA) against the NS3 protease, NS5A and the NS5B polymerase. In combination, these inhibitors have since 2014 permitted interferon-free therapy with cure rates above 90% among patients with chronic HCV infection; however, viral resistance represents a challenge. Worldwide control of HCV will most likely require the development of a prophylactic vaccine, and numerous candidates have been pursued. Research characterizing features critical for antibody-based virus neutralization and T cell based virus elimination from infected cells is essential for this effort. If the world community promotes an ambitious approach by applying current DAA broadly, continues to develop

  12. HCV亚基因复制子支持细胞的筛选%Replication of HCV Subgenomic Replicons in Two New Cell Lines

    Institute of Scientific and Technical Information of China (English)

    邬刚; 吴英松; 董文其; 陈白虹; 李明

    2006-01-01

    目的寻找新的支持HCV亚基因复制子复制的支持细胞,为研究HCV复制机制、HCV与宿主间的关系,及建立HCV缺陷病毒培养体系提供材料储备.方法质粒pNNeo3-5B含HCV-N亚基因复制子cDNA,该复制子RNA能在Huh7细胞中有效复制.缺失其中的BsaB Ⅰ-Hpa Ⅰ片段(含NS5B的GDD基序),生成缺陷型复制子质粒pNNeo3-5B(△).将以上两个质粒线性化后体外转录出复制子RNA:rNNeo3-5B和rNNeo3-5B(△).将rNNeo3-5B电转染Huh7、SMMC7721、HepG2、BEL7402、Lo2、CBRH7919、BHIK21、Vero E6、293和293T等细胞,以G418筛选3周,观察克隆形成情况,并检测克隆中HCV特异性RNA和蛋白.结果在CBRH7919和BHK21获得G418抗性克隆,转染rNNeo3-5B(△)的细胞和未转染复制子的细胞则全部死亡.RT-PCR检测证实各克隆都含有HCV 5'NTR和新霉素磷酸转移酶基因,间接免疫荧光和Western blot检测证实有大量表达的HCV NS3和NS5A蛋白,未转染复制子的细胞全为阴性.结论本研究证实CBRH7919和BHK21能支持HCV复制子的自主复制,可作为建立HCV病毒培养体系的备选材料.

  13. Safety and efficacy of ledipasvir‐sofosbuvir in black patients with hepatitis C virus infection: A retrospective analysis of phase 3 data

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    Jeffers, Lennox J.; Ravendhran, Natarajan; Shiffman, Mitchell L.; Poulos, John; Sulkowski, Mark S.; Gitlin, Norman; Workowski, Kimberly; Zhu, Yanni; Yang, Jenny C.; Pang, Phillip S.; McHutchison, John G.; Muir, Andrew J.; Howell, Charles; Kowdley, Kris; Afdhal, Nezam; Reddy, K. Rajender

    2015-01-01

    Black patients chronically infected with genotype 1 hepatitis C virus (HCV) have historically had lower rates of response to interferon‐based treatment than patients of other races. In the phase 3 ION program, the single‐tablet regimen of the NS5A inhibitor ledipasvir and NS5B nucleotide polymerase inhibitor sofosbuvir was shown to be safe and highly effective in the general population. The aim of this study was to evaluate the safety and efficacy of ledipasvir/sofosbuvir in black patients using data from the three open‐label ION clinical trials, which evaluated the safety and efficacy of 8, 12, and 24 weeks of ledipasvir/sofosbuvir with or without ribavirin for the treatment of treatment‐naïve and treatment‐experienced patients with genotype 1 HCV, including those with compensated cirrhosis. The primary endpoint was sustained virologic response at 12 weeks after the end of therapy (SVR12). For our analysis, rates of SVR12, treatment‐emergent adverse events, and graded laboratory abnormalities were analyzed in black versus non‐black patients. Of the 1949 patients evaluated, 308 (16%) were black. On average, black patients were older, had higher body mass index, were more likely to be IL28B non‐CC, and had a lower serum alanine aminotransferase at baseline than non‐black patients. Overall, 95% of black and 97% of non‐black patients achieved SVR12. The rate of relapse was 3% in black patients as compared with 2% in non‐black patients. The most common adverse events included fatigue, headache, nausea, and insomnia. The majority of adverse events occurred more frequently in the ribavirin‐containing arms of the studies. No differences were observed in overall safety by race. Conclusion: A once‐daily dosage of ledipasvir/sofosbuvir was similarly effective in black and non‐black patients with genotype 1 HCV infection. The addition of ribavirin did not appear to increase SVR12 but was associated with higher rates of adverse events. (Hepatology

  14. Epidemic history of major genotypes of hepatitis C virus in Uruguay.

    Science.gov (United States)

    Castells, M; Bello, G; Ifrán, S; Pereyra, S; Boschi, S; Uriarte, R; Cristina, J; Colina, R

    2015-06-01

    Worldwide, more than 170 million people are chronically infected with the hepatitis C virus (HCV) and every year die more than 350,000 people from HCV-related liver diseases. Recently, HCV was reclassified into seven major genotypes and 67 subtypes. Some subtypes as 1a, 1b and 3a, have become epidemic as a result of the new parenteral transmission routes and are responsible for most HCV infections in Western countries. HCV 1a subtype have been sub-categorized into two separate sub clades. Recent studies based on the analysis of NS5B genome region, reveal that HCV epidemics in Argentina and Brazil are characterized by multiple introductions events of subtypes 1a, 1b and 3a, followed by subsequent local dispersion. There is no data about HCV genotypes circulating in Uruguay and their evolutionary and demographic history. To this end, a total of 153 HCV NS5B gene sequences were obtained from Uruguayan patients between 2005 and 2011. 86 (56%) sequences grouped with subtype 1a, 40 (26%) with subtype 3a and 27 (18%) with subtype 1b. Furthermore, subtype 1a sequences were distributed among both clades, 1 (n=62, 72%) and 2 (n=24, 28%). Four local HCV clades were found: UY-1a(I), UY-1a(II), UY-1a(III) and UY-3a; comprising a 39% of all HCV viruses analyzed in this study. HCV epidemic in Uruguay has been driving by multiple introductions of subtypes 1a, 1b and 3a and by local dissemination of a few country-specific strains. The evolutionary and demographic history of the major Uruguayan HCV clade UY-1a(I) was reconstructed under two different molecular clock rate models and displayed an epidemic history characterized by an initial phase of rapid expansion followed by a more recent reduction of growth rate since 2000-2005. This is the first comprehensive study about the molecular epidemiology and epidemic history of HCV in Uruguay.

  15. Daclatasvir with sofosbuvir and ribavirin for hepatitis C virus infection with advanced cirrhosis or post‐liver transplantation recurrence

    Science.gov (United States)

    Schiff, Eugene R.; Vierling, John M.; Landis, Charles; Fontana, Robert J.; Yang, Rong; McPhee, Fiona; Hughes, Eric A.; Noviello, Stephanie; Swenson, Eugene S.

    2016-01-01

    Chronic hepatitis C virus (HCV) infection with advanced cirrhosis or post‐liver transplantation recurrence represents a high unmet medical need with no approved therapies effective across all HCV genotypes. The open‐label ALLY‐1 study assessed the safety and efficacy of a 60‐mg once‐daily dosage of daclatasvir (pan‐genotypic NS5A inhibitor) in combination with sofosbuvir at 400 mg once daily (NS5B inhibitor) and ribavirin at 600 mg/day for 12 weeks with a 24‐week follow‐up in two cohorts of patients with chronic HCV infection of any genotype and either compensated/decompensated cirrhosis or posttransplantation recurrence. Patients with on‐treatment transplantation were eligible to receive 12 additional weeks of treatment immediately after transplantation. The primary efficacy measure was sustained virologic response at posttreatment week 12 (SVR12) in patients with a genotype 1 infection in each cohort. Sixty patients with advanced cirrhosis and 53 with posttransplantation recurrence were enrolled; HCV genotypes 1 (76%), 2, 3, 4, and 6 were represented. Child‐Pugh classifications in the advanced cirrhosis cohort were 20% A, 53% B, and 27% C. In patients with cirrhosis, 82% (95% confidence interval [CI], 67.9%‐92.0%) with genotype 1 infection achieved SVR12, whereas the corresponding rates in those with genotypes 2, 3, and 4 were 80%, 83%, and 100%, respectively; SVR12 rates were higher in patients with Child‐Pugh class A or B, 93%, versus class C, 56%. In transplant recipients, SVR12 was achieved by 95% (95% CI, 83.5%‐99.4%) and 91% of patients with genotype 1 and 3 infection, respectively. Three patients received peritransplantation treatment with minimal dose interruption and achieved SVR12. There were no treatment‐related serious adverse events. Conclusion: The pan‐genotypic combination of daclatasvir, sofosbuvir, and ribavirin was safe and well tolerated. High SVR rates across multiple HCV genotypes were achieved by patients with

  16. Spatio-temporal tracking and phylodynamics of an urban dengue 3 outbreak in Sao Paulo, Brazil.

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    Adriano Mondini

    Full Text Available The dengue virus has a single-stranded positive-sense RNA genome of approximately 10.700 nucleotides with a single open reading frame that encodes three structural (C, prM, and E and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins. It possesses four antigenically distinct serotypes (DENV 1-4. Many phylogenetic studies address particularities of the different serotypes using convenience samples that are not conducive to a spatio-temporal analysis in a single urban setting. We describe the pattern of spread of distinct lineages of DENV-3 circulating in São José do Rio Preto, Brazil, during 2006. Blood samples from patients presenting dengue-like symptoms were collected for DENV testing. We performed M-N-PCR using primers based on NS5 for virus detection and identification. The fragments were purified from PCR mixtures and sequenced. The positive dengue cases were geo-coded. To type the sequenced samples, 52 reference sequences were aligned. The dataset generated was used for iterative phylogenetic reconstruction with the maximum likelihood criterion. The best demographic model, the rate of growth, rate of evolutionary change, and Time to Most Recent Common Ancestor (TMRCA were estimated. The basic reproductive rate during the epidemics was estimated. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide-long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000-2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1, closely related to the remaining 22 isolates (lineage 2. We assumed that these lineages appeared before 2006 in different occasions. By transforming the inferred exponential growth rates into the basic reproductive rate, we obtained values for lineage 1 of R(0 = 1.53 and values for

  17. 抗丙型肝炎病毒药索非布韦的临床研究进展

    Institute of Scientific and Technical Information of China (English)

    陈丹; 汤姝岚

    2015-01-01

    目的:综述抗丙型肝炎病毒(HCV)药索非布韦的临床研究进展.方法:以“Sofosbuvir"“索非布韦”“索氟布韦”“丙型肝炎”和“NS5B聚合酶抑制剂”等为关键词,组合检索2010年1月至2015年2月PubMed、万方、维普、中国知网等数据库,对索非布韦的临床药理(作用机制、药动学、药物相互作用、耐药性)、适用特殊人群、临床试验及安全性评价等内容进行综述.结果:共检索到相关文献76条,其中有效文献20条.作用机制方面,索非布韦通过与HCV的RNA核苷酸竞争HCV特异性NS5B聚合酶活化位点而发挥抗病毒作用,给药剂量为400 mg/d时对病毒的抑制作用最佳.临床应用方面,索非布韦适用于HCV初治患者和既往干扰素治疗失败的患者,不同HCV基因型感染可采用不同的给药方案;索非布韦对HCV感染合并肝硬化、肝脏移植和人类免疫缺陷病毒感染患者也有效,且给药时无需调整剂量.此外,索非布韦的耐药性较好、药物相互作用少、不良事件发生率较低.结论:索非布韦用于慢性HCV感染治疗,具有病毒学应答率高、适用人群广、不良反应发生率低、给药方式简单等特点,相信在未来会拥有良好的应用前景.

  18. Hepatitis C: sexual or intrafamilial transmission? Epidemiological and phylogenetic analysis of hepatitis C virus in 24 infected couples Hepatite C: transmissão sexual ou intrafamiliar? Análise epidemiológica e filogenética do vírus da hepatite C em 24 casais infectados

    Directory of Open Access Journals (Sweden)

    Norma de Paula Cavalheiro

    2009-06-01

    Full Text Available The role of sexual or intrafamilial transmission of hepatitis C is controversial. A phylogenetic analysis was performed on the non-structural region 5B of the hepatitis C virus (NS5B-HCV. High percentages of homology (mean of 98.3% were shown between the couples. Twenty (83.3% of the 24 men but only two of the women (8.3% reported having had sexually transmitted diseases during their lives. The risk factors for HCV acquisition were blood transfusion (10 couples, use of illegal injected drugs (17, use of inhalants (15, acupuncture (5 and tattoos (5. The shared use of personal hygiene items included toothbrushes between six couples (25%, razor blades between 16 (66.7%, nail clippers between 21 (87.5% and manicure pliers between 14 (58.3%. The high degree of similarity of the hepatitis C virus genome supports the hypothesis of hepatitis C virus transmission between these couples. The shared use of personal hygiene items suggests the possibility of intrafamilial transmission of infection.O papel da transmissão sexual ou intrafamiliar da hepatite C é controverso. Foi feita análise filogenética, região não estrutural 5B do vírus da hepatite C (NS5B-HCV. Altas percentagens de homologia com média de 98,3% foi revelada entre os casais. Vinte (83,3% de 24 homens, contra apenas duas (8,3% mulheres reportaram doença sexualmente transmisível durante suas vidas. Os fatores de risco para aquisição da doença foram: transfusão de sangue para 10 casais, uso de drogas ilícitas injetáveis para 17, inalatórias para 15, acupuntura em 5 e tatuagens para 5. O compartilhamento de utensílios de higiene pessoal incluem: escova de dente para seis (25% dos casais, lâmina de barbear para 16 (66,7%, cortador de unhas para 21 (87,5% e alicate de manicure para 14 (58,3%. O alto grau de similaridade genômica entre os vírus da hepatite C suporta a hipótese de transmissão entre os casais. O uso compartilhado de utensílios de higiene pessoal sugere a

  19. Hepatitis C and B Virus Infection in Chinese Patients with Extrahepatic Bile Duct Carcinoma

    Institute of Scientific and Technical Information of China (English)

    CHENMingyi; HUANGZhiqiang; CHENLezhen; GAOYabing; PENGRuiyun; WANGDewen

    2002-01-01

    Objective:In China, the incidence of extrahepatic bile duct carcinoma (EBDC) tends to increase over the past decades. The etiology of the noted increase in EBDC is not identified. Approximately, in a half of the overall Chinese patients with EBDC, the causative factors in the development of EBDC have not been demonstrated. There is a high prevalence of hepatitis C virus (HCV) or hepatitis B virus (HBV)in China, both of which can induce malignant transformation of infected cells and strongly associated with hepatocellular carcinoma (HCC).In this study,EBDC tissues from Chinese patients were examined for the presence of HCV and HBV infection to investigate further the potential causes of EBDC. Methods:HCV NS5 protein and HBsAg were detected by labeled streptavidin biotin (LSAB) method; HCV RNA and HBV DNA were detected by in situ polymerase chain reaction (IS-PCR) in formalin fixed, paraffin embedded specimens from 51 Chinese patients with EBDC. HCV RNA and HBV DNA were detected by IS-PCR in 34 Chinese patients with specimens of benign lesions of hepatobiliary tract(control group). Results:In 51 case tissue sections of EBDC, NS5 protein was detected in 14 (27.5%), and HBsAg in 5 (9.8%), HCV RNA in 18(35.4%) and HBV DNA in 8 (15.9%), respectively, of which HCV and HBV co-infection was detected in 2 (3.9%). In 34 case tissue sections of the control group, HCV RNA was detected in 2 (5.9%), and HBV DNA in 3 (8.8%).Conclusion:In this study using standard histochemical and PCR techniques,HCV and HBV and HBV presence in EBDC tissues than would be expected on serologic grounds.The detectable rate of HCV RNA in EBDC tissues was significantly higher than in control group(x2=9.808,P=0.002).As a result ,this study indicates that there is a correlation between the presence of HCV infection and EBDC,and HCV infection has possible ctiologic significance in the development of EBDCin China.While HBV DNA was detecled in EBDC tissues with the difference in the detectable rate of HBV

  20. The Novel Cyclophilin Inhibitor CPI-431-32 Concurrently Blocks HCV and HIV-1 Infections via a Similar Mechanism of Action.

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    Philippe A Gallay

    Full Text Available HCV-related liver disease is the main cause of morbidity and mortality of HCV/HIV-1 co-infected patients. Despite the recent advent of anti-HCV direct acting antivirals (DAAs, the treatment of HCV/HIV-1 co-infected patients remains a challenge, as these patients are refractory to most therapies and develop liver fibrosis, cirrhosis and liver cancer more often than HCV mono-infected patients. Until the present study, there was no suitable in vitro assay to test the inhibitory activity of drugs on HCV/HIV-1 co-infection. Here we developed a novel in vitro "co-infection" model where HCV and HIV-1 concurrently replicate in their respective main host target cells--human hepatocytes and CD4+ T-lymphocytes. Using this co-culture model, we demonstrate that cyclophilin inhibitors (CypI, including a novel cyclosporin A (CsA analog, CPI-431-32, simultaneously inhibits replication of both HCV and HIV-1 when added pre- and post-infection. In contrast, the HIV-1 protease inhibitor nelfinavir or the HCV NS5A inhibitor daclatasvir only blocks the replication of a single virus in the "co-infection" system. CPI-431-32 efficiently inhibits HCV and HIV-1 variants, which are normally resistant to DAAs. CPI-431-32 is slightly, but consistently more efficacious than the most advanced clinically tested CypI--alisporivir (ALV--at interrupting an established HCV/HIV-1 co-infection. The superior antiviral efficacy of CPI-431-32 over ALV correlates with its higher potency inhibition of cyclophilin A (CypA isomerase activity and at preventing HCV NS5A-CypA and HIV-1 capsid-CypA interactions known to be vital for replication of the respective viruses. Moreover, we obtained evidence that CPI-431-32 prevents the cloaking of both the HIV-1 and HCV genomes from cellular sensors. Based on these results, CPI-431-32 has the potential, as a single agent or in combination with DAAs, to inhibit both HCV and HIV-1 infections.

  1. Hepatitis C Virus Genotypes

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    Kayhan Azadmanesh

    2005-09-01

    throughout the viral genome(9.Genomic Organization of HCVThis virus has a positive-sense single-stranded RNA genome of about 10 kb containing one long open reading frame (ORF. All its proteins are encoded in a single open reading frame that encodes a polyprotein of approximately 3010 amino acids(10 (Fig. 1, with genes coding for structural proteins situated towards the N-terminus of the genome and non-structural genes located near the C-terminus. The structural genes code for the capsid protein (C, or Core and the envelope glycoproteins (E1, E2. The first 27 amino acids of the E2 gene constitute the hypervariable region 1 (HVR1, which is the most variable region of the genome and appears to be involved in virus evasion of the immune system and disease progression. The non-structural genes code for a protease (NS2, NS3 and its cofactor (NS4A, a helicase (NS3, a protein of unknown function (NS4B, a phosphoprotein (NS5A, and an RNA-dependent RNA polymerase (NS5B. In addition, the HCV genome has 5'-UTR and 3'- UTR untranslated regions (UTRs that are involved in control of viral translation. Correspondence:Malek H. Ahmadipour, Department of Hepatitis and AIDS,Pasteur Institute of Iran, Tehran, IranTel: +98 21 6696 92 91Fax: +98 21 6646 51 32E-mail: m_ahmadipour@yahoo.com It has a complicated secondary structure comprised of at least seven stem-loops important for ribosome entry and presumably, for viral RNA replication(11, 12. The translation of HCV RNA begins at the internal ribosome entry site (IRES and at the 40S ribosomal subunit in the absence of external factors, which makes the HCV translation efficient(13. Thus, the secondary structure of IRES plays an important role in HCV replication(14, and single nucleotide changes within this region could alter viral replication characteristics. HCV replication via RNA-dependent RNApolymerase is very error-prone and generates mutations at an estimated rate of 10-5 mutations per nucleotide per replication. This high mutation rate is

  2. HIV and HCV coinfection: prevalence, associated factors and genotype characterization in the Midwest Region of Brazil.

    Science.gov (United States)

    Freitas, Solange Zacalusni; Teles, Sheila Araújo; Lorenzo, Paulo Cesar; Puga, Marco Antonio Moreira; Tanaka, Tayana Serpa Ortiz; Thomaz, Danilo Yamamoto; Martins, Regina Maria Bringel; Druzian, Angelita Fernandes; Lindenberg, Andréa Siqueira Campos; Torres, Marina Sawada; Pereira, Sérgio A; Villar, Livia Melo; Lampe, Elisabete; Motta-Castro, Ana Rita Coimbra

    2014-01-01

    A cross-sectional study on prevalence, associated factors and genotype distribution of HCV infection was conducted among 848 HIV-infected patients recruited at reference centers in the Midwest Region of Brazil. The prevalence rate of HIV-HCV coinfection was 6.9% (95% CI: 5.2 to 8.6). In multivariable analysis, increasing age, use of illicit drugs (injection and non-injection), a history of blood transfusion before 1994, and the absence of a steady partnership were significant independent associated factors for HIV-HCV coinfection. The phylogenetic analysis based on the NS5B region revealed the presence of two major circulating genotypes of HCV: genotypes 1 (58.3%) and 3 (41.7%). The prevalence of HIV-HCV coinfection was lower than those reported in studies conducted with HIV-infected patients in different regions of Brazil, due to the fact that illicit drug use is not a frequent mode of HIV transmission in this region of Brazil. Serologic screening of HIV-patients for HCV before initiating antiretroviral treatment, a comprehensive identification of associated factors, and the implementation of effective harm reduction programs are highly recommended to provide useful information for treatment and to prevent HCV coinfection in these patients.

  3. Molecular characterization of HCV in a Swedish county over 8 years (2002–2009 reveals distinct transmission patterns

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    Josefine Ederth

    2016-02-01

    Full Text Available Background: Hepatitis C virus (HCV is a major public health concern and data on its molecular epidemiology in Sweden is scarce. We carried out an 8-year population-based study of newly diagnosed HCV cases in one of Sweden's centrally situated counties, Södermanland (D-county. The aim was to characterize the HCV strains circulating, analyze their genetic relatedness to detect networks, and in combination with demographic data learn more about transmission. Methods: Molecular analyses of serum samples from 91% (N=557 of all newly notified cases in D-county, 2002–2009, were performed. Phylogenetic analysis (NS5B gene, 300 bp was linked to demographic data from the national surveillance database, SmiNet, to characterize D-county transmission clusters. The linear-by-linear association test (LBL was used to analyze trends over time. Results: The most prevalent subtypes were 1a (38% and 3a (34%. Subtype 1a was most prevalent among cases transmitted via sexual contact, via contaminated blood, or blood products, while subtype 3a was most prevalent among people who inject drugs (PWIDs. Phylogenetic analysis revealed that the subtype 3a sequences formed more and larger transmission clusters (50% of the sequences clustered, while the 1a sequences formed smaller clusters (19% of the sequences clustered, possibly suggesting different epidemics. Conclusion: We found different transmission patterns in D-county which may, from a public health perspective, have implications for how to control virus infections by targeted interventions.

  4. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

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    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  5. Solvation mechanisms of nedocromil sodium from activation energy and reaction enthalpy measurements of dehydration and dealcoholation.

    Science.gov (United States)

    Richards, Alison C; McColm, Ian J; Harness, J Barrie

    2002-04-01

    Two independent athermal methods of analysis have been used to determine the activation energies associated with the dehydration of nedocromil sodium hydrates. For the highest temperature reaction, monohydrate to the anhydrate, the differences in the measured activation energies indicate a three-dimensional nucleation mechanism in the bulk of the crystal with subsequent three-dimensional anhydrate crystal growth. The number of critical nuclei varies inversely with heating rate. Measured enthalpy values for successive removal of water molecules at 31.7 +/- 1.0, 91.3 +/- 0.8, and 193 +/- 0.6 degrees C are the same, within experimental error, at 21.6 +/- 2.6 kJ (mol H(2)O)(-1), as determined from differential thermal analysis traces. This result implies that an earlier concept of "strong" and "weak" water binding is not relevant and temperatures at which H(2)O molecules are removed is related to nucleation effects and not bond energies. The low temperature shoulder on the 91.3 degrees C peak is identified as an effect arising from open pan analysis conditions. The appearance of "transient" peaks in the conditioning stage of nedocromil sodium trihydrate thermal analysis experiments have been investigated and an explanation based on the presence of alcoholates [(NS)(4) small middle dot 5CH(3)OH, (NS)(5) small middle dot 9C(2)H(5)OH, and (NS)(2) small middle dot C(3)H(7)OH] in the preparations is proposed.

  6. Detection of novel insect flavivirus sequences integrated in Aedes albopictus (Diptera: Culicidae in Northern Italy

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    Tenorio Antonio

    2009-07-01

    Full Text Available Abstract The presence of DNA sequences integrated from a new flavivirus related to Cell Fusing Agent and Kamiti River Virus was identified in wild Aedes albopictus mosquito populations from the provinces of Trentino and Padova, Northern Italy. Field work was developed during August–October 2007 with BG-traps, and mosquitoes were screened for flavivirus and alphavirus. No alphavirus was detected, indicating that Chikungunya virus is not present in these mosquitoes in Trentino and Padova area. However, 21% of the pools were positive for flavivirus, further recognised with BLAST as similar to Kamiti River Virus. Phylogenetical analysis with 708 nucleotides from the NS5 gene identified this virus as a new member of the insect flavivirus clade, together with others like Kamiti River Virus, Cell Fusing Agent or Culex flavivirus, and in the group of those transmitted by Aedes. Furthermore, the treatment with RNAse, indicated that this flavivirus should be integrated in the genome of Ae. albopictus. These results propose that these sequences are transmitted by both sexes, and with different prevalence in the studied populations, and support the idea of a widespread distribution of integrated genomes in several mosquitoes from different areas, as first demonstrated with Cell Silent Agent. Evolutionary implications of this discovery and application in flavivirus phylogeny are discussed.

  7. Faldaprevir for the Treatment of Hepatitis C

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    Tatsuo Kanda

    2015-03-01

    Full Text Available The current treatments for chronic hepatitis C virus (HCV genotype 1 infection are combinations of direct-acting antivirals, and faldaprevir is one of the new generation of HCV NS3/4A protease inhibitors. At the end of 2013, the US Food and Drug Administration (FDA approved the HCV NS3/4A protease inhibitor simeprevir and the HCV NS5B polymerase inhibitor sofosbuvir. Simeprevir or sofosbuvir in combination with pegylated interferon and ribavirin are available for clinical use. Faldaprevir, another HCV NS3/4A protease inhibitor that also has fewer adverse events than telaprevir or boceprevir, is under development. Of interest, faldaprevir in combination with pegylated interferon and ribavirin, and interferon-free treatment with faldaprevir in combination with deleobuvir plus ribavirin provides high sustained virological response rates for HCV genotype 1 infection. The aim of this article is to review these data concerning faldaprevir. Faldaprevir in combination with pegylated interferon and ribavirin treatment appears to be associated with fewer adverse events than telaprevir or boceprevir in combination with pegylated interferon and ribavirin, and may be one of the therapeutic options for treatment-naive patients with HCV genotype 1. The interferon-free combination of faldaprevir and deleobuvir with ribavirin was effective for HCV genotype 1 infection and may hold promise for interferon-ineligible and interferon-intolerant patients.

  8. Sofosbuvir: an all-around drug in hepatitis C treatment new era – The first phase

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    Alessia Ciancio

    2015-04-01

    Full Text Available Chronic hepatitis C virus (HCV infection is a slowly progressive disease affecting more than 185 million people worldwide. For many years, the combination of pegylated interferon (Peg-IFN plus ribavirin (RBV has been the backbone of treatment for patients infected with HCV. More than two years ago, the first generation direct-acting antiviral agents (DAAs – the protease inhibitors boceprevir and telaprevir – were approved for treatment of genotype 1 patients, doubling the cure rate. The new DAAs that have been developed, are effective for multiple genotypes, improve rates of sustained viral response with fewer side effects, simplify dosing and drug-drug interactions, and in some patients, offer the promise of interferon-free and/or ribavirin-free therapy. These new agents include the recently approved second generation protease inhibitor, the HCV NS5B polymerase inhibitor sofosbuvir and the NS3/4A protease inhibitor simeprevir as well as several other agents that are currently in later phases of development. With sofosbuvir-based regimens, successful interferon-free treatment is now available across all genotypes. In fact sofosbuvir is very effective in combination with Peg-interferon and ribavirine or with ribavirine alone or with other direct anti-viral agents. The following assessment evaluates the evidence on the clinical effectiveness and harms of sofosbuvir for the treatment of chronic hepatitis C.http://dx.doi.org/10.7175/rhc.v6i2.1164

  9. Closed And Open String Theories In Non-critical Backgrounds

    CERN Document Server

    Murthy, S

    2004-01-01

    This thesis is a study of closed and open string theories in low dimensional spacetimes, and the various relations between these theories. In particular, we focus on the theory of the two-dimensional black hole. We first study closed strings in the background of the Euclidean two-dimensional black hole (SL2( R )/U(1)) tensored with flat space, using the duality relating these theories to non-critical superstrings described by the supersymmetric sine-Liouville interaction on the worldsheet. We point out a subtlety in their geometric interpretation, and clarify the symmetry structure of the theories based on the understanding of these theories as near horizon limits of wrapped NS5-branes. In one such example (cigar × R6 ), we use the brave description to understand the enhancement of the global symmetry in the coset theory from U(1) to SO(3) under which the sine-Liouville and cigar interactions are related. In the same example, a worldsheet description of the moduli space R4/Z2 is presented. W...

  10. Discovery of a candidate quiescent low-mass X-ray binary in the globular cluster NGC 6553

    CERN Document Server

    Guillot, Sebastien; Brown, Edward F; Pavlov, George G; Zavlin, Vyacheslav E

    2011-01-01

    This paper reports the search for quiescent low-mass X-ray binaries (qLMXBs) in the globular cluster (GC) NGC 6553 using an XMM-Newton observation designed specifically for that purpose. The analysis leading to the spectral identification of one candidate qLMXB in the core of the cluster, based on the consistency of the spectrum with a neutron star H-atmosphere model at the distance of NGC 6553, is described in this paper. Specifically, the best-fit radius found using the three XMM European Photon Imaging Camera (EPIC) spectra is R_NS=6.3(+2.3)(-0.8) km (for M_NS=1.4 Msun) and the best-fit temperature is kTeff=136(+21)(-34) eV. Both physical parameters are in accordance with typical values of previously identified qLMXBs in GC and in the field, i.e. R_NS~5-20 km and kTeff~50-150 eV. A power-law (PL) component with a photon index Gamma=2.1(+0.5)(-0.8) is also required for the spectral fit and contributes to ~33% of the total flux of the X-ray source. A detailed analysis supports the hypothesis that the PL comp...

  11. Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Naïve Patients

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    Nadia Marascio

    2016-08-01

    Full Text Available Naturally occurring resistance-associated substitutions (RASs can negatively impact the response to direct-acting antivirals (DAAs agents-based therapies for hepatitis C virus (HCV infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014 project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L, telaprevir (V36L, I132V, simeprevir (V36L, and grazoprevir (V36L, V170I. Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N. This mutation could be important for treatment strategies in cases of previous therapy failure.

  12. Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Naïve Patients.

    Science.gov (United States)

    Marascio, Nadia; Pavia, Grazia; Strazzulla, Alessio; Dierckx, Tim; Cuypers, Lize; Vrancken, Bram; Barreca, Giorgio Settimo; Mirante, Teresa; Malanga, Donatella; Oliveira, Duarte Mendes; Vandamme, Anne-Mieke; Torti, Carlo; Liberto, Maria Carla; Focà, Alfredo; The Sinergie-Umg Study Group

    2016-08-27

    Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

  13. An unprecedented binuclear cadmium dithiocarbamate adduct: bis[μ2-N-(2-hydroxyethyl-N-isopropylcarbamodithioato-κ3S:S,S′]bis{[N-(2-hydroxyethyl-N-isopropylcarbamodithioato-κ2S,S′](3-{(1E-[(E-2-(pyridin-3-ylmethylidenehydrazin-1-ylidene]methyl}pyridine-κNcadmium]} dihydrate

    Directory of Open Access Journals (Sweden)

    Hadi D. Arman

    2016-09-01

    Full Text Available The asymmetric unit in the title binuclear compound, [Cd(C6H12NOS22(C12H10N4]2·2H2O, comprises a CdII atom, two dithiocarbamate (dtc anions, a monodentate 3-pyridinealdazine ligand and a lattice water molecule. The binuclear molecule is constructed by the application of inversion symmetry. One dtc ligand simultaneously chelates one cadmium atom and bridges the centrosymmetric mate, while the other dtc ligand is chelating only. This leads to a centrosymmetric [Cd(dtc2]2 core to which are appended two 3-pyridinealdazine ligands. The resulting NS5 donor set is based on an octahedron. The three-dimensional molecular packing is sustained by hydroxyl-O—H(hydroxyl and water-O—H...O(hydroxyl hydrogen bonding, leading to supramolecular layers parallel to (101 which are connected by water-O—H...N(pyridyl hydrogen bonding; additional C—H...O, S π(chelate ring interactions are also evident. The retention of the central [Cd(dtc2]2 core upon adduct formation is unprecedented in the structural chemistry of the zinc-triad dithiocarbamates.

  14. Bovine viral diarrhea virus type 2 in vivo infection modulates TLR4 responsiveness in differentiated myeloid cells which is associated with decreased MyD88 expression.

    Science.gov (United States)

    Schaut, Robert G; McGill, Jodi L; Neill, John D; Ridpath, Julia F; Sacco, Randy E

    2015-10-02

    Symptoms of bovine viral diarrhea virus (BVDV) infection range from subclinical to severe, depending on strain virulence. Several in vitro studies showed BVDV infection impaired leukocyte function. Fewer studies have examined the effects of in vivo BVDV infection on monocyte/macrophage function, especially with strains of differing virulence. We characterized cytokine production by bovine myeloid cells isolated early or late in high (HV) or low virulence (LV) BVDV2 infection. Given BVDV infection may enhance susceptibility to secondary bacterial infection, LPS responses were examined as well. Monocytes from HV and LV infected calves produced higher levels of cytokines compared to cells from controls. In contrast, monocyte-derived macrophage cytokine levels were generally reduced. Modulated cytokine expression in HV BVDV2 macrophages was associated with decreased MyD88 expression, likely due to its interaction with viral NS5A. These data and those of others, suggest that certain Flaviviridae may have evolved strategies for subverting receptor signaling pathways involving MyD88.

  15. Genetic Variability of Bovine Viral Diarrhea Virus and Evidence for a Possible Genetic Bottleneck during Vertical Transmission in Persistently Infected Cattle.

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    Natalie Dow

    Full Text Available Bovine viral diarrhea virus (BVDV, a Pestivirus in the family Flaviviridae, is an economically important pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI cattle, which shed large quantities of virus throughout life. Despite the absence of an acquired immunity against BVDV in these PI cattle there are strong indications of viral variability that are of clinical and epidemiological importance. In this study the variability of E2 and NS5B sequences in multiple body compartments of PI cattle were characterized using clonal sequencing. Phylogenetic analyses revealed that BVDV exists as a quasispecies within PI cattle. Viral variants were clustered by tissue compartment significantly more often than expected by chance alone with the central nervous system appearing to be a particularly important viral reservoir. We also found strong indications for a genetic bottleneck during vertical transmission from PI animals to their offspring. These quasispecies analyses within PI cattle exemplify the role of the PI host in viral propagation and highlight the complex dynamics of BVDV pathogenesis, transmission and evolution.

  16. Genetic Variability of Bovine Viral Diarrhea Virus and Evidence for a Possible Genetic Bottleneck during Vertical Transmission in Persistently Infected Cattle.

    Science.gov (United States)

    Dow, Natalie; Chernick, Adam; Orsel, Karin; van Marle, Guido; van der Meer, Frank

    2015-01-01

    Bovine viral diarrhea virus (BVDV), a Pestivirus in the family Flaviviridae, is an economically important pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI) cattle, which shed large quantities of virus throughout life. Despite the absence of an acquired immunity against BVDV in these PI cattle there are strong indications of viral variability that are of clinical and epidemiological importance. In this study the variability of E2 and NS5B sequences in multiple body compartments of PI cattle were characterized using clonal sequencing. Phylogenetic analyses revealed that BVDV exists as a quasispecies within PI cattle. Viral variants were clustered by tissue compartment significantly more often than expected by chance alone with the central nervous system appearing to be a particularly important viral reservoir. We also found strong indications for a genetic bottleneck during vertical transmission from PI animals to their offspring. These quasispecies analyses within PI cattle exemplify the role of the PI host in viral propagation and highlight the complex dynamics of BVDV pathogenesis, transmission and evolution.

  17. Discovery of a Second Transient Low-Mass X-ray Binary in the Globular Cluster NGC 6440

    CERN Document Server

    Heinke, C O; Cohn, H N; Lugger, P M; Budac, S A; Servillat, M; Linares, M; Strohmayer, T E; Markwardt, C B; Wijnands, R; Swank, J H; Knigge, C; Bailyn, C; Grindlay, J E

    2009-01-01

    We have identified a new transient luminous low-mass X-ray binary, NGC 6440 X-2, with Chandra/ACIS, RXTE/PCA, and Swift/XRT observations of the globular cluster NGC 6440. The discovery outburst (July 28-31, 2009) peaked at L_X~1.5*10^36 ergs/s, and lasted for 21, B>22 in quiescence from archival HST imaging, g'>22 during the third outburst from Gemini-South GMOS imaging, and J>~18.5$ and K>~17 during the second outburst from CTIO 4-m ISPI imaging. Archival Chandra X-ray images of the core do not detect the quiescent counterpart, and place a bolometric luminosity limit of L_{NS}< 5.6*10^31 ergs/s (one of the lowest measured) for a hydrogen atmosphere neutron star. A followup Chandra observation finds marginal evidence of enhanced quiescent emission at L_X (0.5-10 keV)~6*10^31 ergs/s 10 days into quiescence. NGC 6440 X-2 currently shows the shortest recurrence time (32 days) of any known X-ray transient, although regular outbursts were not visible in the bulge scans before early 2009. Fast, low-luminosity tr...

  18. Molecular epidemiology of hepatitis C infection in Cyprus within the general population and high-risk cohorts

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    Kostrikis Leondios G

    2011-10-01

    Full Text Available Abstract Background Initial data on the molecular epidemiology of HCV infection in Cyprus showed a highly polyphyletic infection and multiple points of introduction into the general population. The continuation and expansion of this investigation is presented here including high risk groups. Findings The samples include additional subjects from the general population, a group of inmates and HIV/HCV coinfected individuals, whose strains were amplified by RT-PCR and sequenced in partial Core-E1 and NS5B regions. The results confirm the broad genotype distribution and polyphyletic infection on the island, and no new subtypes were found. Monophyletic clusters between strains of the prisoners and the injecting drug users imply sharing of infected equipment, and highlight the risk of widespread transmission in these cohorts, although no spill-over to the general population was observed. Conclusions The results of this study underline the impact of population movements and high-risk population groups on the changing molecular epidemiology of HCV, with strains moving to Europe from Asia, Africa and Eastern Europe by means of immigration and modern transmission routes.

  19. Intrinsically Disordered Side of the Zika Virus Proteome

    Directory of Open Access Journals (Sweden)

    Rajanish Giri

    2016-11-01

    Full Text Available Over the last few decades, concepts of protein intrinsic disorder have been implicated in different biological processes. Recent studies have suggested that intrinsically disordered proteins (IDPs provide structural plasticity and functional diversity to viral proteins that are involved in rapid replication and immune evasion in host cells. In case of Zika virus, the roles of protein intrinsic disorder in mechanisms of pathogenesis are not completely understood. In this study, we have analyzed the prevalence of intrinsic disorder in Zika virus proteome (strain MR 766. Our analyses revealed that Zika virus polyprotein is enriched with intrinsically disordered protein regions (IDPRs and this finding is consistent with previous reports on the involvement of IDPs in shell formation and virulence of the Flaviviridae family. We found abundant IDPRs in Capsid, NS2B, NS3, NS4A, and NS5 proteins that are involved in mature particle formation and replication. In our view, the intrinsic disorder-focused analysis of ZIKV proteins could be important for the development of new disorder-based drugs.

  20. The Giant Inflaton

    Science.gov (United States)

    DeWolfe, Oliver; Kachru, Shamit; Verlinde, Herman

    2004-05-01

    We investigate a new mechanism for realizing slow roll inflation in string theory, based on the dynamics of p anti-D3 branes in a class of mildly warped flux compactifications. Attracted to the bottom of a warped conifold throat, the anti-branes then cluster due to a novel mechanism wherein the background flux polarizes in an attempt to screen them. Once they are sufficiently close, the M units of flux cause the anti-branes to expand into a fuzzy NS5-brane, which for rather generic choices of p/M will unwrap around the geometry, decaying into D3-branes via a classical process. We find that the effective potential governing this evolution possesses several epochs that can potentially support slow-roll inflation, provided the process can be arranged to take place at a high enough energy scale, of about one or two orders of magnitude below the Planck energy; this scale, however, lies just outside the bounds of our approximations.

  1. The Giant Inflaton

    CERN Document Server

    De Wolfe, O; Verlinde, H; Wolfe, Oliver De; Kachru, Shamit; Verlinde, Herman

    2004-01-01

    We investigate a new mechanism for realizing slow roll inflation in string theory, based on the dynamics of p anti-D3 branes in a class of mildly warped flux compactifications. Attracted to the bottom of a warped conifold throat, the anti-branes then cluster due to a novel mechanism wherein the background flux polarizes in an attempt to screen them. Once they are sufficiently close, the M units of flux cause the anti-branes to expand into a fuzzy NS5-brane, which for rather generic choices of p/M will unwrap around the geometry, decaying into D3-branes via a classical process. We find that the effective potential governing this evolution possesses several epochs that can potentially support slow-roll inflation, provided the process can be arranged to take place at a high enough energy scale, of about one or two orders of magnitude below the Planck energy; this scale, however, lies just outside the bounds of our approximations.

  2. Elliptic Genus of E-strings

    CERN Document Server

    Kim, Joonho; Lee, Kimyeong; Park, Jaemo; Vafa, Cumrun

    2014-01-01

    We study a family of 2d N=(0,4) gauge theories which describes at low energy the dynamics of E-strings, the M2-branes suspended between a pair of M5 and M9 branes. The gauge theory is engineered using a duality with type IIA theory, leading to the D2-branes suspended between an NS5-brane and 8 D8-branes on an O8-plane. We compute the elliptic genus of this family of theories, and find agreement with the known results for single and two E-strings. The partition function can in principle be computed for arbitrary number of E-strings, and we compute them explicitly for low numbers. We test our predictions against the partially known results from topological strings, as well as from the instanton calculus of 5d Sp(1) gauge theory. Given the relation to topological strings, our computation provides the all genus partition function of the refined topological strings on the canonical bundle over 1/2 K3.

  3. New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory.

    Science.gov (United States)

    Roszak, Aleksander W; Moulisová, Vladimíra; Reksodipuro, Adhie D P; Gardiner, Alastair T; Fujii, Ritsuko; Hashimoto, Hideki; Isaacs, Neil W; Cogdell, Richard J

    2012-02-15

    Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.

  4. Inflation from Supergravity with Gauged R-symmetry in de Sitter Vacuum

    CERN Document Server

    Antoniadis, I; Isono, H; Knoops, R

    2016-01-01

    We study the cosmology of a recent model of supersymmetry breaking, in the presence of a tuneable positive cosmological constant, based on a gauged shift symmetry of a string modulus that can be identified with the string dilaton. The minimal spectrum of the `hidden' supersymmetry breaking sector consists then of a vector multiplet that gauges the shift symmetry of the dilaton multiplet and when coupled to the MSSM leads to a distinct low energy phenomenology depending on one parameter. Here we study the question if this model can also lead to inflation by identifying the dilaton with the inflaton. We find that this is possible if the K\\"ahler potential is modified by a term that has the form of NS5-brane instantons, leading to an appropriate inflationary plateau around the maximum of the scalar potential, depending on two extra parameters. This model is consistent with present cosmological observations without modifying the low energy particle phenomenology associated to the minimum of the scalar potential.

  5. Drug: D10105 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D10105 Drug Daclatasvir hydrochloride (JAN); Daclatasvir dihydrochloride (USAN) C40H50N8O6. 2HCl 810....3387 811.7969 D10105.gif Treatment of hepatitis C [DS:H00413] HCV NS5A inhibitor [CPD:C182...92] ko05160 Hepatitis C CAS: 1009119-65-6 PubChem: 135626823 LigandBox: D10105 ATOM 56 1 C8x C 21.4200 -21.4...900 2 C8y C 21.4200 -22.8900 3 C8x C 22.6100 -23.5900 4 C8x C 23.8700 -22.8900 5 ...C8y C 23.8700 -21.4900 6 C8x C 22.6100 -20.7900 7 C8y C 25.0600 -20.7900 8 C8x C 26.3200 -21.4900 9 C8x C 27.5800 -20.7900 10

  6. Ring Relations and Mirror Map from Branes arXiv

    CERN Document Server

    Assel, Benjamin

    We study the space of vacua of three-dimensional $\\mathcal{N}=4$ theories from a novel approach building on the type IIB brane realization of the theory and in which the insertion of local chiral operators in the path integral is obtained from integrating out light modes in appropriate brane setups. Most of our analysis focuses on abelian quiver theories which can be realized as the low-energy theory of D3-D5-NS5 brane arrays. Their space of vacua contains a Higgs branch, parametrized by the vevs of half-BPS meson operators, and a Coulomb branch, parametrized by the vevs of half-BPS monopole operators. We show that the Higgs operators are inserted by adding F1 strings and D3 branes, while the Coulomb operators are inserted by adding D1 strings and D3 branes, with specific orientations, to the initial brane setup of the theory. This approach has two main advantages. First the ring relations describing the Higgs and Coulomb branches can be derived by looking at specific brane setups with multiple interpretation...

  7. Lymphotoxin signaling is initiated by the viral polymerase in HCV-linked tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Yannick Simonin

    2013-03-01

    Full Text Available Exposure to hepatitis C virus (HCV typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-β in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKβ inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-β pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis.

  8. Isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14, from domestic rabbits.

    Science.gov (United States)

    Lau, Susanna K P; Woo, Patrick C Y; Yip, Cyril C Y; Fan, Rachel Y Y; Huang, Yi; Wang, Ming; Guo, Rongtong; Lam, Carol S F; Tsang, Alan K L; Lai, Kenneth K Y; Chan, Kwok-Hung; Che, Xiao-Yan; Zheng, Bo-Jian; Yuen, Kwok-Yung

    2012-05-01

    We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 10(8) copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5'-UCUAAAC-3'. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits.

  9. DNA Typing of Sporothrix schenckii by Analysis of Ribosomal-DNA Regions%利用核糖体基因进行申克孢子丝菌基因分型

    Institute of Scientific and Technical Information of China (English)

    张振颖; 刘晓明; 高晓蓉; 杨国玲; 金礼吉; 安利佳

    2005-01-01

    目的利用探针与DNA印迹法对申克孢子丝菌进行种内分型,探讨其基因型特征与菌种来源及临床表现的关系.方法CTAB法提取来源于不同地区31株孢子丝菌临床分离株及1株标准株的基因组DNA,以真菌通用引物ITS4、NS5扩增标准株的rDNA序列作为探针,与经限制性内切酶Apal酶切后的基因组DNA进行印迹杂交.结果杂交后形成多种清晰而稳定的带型,根据带型将31株孢子丝菌分为15种基因型(A-O型),其中A、B、C三型占51.61%.结论探针与DNA印迹法是孢子丝菌种内分型较为敏感而可靠的方法,该方法所分基因型的不同与菌种的地区来源及临床表现有一定的相关性.

  10. Factors associated with the response to interferon-based antiviral therapies for chronic hepatitis C

    Institute of Scientific and Technical Information of China (English)

    Hirayuki; Enomoto; Shuhei; Nishiguchi

    2015-01-01

    Hepatitis C virus(HCV) infection is a major health concern worldwide. Interferon-α(IFN-α) therapy has been the main antiviral treatment for more than 20 years. Because of its established antitumor effects, IFNbased treatments for chronic HCV infection still have a clinical impact, particularly for patients with high risk conditions of developing hepatocellular carcinoma, such as older age and advanced liver fibrosis. As a result of exhaustive research, several viral factors, including NS5 A amino acid mutations such as the IFN sensitivitydetermining region and the IFN/ribavirin resistancedetermining region, and mutations of amino acids in the core protein region(core 70 and 91) were shown to be associated with the response to IFN-α treatment. In addition, among the host factors related to the response to IFN-α treatment, polymorphisms of the interleukin-28 B gene were identified to be the most important factor. In this article, we review the factors associated with the efficacy of IFN-α treatment for chronic HCV infection. In addition, our recent findings regarding the possible involvement of anti-IFN-α neutralizing antibodies in a non-response to pegylated-IFN-α treatment are also described.

  11. Hepatitis B and C prevalences among blood donors in the south region of Brazil

    Directory of Open Access Journals (Sweden)

    H. C. F. F. Vasconcelos

    1994-12-01

    Full Text Available The prevalence of hepatitis B and C infection has been determined in a seroepidemiological survey among blood donors from the south of Brazil (Florianópolis, State of Santa Catarina. These markers has also been correlated with the levels of alanine aminotransferase (ALT, a surrogate marker to prevent post-transfusion hepatitis. Sera from 5000 donors were randomly collected in the period of April to November 1991. The prevalences of HBsAg, anti-HBs and anti-HBc were respectively 0.78, 7.02 and 13.98. The anti-HCV prevalence after confirmation testing with line immunoassay (LIA, was 1.14. Normal values of ALT ( = 70 U/ml in 2.48. The positivity of anti-HCV antibodies increased with the elevation of ALT levels. This correlation was not observed in relation to HBsAg. There exists a diversity in the recognition of HCV epitopes among HCV positive donors. Via the confirmation test used, we could observe that 94.7 of donors recognize the structural core antigen. Besides that, we observed that 5.26 of the HCV reactive sera recognized only epitopes located in the NS4 and/or NS5 region, indicating the importance of these epitopes for the improvement of assays.

  12. N=(4,4) Gauged Linear Sigma Models for Defect Five-branes

    CERN Document Server

    Kimura, Tetsuji

    2015-01-01

    We study two-dimensional ${\\cal N}=(4,4)$ gauged linear sigma model (GLSM). Its low energy effective theory is a nonlinear sigma model whose target space gives rise to a configuration of five-branes in string theory. In this article we focus on sigma models for NS5-branes, KK5-branes and an exotic $5^2_2$-brane. In particular, we carefully analyze the GLSM for an exotic $5^2_2$-brane whose background configuration is multi-valued. The exotic $5^2_2$-brane is a concrete example of nongeometric configuration in string theory. We find that the exotic feature originates from the string winding coordinate in a very clear way. In order to complete this analysis, we propose a duality transformation formula which converts an ${\\cal N}=(2,2)$ chiral superfield in F-term to a twisted chiral superfield coupled to an unconstrained complex superfield. This article is a short review based on arXiv:1304.4061 in collaboration with Shin Sasaki.

  13. Semi-doubled Sigma Models for Five-branes

    CERN Document Server

    Kimura, Tetsuji

    2015-01-01

    We study two-dimensional ${\\cal N}=(2,2)$ gauge theory and its dualized system in terms of complex (linear) superfields and their alternatives. Although this technique itself is not new, we can obtain a new model, the so-called "semi-doubled" GLSM. Similar to doubled sigma model, this involves both the original and dual degrees of freedom simultaneously, whilst the latter only contribute to the system via topological interactions. Applying this to the ${\\cal N}=(4,4)$ GLSM for H-monopoles, i.e., smeared NS5-branes, we obtain its T-dualized systems in quite an easy way. As a bonus, we also obtain the semi-doubled GLSM for an exotic $5^3_2$-brane whose background is locally nongeometric. In the low energy limit, we construct the semi-doubled NLSM which also generates the conventional string worldsheet sigma models. In the case of the NLSM for $5^3_2$-brane, however, we find that the Dirac monopole equation does not make sense any more because the physical information is absorbed into the divergent part via the ...

  14. Hyper-K\\"{a}hler with Torsion, T-duality, and Defect (p,q) Five-branes

    CERN Document Server

    Kimura, Tetsuji; Yata, Masaya

    2014-01-01

    We investigate the five-branes interpretation of a hyper-K\\"{a}hler geometry with torsion (HKT). This geometry is obtained by a conformal transformation of the Taub-NUT space which represents a single Kaluza-Klein five-brane. This HKT would represent an NS5-brane on the Taub-NUT space. In order to explore the HKT further, we compactify one transverse direction, and study the $O(2,2;{\\mathbb Z}) = SL(2,{\\mathbb Z}) \\times SL(2,{\\mathbb Z})$ monodromy structure associated with two-torus. Performing the conjugate transformation, we obtain a new solution whose physical interpretation is the defect $(p,q)$ five-branes on the ALG space. Throughout this analysis, we understand that the HKT represents the coexistent state of two kinds of five-branes. This situation is different from composite states such as $(p,q)$ five-branes or $(p,q)$ seven-branes in type IIB theory. We also study the T-dualized system of the HKT. We again find a new solution which also indicates the defect $(p,q)$ five-branes on another ALG space...

  15. Phase transitions of large N orbifold gauge theories

    Energy Technology Data Exchange (ETDEWEB)

    Hikida, Y.

    2006-10-15

    We study the phase structures of N=4 SU(N) super Yang-Mills theories on R x S{sup 3}/Z{sub k} with large N. The theory has many vacua labelled by the holonomy matrix along the non-trivial cycle on S{sup 3}/Z{sub k}, and for the fermions the periodic and the anti-periodic boundary conditions can be assigned along the cycle. We compute the partition functions of the orbifold theories and observe that phase transitions occur even in the zero 't Hooft coupling limit. With the periodic boundary condition, the vacua of the gauge theory are dual to various arrangements of k NS5-branes. With the anti-periodic boundary condition, transitions between the vacua are dual to localized tachyon condensations. In particular, the mass of a deformed geometry is compared with the Casimir energy for the dual vacuum. We also obtain an index for the supersymmetric orbifold theory. (orig.)

  16. Cell culture system of a hepatitis C genotype 3a and 2a chimera

    DEFF Research Database (Denmark)

    2015-01-01

    A robust and genetically stable cell culture system for Hepatitis C Virus (HCV) genotype 3a is provided. A genotype 3a/2a (S52/JFH1) recombinant containing the structural genes (Core, E1, E2), p7 and NS2 of strain S52 was constructed and characterized in Huh7.5 cells. S52/JFH1 and J6/JFH viruses...... passaged in cell culture had comparable growth kinetics and yielded similar peak HCV RNA titers and infectivity titers. Direct genome sequencing of cell culture derived S52/JFH1 viruses identified putative adaptive mutations in Core, E2, p7, NS3, and NS5A; clonal analysis revealed that all genomes analyzed...... exhibited different combinations of these mutations. Finally, viruses resulting from transfection with RNA transcripts of five S52/JFH1 recombinants containing these combinations of putative adaptive mutations performed as efficiently as J6/JFH viruses in Huh7.5 cells and were all genetically stable after...

  17. The hidden seasonality of the rare biosphere in coastal marine bacterioplankton

    KAUST Repository

    Alonso-Sáez, Laura

    2015-04-08

    Summary: Rare microbial taxa are increasingly recognized to play key ecological roles, but knowledge of their spatio-temporal dynamics is lacking. In a time-series study in coastal waters, we detected 83 bacterial lineages with significant seasonality, including environmentally relevant taxa where little ecological information was available. For example, Verrucomicrobia had recurrent maxima in summer, while the Flavobacteria NS4, NS5 and NS2b clades had contrasting seasonal niches. Among the seasonal taxa, only 4 were abundant and persistent, 20 cycled between rare and abundant and, remarkably, most of them (59) were always rare (contributing <1% of total reads). We thus demonstrate that seasonal patterns in marine bacterioplankton are largely driven by lineages that never sustain abundant populations. A fewer number of rare taxa (20) also produced episodic \\'blooms\\', and these events were highly synchronized, mostly occurring on a single month. The recurrent seasonal growth and loss of rare bacteria opens new perspectives on the temporal dynamics of the rare biosphere, hitherto mainly characterized by dormancy and episodes of \\'boom and bust\\', as envisioned by the seed-bank hypothesis. The predictable patterns of seasonal reoccurrence are relevant for understanding the ecology of rare bacteria, which may include key players for the functioning of marine ecosystems. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  18. Hepatitis C virus proteins do not directly trigger fibrogenic events in cultured human liver myofibroblasts.

    Science.gov (United States)

    Tan, K; Guibert, C; Neaud, V; Rosenbaum, J

    2003-11-01

    Although liver fibrosis is the major complication of hepatitis C virus (HCV) infection, the mechanisms of fibrogenesis in this setting are not completely understood. The aim of this study was to test the direct effect of HCV proteins on signalling- and fibrosis-related events in cultured human liver myofibroblasts, the effector cells of liver fibrogenesis. Cultured myofibroblasts were exposed to recombinant HCV core, a structural protein, and nonstructural proteins (NS) 3, NS 4 and NS 5. HCV proteins did not significantly increase DNA synthesis in myofibroblasts. We then examined if these proteins affected early signalling events. None of the HCV proteins affected the phosphorylation of the mitogen activated protein kinases/extracellular regulated kinases 1 and 2, or of the phosphatidylinositol 3-kinase target, Akt. HCV proteins had also no effect on intracellular calcium concentration. In other experiments, fibrogenesis-related parameters were measured. None of the HCV proteins had any effect on the secretion of type I collagen, tissue inhibitor of matrix metalloproteinases type 1, gelatinase or urokinase. Alpha-smooth muscle actin expression was also not modified. In summary, our experiments do not support a direct effect of these HCV proteins on fibrogenic cells.

  19. A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors.

    Science.gov (United States)

    Jaffrelo, Loic; Chabas, Sandrine; Reigadas, Sandrine; Pflieger, Aude; Wychowski, Czeslaw; Rumi, Julie; Ventura, Michel; Toulmé, Jean-Jacques; Staedel, Cathy

    2008-09-01

    We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.

  20. Early events in the generation of autophagosomes are required for the formation of membrane structures involved in hepatitis C virus genome replication.

    Science.gov (United States)

    Mohl, Bjorn-Patrick; Bartlett, Christopher; Mankouri, Jamel; Harris, Mark

    2016-03-01

    Hepatitis C virus (HCV) infection has been shown to induce autophagy but the mechanisms underpinning this process remain to be elucidated. Induction of autophagy requires the class III phosphatidylinositol 3-kinase, Vps34, which produces phosphatidylinositol 3-phosphate (PI3P) within the endoplasmic reticulum (ER) membrane. This recruits proteins with PI3P binding domains such as the double-FYVE-containing protein 1 (DFCP1). DFCP1 generates cup-shaped protrusions from the ER membrane, termed omegasomes, which provide a platform for the production of autophagosomes. Here we present data demonstrating that both Vps34 and DFCP1 are required for HCV genome replication, in the context of both a subgenomic replicon and virus infection, but did not affect virus entry or initial translation. Using live cell fluorescence microscopy we demonstrated that early during HCV infection the nascent viral genome replication complexes (identified by using non-structural protein NS5A as a marker) transiently colocalize with DFCP1-positive punctae (omegasomes), before the two structures move apart from each other. This observation is reminiscent of the transient association of LC3 and DFCP1 during omegasome formation, and therefore we propose that omegasomes are utilized by HCV to generate the double-membrane vesicles which are the hallmark of HCV replication complexes.

  1. PI3K-Akt signaling pathway upregulates hepatitis C virus RNA translation through the activation of SREBPs.

    Science.gov (United States)

    Shi, Qing; Hoffman, Brett; Liu, Qiang

    2016-03-01

    Hepatitis C virus (HCV) activates PI3K-Akt signaling to enhance entry and replication. Here, we found that this pathway also increased HCV translation. Knocking down the three Akt isoforms significantly decreased, whereas ectopic expression increased HCV translation. HCV translation upregulation by Akt required their kinase activities because Akt kinase-dead mutants downregulated HCV translation; and was dependent on PI3K activity since it was sensitive to PI3K inhibitor wortmannin. The viral 3'UTR was not involved in translation upregulation by Akt. HCV NS5A increased Akt phosphorylation/activity and HCV translation in the absence of the viral 3'UTR. Sterol regulatory element-binding proteins (SREBPs) were the downstream effectors of the PI3K-Akt pathway in regulating HCV translation because Akt1 and Akt2 activated both SREBP-1 and SREBP-2, whereas Akt3 upregulated SREBP-1. Knocking down SREBPs significantly decreased, while ectopic expression of SREBPs increased HCV translation. Taken together, we showed that the PI3K-Akt signaling pathway positively regulates HCV translation through SREBPs.

  2. Hepatitis C virus and host cell lipids: an intimate connection.

    Science.gov (United States)

    Alvisi, Gualtiero; Madan, Vanesa; Bartenschlager, Ralf

    2011-01-01

    Hepatitis C virus (HCV) is a major human pathogen, persistently infecting more than 170 million individuals worldwide. The recent establishment of fully permissive culture systems allowed unraveling the close link between host cell lipids and HCV, at each step of the viral replication cycle. HCV entry is triggered by the timely coordinated interaction of virus particles with cell surface receptors, including the low-density lipoprotein receptor. Viral RNA replication strictly depends on fatty acids and cholesterol biosynthesis. This process occurs on modified intracellular membranes, forming a membranous web. Their biogenesis is induced by the viral nonstructural proteins (NS) 4B and NS5A and requires the activity of cellular lipid kinases belonging to the phosphatidylinositol-4-kinase III family. A hallmark of HCV-induced membranes is thus the presence of phosphatidylinositol-4-phosphate (PI4P), which is synthesized by these kinases. Intriguingly, certain recently identified HCV dependency factors selectively bind to PI derivatives, suggesting a crucial role for PIPs in viral RNA replication and assembly. The latter occurs on the surface of lipid droplets and is tightly connected to the very low density lipoprotein pathway leading to the formation of unique lipoviro particles. Thus, HCV exploits lipid metabolism in many ways and may therefore serve as a model system to gain insights into membrane biogenesis, lipid droplet formation and lipid trafficking.

  3. The Complement System in Flavivirus Infections

    Science.gov (United States)

    Conde, Jonas N.; Silva, Emiliana M.; Barbosa, Angela S.; Mohana-Borges, Ronaldo

    2017-01-01

    The incidence of flavivirus infections has increased dramatically in recent decades in tropical and sub-tropical climates worldwide, affecting hundreds of millions of people each year. The Flaviviridae family includes dengue, West Nile, Zika, Japanese encephalitis, and yellow fever viruses that are typically transmitted by mosquitoes or ticks, and cause a wide range of symptoms, such as fever, shock, meningitis, paralysis, birth defects, and death. The flavivirus genome is composed of a single positive-sense RNA molecule encoding a single viral polyprotein. This polyprotein is further processed by viral and host proteases into three structural proteins (C, prM/M, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) that are involved in viral replication and pathogenicity. The complement system has been described to play an important role in flavivirus infection either by protecting the host and/or by influencing disease pathogenesis. In this mini-review, we will explore the role of complement system inhibition and/or activation against infection by the Flavivirus genus, with an emphasis on dengue and West Nile viruses. PMID:28261172

  4. Oscillating supertubes and neutral rotating black hole microstates

    CERN Document Server

    Mathur, Samir D

    2014-01-01

    The construction of neutral black hole microstates is an important problem, with implications for the information paradox. In this paper we conjecture a construction of non-supersymmetric supergravity solutions describing D-brane configurations which carry mass and angular momentum, but no other conserved charges. We first study a classical string solution which locally carries dipole winding and momentum charges in two compact directions, but globally carries no net winding or momentum charge. We investigate its backreaction in the D1-D5 duality frame, where this object becomes a supertube which locally carries oscillating dipole D1-D5 and NS1-NS5 charges, and again carries no net charge. In the limit of an infinite straight supertube, we find an exact supergravity solution describing this object. We conjecture that a similar construction may be carried out based on a class of two-charge non-supersymmetric D1-D5 solutions. These results are a step towards demonstrating how neutral black hole microstates may ...

  5. Oscillating supertubes and neutral rotating black hole microstates

    Energy Technology Data Exchange (ETDEWEB)

    Mathur, Samir D.; Turton, David [Department of Physics, The Ohio State University,191 W Woodruff Ave, Columbus, OH 43210 (United States)

    2014-04-10

    The construction of neutral black hole microstates is an important problem, with implications for the information paradox. In this paper we conjecture a construction of non-supersymmetric supergravity solutions describing D-brane configurations which carry mass and angular momentum, but no other conserved charges. We first study a classical string solution which locally carries dipole winding and momentum charges in two compact directions, but globally carries no net winding or momentum charge. We investigate its backreaction in the D1-D5 duality frame, where this object becomes a supertube which locally carries oscillating dipole D1-D5 and NS1-NS5 charges, and again carries no net charge. In the limit of an infinite straight supertube, we find an exact supergravity solution describing this object. We conjecture that a similar construction may be carried out based on a class of two-charge non-supersymmetric D1-D5 solutions. These results are a step towards demonstrating how neutral black hole microstates may be constructed in string theory.

  6. An outbreak of imported dengue fever from Myanmar to the border of China, with its viral molecular epidemiological features%云南中缅边境一起输入性登革热暴发的分子流行病学研究

    Institute of Scientific and Technical Information of China (English)

    张海林; 付士红; 邓掌; 袁军; 姜红月; 李铭华; 高晓艳; 王静林; 刘永华

    2013-01-01

    Objective To understand the epidemiologic characteristics of dengue fever,imported from Myanmar to the border of Yunnan province,China.Viral molecular epidemiologic features were also studied.Methods Questionnaires were used on each diagnosed,suspected dengue fever,case or unknown cases with fever when coming from Myanmar entering the port and hospitals in Ruili city of Yunnan province.Serum samples of these patients were collected to detect IgM antibody against dengue virus and RT-PCR assay.Homology and phylogenetic tree based on the whole nucleotide sequence of PrM-C and NS5 gene of dengue virus were further analyzed.Results A total of 103 sera were collected from patients at acute stage in Ruili city in July to November 2008.Among them,49 cases were confirmed for dengue fever according to IgM and nucleic acid testings.Except one,other 48 cases were all imported into Ruili,from Myanmar.Of those,18 patients were residents from Mujie city of Myanmar and hospitalized in Ruili and the rest 30 patients were Chinese citizens who had finished business and returned from Myanmar.Two isolates of serum samples from the imported cases were identified and both homology and phylogenetic analysis were performed,using the nucleotide sequences of PrM and NS5 genes.They were divided into dengue type 1 (RLB61) and dengue type 3 (RLC31) and were closer to the dengue virus strains isolated from Southeast Asia countries.Conclusion It is confirmed that an epidemic of dengue fever which was imported from Myanmar to Ruili city of Yunnan province,China.Evidence also showed that both type Ⅰ and Ⅲ epidemic strains of dengue virus did exist in Mujie city of Myanmar in 2008.%目的 调查云南中缅边境一起输入性登革热流行状况及其流行病毒株的分子流行病学特点.方法 采集医院就诊和口岸入境人员中登革热、疑似登革热和不明原因发热患者血清标本并进行流行病学调查,采用ELISA检测登革病毒IgM抗体,RT-PCR检测登革

  7. Stability, Tunneling and Flux Changing de Sitter Transitions in the Large Volume String Scenario

    CERN Document Server

    de Alwis, S; Hatefi, E; Quevedo, F

    2013-01-01

    We study the non-perturbative stability of the Large Volume Scenario (LVS) of IIB string compactifications, by analysing transitions mediated by the Brown-Teitelboim (BT) brane nucleations and by Coleman De Luccia tunneling (CDL). We find that, as long as the effective field theory description holds, the LVS AdS minima are stable despite being non-supersymmetric. This opens the possibility of having a CFT dual. Metastable de Sitter vacua behave differently depending on the uplifting mechanism. We find explicit expressions for the different decay rates in terms of exponentials of the volume. Among the transitions of dS to dS those with increasing volume and decreasing vacuum energy are preferred, though dS decays to AdS (big-crunch sinks) have higher probability. Transitions via the CDL mechanism to decompactification are exponentially suppressed compared to these. The BT decays correspond to flux/D3 brane transitions mediated by the nucleation of D5/NS5 branes. We compare our results with previous analysis fo...

  8. Indian National Association for Study of the Liver (INASL) Guidance for Antiviral Therapy Against HCV Infection: Update 2016.

    Science.gov (United States)

    Puri, Pankaj; Saraswat, Vivek A; Dhiman, Radha K; Anand, Anil C; Acharya, Subrat K; Singh, Shivaram P; Chawla, Yogesh K; Amarapurkar, Deepak N; Kumar, Ajay; Arora, Anil; Dixit, Vinod K; Koshy, Abraham; Sood, Ajit; Duseja, Ajay; Kapoor, Dharmesh; Madan, Kaushal; Srivastava, Anshu; Kumar, Ashish; Wadhawan, Manav; Goel, Amit; Verma, Abhai; Shalimar; Pandey, Gaurav; Malik, Rohan; Agrawal, Swastik

    2016-06-01

    India contributes significantly to the global burden of HCV. While the nucleoside NS5B inhibitor sofosbuvir became available in the Indian market in March 2015, the other directly acting agents (DAAs), Ledipasvir and Daclatasvir, have only recently become available in the India. The introduction of these DAA in India at a relatively affordable price has led to great optimism about prospects of cure for these patients as not only will they provide higher efficacy, but combination DAAs as all-oral regimen will result in lower side effects than were seen with pegylated interferon alfa and ribavirin therapy. Availability of these newer DAAs has necessitated revision of INASL guidelines for the treatment of HCV published in 2015. Current considerations for the treatment of HCV in India include the poorer response of genotype 3, nonavailability of many of the DAAs recommended by other guidelines and the cost of therapy. The availability of combination DAA therapy has simplified therapy of HCV with decreased reliance of evaluation for monitoring viral kinetics or drug related side effects.

  9. Exact Thresholds and Instanton Effects in String Theory

    CERN Document Server

    Obers, N A

    2001-01-01

    In this lecture we summarize some recent work on the understanding of instanton effects in string theories with 16 supersymmetries. In particular, we consider F^4 couplings using the duality between the heterotic string on T^4 and type IIA on K_3 at an orbifold point, as well as higher and lower dimensional versions of this string-string duality. At the perturbative level a non-trivial test of the duality, requiring several miraculous identities, is presented by matching a purely one-loop heterotic amplitude to a purely tree-level type II result. A wide variety of non-perturbative effects is shown to occur in this setting, including D-brane instantons for type IIA on K_3 x S^1 and NS5-brane instantons for type IIA on K_3 x T^2. Moreover, the analysis of the three-dimensional case, which possesses a non-perturbative SO(8,24,Z) U-duality, reveals the presence of Kaluza-Klein 5-brane instanton effects, both on the heterotic and the type II side.

  10. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Brun Jensen, Sanne;

    2012-01-01

    viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome......Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture...... patient isolates representing HCV genotypes 1-7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8...

  11. Efficacy and safety of 8 weeks versus 12 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin in patients with hepatitis C virus genotype 1 mono-infection and HIV/hepatitis C virus co-infection (C-WORTHY)

    DEFF Research Database (Denmark)

    Sulkowski, Mark; Hezode, Christophe; Gerstoft, Jan;

    2015-01-01

    BACKGROUND: Both hepatitis C virus (HCV) mono-infected and HIV/HCV co-infected patients are in need of safe, effective, all-oral HCV regimens. In a phase 2 study we aimed to assess the efficacy and safety of grazoprevir (MK-5172; HCV NS3/4A protease inhibitor) and two doses of elbasvir (MK-8742......; HCV NS5A inhibitor) in patients with HCV mono-infection and HIV/HCV co-infection. METHODS: The C-WORTHY study is a phase 2, multicentre, randomised controlled trial of grazoprevir plus elbasvir with or without ribavirin in patients with HCV; here, we report findings for previously untreated (genotype...... carcinoma, or decompensated liver disease. In part A of the study we randomly assigned HCV-mono-infected patients to receive 12 weeks of grazoprevir (100 mg) plus elbasvir (20 mg or 50 mg) with or without ribavirin (arms A1-3); in part B we assigned HCV-mono-infected patients to 8 or 12 weeks of grazoprevir...

  12. Effects of the CCR5-Delta32 mutation on hepatitis C virus-specific immune responses in patients with haemophilia.

    Science.gov (United States)

    Ahlenstiel, Golo; Woitas, Rainer P; Iwan, Agathe; Nattermann, Jacob; Feldmann, Georg; Rockstroh, Jürgen K; Oldenburg, Johannes; Kupfer, Bernd; Sauerbruch, Tilman; Spengler, Ulrich

    2009-01-01

    In hepatitis C virus (HCV) infection antiviral T cells express the CC chemokine receptor 5 (CCR5). Their recruitment to the liver is an important step in the immune response. A 32 base pair deletion in the CCR5 gene leads to reduced expression and total loss of CCR5 in CCR5-Delta32 heterozygous and homozygous subjects, respectively. However, the role of this mutation for antiviral immunity remains unclear. Here, we analysed proliferation, IFN-gamma and IL-4 secretion (ELISpot) induced by the HCV antigens core, NS3, NS4, and NS5a in 21 anti-HCV-positive haemophiliac patients in relationship to their CCR5 genotypes (CCR5 wildtype n = 10, CCR5-Delta32 heterozygous n = 5 and CCR5-Delta32 homozygous n = 6). Furthermore, T cell migration in response to the CCR5 ligands CCL3, -4 and -5 was studied. Overall IFN-gamma responses to HCV proteins were only slightly greater in CCR5 wild-type patients than in CCR5-Delta32 carriers (0.6 versus 0.24 SFC/10(4) PBMC; p = 0.043). This difference was consistently seen with all tested HCV antigens. In contrast, neither T cell migration, nor PBMC proliferation, nor IL-4 production differed between CCR5 genotypes. Interruption of the CCR5 signalling pathway due to CCR5-Delta32 may potentially result in subtle reduction of HCV specific IFN-gamma responses in anti-HCV-positive haemophiliac patients.

  13. HIV AND HCV COINFECTION: PREVALENCE, ASSOCIATED FACTORS AND GENOTYPE CHARACTERIZATION IN THE MIDWEST REGION OF BRAZIL

    Directory of Open Access Journals (Sweden)

    Solange Zacalusni Freitas

    2014-12-01

    Full Text Available A cross-sectional study on prevalence, associated factors and genotype distribution of HCV infection was conducted among 848 HIV-infected patients recruited at reference centers in the Midwest Region of Brazil. The prevalence rate of HIV-HCV coinfection was 6.9% (95% CI: 5.2 to 8.6. In multivariable analysis, increasing age, use of illicit drugs (injection and non-injection, a history of blood transfusion before 1994, and the absence of a steady partnership were significant independent associated factors for HIV-HCV coinfection. The phylogenetic analysis based on the NS5B region revealed the presence of two major circulating genotypes of HCV: genotypes 1 (58.3% and 3 (41.7%. The prevalence of HIV-HCV coinfection was lower than those reported in studies conducted with HIV-infected patients in different regions of Brazil, due to the fact that illicit drug use is not a frequent mode of HIV transmission in this region of Brazil. Serologic screening of HIV-patients for HCV before initiating antiretroviral treatment, a comprehensive identification of associated factors, and the implementation of effective harm reduction programs are highly recommended to provide useful information for treatment and to prevent HCV coinfection in these patients.

  14. 干扰素刺激基因ISG20真核表达载体的构建及其抗丙型肝炎病毒复制作用的研究%Expression of interferon-stimulated gene ISG20 and investigation of its antiviral effect on HCV replicon

    Institute of Scientific and Technical Information of China (English)

    贾因棠; 魏来; 蒋栋; 丛旭; 费然

    2006-01-01

    目的:研究IFN-α抗HCV的作用机理及了解ISG20是否参与介导IFN-α对HCV的抑制作用.方法:用RT-PCR法及融合PCR法分别获得野生型及突变型ISG20 cDNA,并将其克隆到真核表达载体pcDNA3.1上,转染含HCV复制子的Huh7细胞进行瞬时表达,通过Northern blot及Western blot分别检测HCVRNA及NS5A蛋白水平,研究表达ISG20对HCV复制子的影响.结果:构建的野生型及突变型ISG20真核表达载体在mRNA水平及蛋白水平的表达均得到证实,并且发现表达野生型ISG20对HCV复制子RNA有抑制作用.结论:成功克隆及表达了ISG20,并对其抗病毒作用进行了初步研究,提示ISG20可能介导IFN-α对HCV的抑制作用.

  15. Black branes on the linear dilaton background

    CERN Document Server

    Clément, G; Clement, Gerard; Gal'tsov, Dmitri

    2004-01-01

    We show that the complete static black p-brane supergravity solution with a single charge contains two and only two branches with respect to behavior at infinity in the transverse space. One branch is the standard family of asymptotically flat black branes, and another is the family of black branes which asymptotically approach the linear dilaton background with antisymmetric form flux (LDB). Such configurations were previously obtained in the near-horizon near-extreme limit of the dilatonic asymptotically flat $p$-branes, and used to describe the thermal phase of field theories involved in the DW/QFT dualities and the thermodynamics of little string theory in the case of the NS5-brane. Here we show by direct integration of the Einstein equations that the asymptotically LDB $p$-branes are indeed exact supergravity solutions, and we prove a new uniqueness theorem for static $p$-brane solutions satisfying cosmic censorship. In the non-dilatonic case, our general non-asymptotically flat p-branes are uncharged bl...

  16. The Future of HCV Therapy: NS4B as an Antiviral Target

    Directory of Open Access Journals (Sweden)

    Hadas Dvory-Sobol

    2010-11-01

    Full Text Available Chronic hepatitis C virus (HCV infection is a major worldwide cause of liver disease, including cirrhosis and hepatocellular carcinoma. It is estimated that more than 170 million individuals are infected with HCV, with three to four million new cases each year. The current standard of care, combination treatment with interferon and ribavirin, eradicates the virus in only about 50% of chronically infected patients. Notably, neither of these drugs directly target HCV. Many new antiviral therapies that specifically target hepatitis C (e.g. NS3 protease or NS5B polymerase inhibitors are therefore in development, with a significant number having advanced into clinical trials. The nonstructural 4B (NS4B protein, is among the least characterized of the HCV structural and nonstructural proteins and has been subjected to few pharmacological studies. NS4B is an integral membrane protein with at least four predicted transmembrane (TM domains. A variety of functions have been postulated for NS4B, such as the ability to induce the membranous web replication platform, RNA binding and NTPase activity. This review summarizes potential targets within the nonstructural protein NS4B, with a focus on novel classes of NS4B inhibitors.

  17. 5-Aminosalicylic acid protection against oxidative damage to synaptosomal membranes by alkoxyl radicals in vitro.

    Science.gov (United States)

    Kanski, J; Lauderback, C; Butterfield, D A

    2001-01-01

    The antioxidant properties of 5-aminosalicylic acid in vitro were evaluated in a synaptosomal membrane system prepared from gerbil cortical synaptosomes using EPR spin labeling and spectroscopic techniques. MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl) and 5-NS (5-nitroxide stearate) spin labels were used to assess changes in protein oxidation and membrane lipid fluidity, respectively. Synaptosomal membranes were subjected to oxidative stress by incubation with 1 mM azo-bis(isobutyronitrile) (AIBN) or 1 mM 2,2'-azobis(amidino propane) dihydrochloride (AAPH) at 37 degrees C for 30 minutes. The EPR analyses of the samples showed significant oxidation of synaptosomal proteins and a decrease in membrane fluidity. 5-Aminosalicylic acid also was evaluated by means of FRAP (the ferric reducing ability of plasma) test as a potential antioxidant. 5-Aminosalicylic acid also showed protection against the oxidation in gerbil cortical synaptosomes system caused by AIBN and AAPH. These results are consistent with the notion of antioxidant protection against free radical induced oxidative stress in synaptosomal membrane system by this agent.

  18. CD8+ T-cell Responses in Flavivirus-Naive Individuals Following Immunization with a Live-Attenuated Tetravalent Dengue Vaccine Candidate.

    Science.gov (United States)

    Chu, Haiyan; George, Sarah L; Stinchcomb, Dan T; Osorio, Jorge E; Partidos, Charalambos D

    2015-11-15

    We are developing a live-attenuated tetravalent dengue vaccine (TDV) candidate based on an attenuated dengue 2 virus (TDV-2) and 3 chimeric viruses containing the premembrane and envelope genes of dengue viruses (DENVs) -1, -3, and -4 expressed in the context of the attenuated TDV-2 genome (TDV-1, TDV-3, and TDV-4, respectively). In this study, we analyzed and characterized the CD8(+) T-cell response in flavivirus-naive human volunteers vaccinated with 2 doses of TDV 90 days apart via the subcutaneous or intradermal routes. Using peptide arrays and intracellular cytokine staining, we demonstrated that TDV elicits CD8(+) T cells targeting the nonstructural NS1, NS3, and NS5 proteins of TDV-2. The cells were characterized by the production of interferon-γ, tumor necrosis factor-α, and to a lesser extent interleukin-2. Responses were highest on day 90 after the first dose and were still detectable on 180 days after the second dose. In addition, CD8(+) T cells were multifunctional, producing ≥2 cytokines simultaneously, and cross-reactive to NS proteins of the other 3 DENV serotypes. Overall, these findings describe the capacity of our candidate dengue vaccine to elicit cellular immune responses and support the further evaluation of T-cell responses in samples from future TDV clinical trials.

  19. Identification of a dengue virus-specific HLA-A*0201-restricted CD8+ T cell epitope.

    Science.gov (United States)

    Wen, Jinsheng; Duan, Zhiliang; Jiang, Lifang

    2010-04-01

    In this study, a combination of epitope-prediction programs and in vitro assays was used to identify dengue virus (DENV)-specific CD8(+) T cell epitopes. Peripheral blood mononuclear cells (PBMCs) isolated from patients who recovered from dengue fever were stimulated with candidate epitope peptides derived from DENV, which were predicted by using SYFPEITHI and RANKpep epitope-prediction programs. The IFN-gamma ELISpot results and the results of intracellular staining of IFN-gamma showed that peptides NS4b_40 (TLYAVATTI), E_256 (QEGAMHTAL), NS3_205 (LPAIVREAI), NS5_210 (SRNSTHEMY), and NS3_207 (AIVREAIKR) could induce the recall response of CD8(+) T cells. Furthermore, the results of the MHC-peptide complex stabilization assay revealed that peptide NS4b_40 (TLYAVATTI) has a high affinity for HLA-A*0201 molecules. The IFN-gamma ELISpot results and staining of intracellular IFN-gamma confirmed that this peptide could induce high-level CD8(+) T cell response in HLA-A*0201 positive PBMCs. Peptide NS4b_40 (TLYAVATTI) was identified as a novel DENV-specific HLA-A*0201-restricted CD8(+) T cell epitope.

  20. Rapid emergence of hepatitis C virus protease inhibitor resistance is expected

    Energy Technology Data Exchange (ETDEWEB)

    Rong, Libin [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory

    2009-01-01

    Approximately 170 million people worldwide are infected with hepatitis C virus (HCV). Current therapy, consisting of pegylated interferon (PEG-IFN) and ribavirin (RBV), leads to sustained viral elimination in only about 45% of patients treated. Telaprevir (VX-950), a novel HCV NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients with chronic hepatitis C genotype 1 infection. However, some patients experience viral breakthrough during dosing, with drug resistant variants being 5%-20% of the virus population as early as day 2 after treatment initiation. Why viral variants appear such a short time after the start of dosing is unclear, especially since this has not been seen with monotherapy for either human immunodeficiency virus or hepatitis B virus. Here, using a viral dynamic model, we explain why such rapid emergence of drug resistant variants is expected when potent HCV protease inhibitors are used as monotherapy. Surprisingly, our model also shows that such rapid emergence need not be the case with some potent HCV NS5B polymerase inhibitors. Examining the case of telaprevir therapy in detail, we show the model fits observed dynamics of both wild-type and drug-resistant variants during treatment, and supports combination therapy of direct antiviral drugs with PEG-IFN and/or RBV for hepatitis C.

  1. Discovery of Novel Viruses in Mosquitoes from the Zambezi Valley of Mozambique

    Science.gov (United States)

    Hayer, Juliette; Abilio, Ana Paula; Mulandane, Fernando Chanisso; Verner-Carlsson, Jenny; Falk, Kerstin I.; Fafetine, Jose M.; Berg, Mikael; Blomström, Anne-Lie

    2016-01-01

    Mosquitoes carry a wide variety of viruses that can cause vector-borne infectious diseases and affect both human and veterinary public health. Although Mozambique can be considered a hot spot for emerging infectious diseases due to factors such as a rich vector population and a close vector/human/wildlife interface, the viral flora in mosquitoes have not previously been investigated. In this study, viral metagenomics was employed to analyze the viral communities in Culex and Mansonia mosquitoes in the Zambezia province of Mozambique. Among the 1.7 and 2.6 million sequences produced from the Culex and Mansonia samples, respectively, 3269 and 983 reads were classified as viral sequences. Viruses belonging to the Flaviviridae, Rhabdoviridae and Iflaviridae families were detected, and different unclassified single- and double-stranded RNA viruses were also identified. A near complete genome of a flavivirus, tentatively named Cuacua virus, was obtained from the Mansonia mosquitoes. Phylogenetic analysis of this flavivirus, using the NS5 amino acid sequence, showed that it grouped with ‘insect-specific’ viruses and was most closely related to Nakiwogo virus previously identified in Uganda. Both mosquito genera had viral sequences related to Rhabdoviruses, and these were most closely related to Culex tritaeniorhynchus rhabdovirus (CTRV). The results from this study suggest that several viruses specific for insects belonging to, for example, the Flaviviridae and Rhabdoviridae families, as well as a number of unclassified RNA viruses, are present in mosquitoes in Mozambique. PMID:27682810

  2. Daclatasvir plus peginterferon alfa and ribavirin for treatment-naive chronic hepatitis C genotype 1 or 4 infection

    DEFF Research Database (Denmark)

    Hézode, Christophe; Hirschfield, Gideon M; Ghesquiere, Wayne

    2015-01-01

    undetectable HCV-RNA at Weeks 4 and 12 (extended rapid virologic response, eRVR) and at 24 weeks post-treatment (sustained virologic response, SVR24) among genotype 1-infected patients. RESULTS: Overall, eRVR was achieved by 54.4% (80/147) of genotype 1-infected patients receiving daclatasvir 20 mg, 54.1% (79......OBJECTIVE: To evaluate the safety and efficacy of daclatasvir, an HCV NS5A inhibitor with pangenotypic activity, administered with peginterferon-alfa-2a/ribavirin. DESIGN: In this Phase 2b double-blind, placebo-controlled study, treatment-naive adults with HCV genotype 1 (N=365) or 4 (N=30......) infection were randomly assigned (2:2:1) to daclatasvir 20 mg or 60 mg, or placebo once daily plus weekly peginterferon-alfa-2a and twice-daily ribavirin. Daclatasvir recipients achieving protocol-defined response (PDR; HCV-RNA

  3. Anti-HCV fragment reagent in facilitating the diagnosis of HCV infection%抗-HCV分片段试剂在辅助诊断HCV感染中的应用

    Institute of Scientific and Technical Information of China (English)

    董小平; 陈筱华; 张洪淼; 方淑环; 孔令光

    2005-01-01

    目的探讨抗-HCV分片段试剂在辅助诊断HCV感染中的效果.方法收集常规初复检抗-HCV不符的临界样本31份,用抗-HCV分片段酶联免疫检测试剂进一步检测抗-HCV-C、NS3、NS4、NS5、膜高变区1(HVR1)抗体.结果 31份初复检不符临界样本分片段检测结果为4份阳性(12.90%),10份不确定,即仅1个抗原片段阳性(32.26%),17份阴性(54.84%).结论对抗-HCV初、复检结果不符的临界样本,用抗-HCV分片段酶联免疫检测试剂进一步作辅助检测,可降低假阳性,提高检测的特异性,具有一定的临床意义.

  4. Comparison Analysis on Reagent of Anti-HCV EIA and Anti-HCV Fragment EIA%抗-HCV EIA试剂与HCV分片段EIA试剂比较分析

    Institute of Scientific and Technical Information of China (English)

    陈莺

    2005-01-01

    [目的]探讨抗-HCV分片段试剂对抗-HCV阳性标本判断价值的可靠性.[方法]用抗-HCVEIA、MEIA及HCV分片段EIA法对23例初筛阳性标本进行检测.[结果]23例中,丙肝分片段不同基因区C区、NS3、NS4和NS5结果阳性分别为18(78.3%)、20(87.0%)、13(56.5%)和12(52.2%),判断阳性18例,可疑3例,阴性2例.23例中,16例各厂家试剂均阳性,不符7例.HCV分片段与进口雅培试剂阳性结果完全相同18例.[结论]用HCV分片段试剂对抗-HCV EIA弱阳性标本的结果判断有一定的临床使用价值.

  5. Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    Science.gov (United States)

    Dixit, Updesh; Pandey, Ashutosh K.; Mishra, Priya; Sengupta, Amitabha; Pandey, Virendra N.

    2016-01-01

    Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3′ NTR and domain IIId of the HCV-IRES in the 5′ NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication. PMID:27106056

  6. The IR obstruction to UV completion for Dante’s Inferno model with higher-dimensional gauge theory origin

    Energy Technology Data Exchange (ETDEWEB)

    Furuuchi, Kazuyuki [Manipal Centre for Natural Sciences, Manipal University,Manipal, Karnataka 576104 (India); Koyama, Yoji [National Center for Theoretical Sciences, National Tsing-Hua University,Hsinchu 30013, Taiwan R.O.C. (China)

    2016-06-21

    We continue our investigation of large field inflation models obtained from higher-dimensional gauge theories, initiated in our previous study http://dx.doi.org/10.1088/1475-7516/2015/02/031. We focus on Dante’s Inferno model which was the most preferred model in our previous analysis. We point out the relevance of the IR obstruction to UV completion, which constrains the form of the potential of the massive vector field, under the current observational upper bound on the tensor to scalar ratio. We also show that in simple examples of the potential arising from DBI action of a D5-brane and that of an NS5-brane that the inflation takes place in the field range which is within the convergence radius of the Taylor expansion. This is in contrast to the well known examples of axion monodromy inflation where inflaton takes place outside the convergence radius of the Taylor expansion. This difference arises from the very essence of Dante’s Inferno model that the effective inflaton potential is stretched in the inflaton field direction compared with the potential for the original field.

  7. Prevalência, fatores de risco e genótipos da hepatite C entre usuários de drogas Prevalencia, factores de riesgo y genotipos de la hepatitis C entre usuarios de drogas Prevalence, risk factors and genotypes of hepatitis C virus infection among drug users, Central-Western Brazil

    Directory of Open Access Journals (Sweden)

    Carmen L R Lopes

    2009-08-01

    Full Text Available OBJETIVO: Estimar a prevalência e fatores associados à infecção pelo vírus da hepatite C em usuários de drogas e identificar os genótipos e subtipos virais circulantes. MÉTODOS: Estudo realizado com 691 usuários de drogas de 26 centros de tratamento de uso de drogas filantrópicos, particulares e públicos de Goiânia (GO e Campo Grande (MS, entre 2005 e 2006. Dados sociodemográficos e fatores de risco para infecção pelo HCV foram obtidos por meio de entrevistas. Amostras sangüíneas foram testadas para a detecção de anticorpos para o HCV. As amostras positivas foram submetidas à detecção do RNA-HCV pela reação em cadeia da polimerase com iniciadores complementares às regiões 5' NC e NS5B do genoma viral e genotipadas pelo line probe assay (LiPA e por seqüenciamento direto, seguido de análise filogenética. Prevalência e odds ratio foram calculados com intervalo de 95% de confiança. Os fatores de risco com pOBJETIVO: Estimar la prevalencia y factores asociados a la infección por el virus de la hepatitis C en usuarios de drogas e identificar los genotipos y subtipos virales circulantes. MÉTODOS: Estudio realizado con 691 usuarios de drogas de 26 centros de tratamiento de uso de drogas filantrópicos, particulares y públicos de Goiania y Campo Grande (Centro-Oeste, entre 2005 y 2006. Datos sociodemográficos y factores de riesgo para infección por el HCV fueron obtenidos por medio de entrevistas. Muestras sanguíneas fueron evaluadas para la detección de anticuerpos para el HCV. Las muestras positivas fueron sometidas a la detección de RNA-HCV por la reacción en cadena de polimerasa con iniciadores complementarios a las regiones 5' NC y NS5B del genoma viral y genotipadas por el line probe assay (LiPA y por secuenciación directa, seguido del análisis filogenético. Prevalencia y odds ratio fueron calculados con intervalo de 95% de confianza. Los factores de riesgo con pOBJECTIVE: To estimate prevalence of

  8. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses

    DEFF Research Database (Denmark)

    Gottwein, J.M.; Scheel, Troels Kasper Høyer; Hoegh, A.M.

    2007-01-01

    BACKGROUND & AIMS: Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a......, which has a high prevalence worldwide. METHODS: Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression...... (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies. RESULTS: S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable...

  9. Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin.

    Directory of Open Access Journals (Sweden)

    José Manuel Cuevas

    Full Text Available We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective pressures.

  10. Predominance of hepatitis C virus genotype 4 infection and rapid transmission between 1935 and 1965 in the Central African Republic.

    Science.gov (United States)

    Njouom, Richard; Frost, Eric; Deslandes, Sylvie; Mamadou-Yaya, Fleurie; Labbé, Annie-Claude; Pouillot, Régis; Mbélesso, Pascal; Mbadingai, Sylvestre; Rousset, Dominique; Pépin, Jacques

    2009-10-01

    The molecular epidemiology of hepatitis C virus (HCV) in the Central African Republic (CAR) is poorly documented. Thus, we conducted phylogenetic analyses of NS5B gene sequences from 58 HCV-infected inhabitants of a remote area of south-west CAR, which indicated that 48 (82.8%) were infected with genotype 4 (HCV-4), five (8.6%) with genotype 2 and five (8.6%) with genotype 1. HCV-4 strains were highly heterogeneous, containing previously described subtypes 4k (48%), 4c (27%), 4r (4%), 4f (4%) and unclassified subtypes (17%). To estimate the epidemic history of these HCV-4 strains, an evolutionary analysis using the coalescent approach was used. The estimated date of the most recent common ancestor of the CAR HCV-4 strains was 1539 (95% confidence intervals, 1317-1697). They exhibited a rapid, exponential spread from 1935 to 1965, simultaneously with what was recently reported in neighbouring Cameroon and Gabon. The hypothesis of a massive iatrogenic transmission during interventions for the control of endemic tropical diseases is discussed.

  11. Preclinical evaluation of multi antigenic HCV DNA vaccine for the prevention of Hepatitis C virus infection

    Science.gov (United States)

    Lee, Hyojin; Jeong, Moonsup; Oh, Jooyeon; Cho, Youngran; Shen, Xuefei; Stone, John; Yan, Jian; Rothkopf, Zachary; Khan, Amir S.; Cho, Byung Mun; Park, Young K.; Weiner, David B.; Son, Woo-Chan; Maslow, Joel N.

    2017-01-01

    Direct-acting antiviral treatment for hepatitis C virus (HCV) infection is costly and does not protect from re-infection. For human and chimpanzees, recovery from acute HCV infection correlates with host CD4+ and CD8+ T cell responses. DNA plasmids targeting the HCV non-structural antigens NS3, NS4, and NS5, were previously reported to induce robust and sustained T cell responses in mice and primates. These plasmids were combined with a plasmid encoding cytokine IL-28B, together named as VGX-6150. The dose-dependent T cell response and safety of VGX-6150 administered intramuscularly and followed by electroporation was assessed in mice. Immune responses plateaued at 20 μg/dose with IL-28B demonstrating significant immunoadjuvant activity. Mice administered VGX-6150 at 40, 400, and 800 μg given either as a single injection or as 14 injections given bi-weekly over 26 weeks showed no vaccine related changes in any clinical parameter compared to placebo recipients. There was no evidence of VGX-6150 accumulation at the injection site or in any organ 1 month following the 14th vaccination. Based on these studies, the approximate lethal dose (ALD) exceeds 800 μg/dose and the NOAEL was 800 μg/dose in mouse. In conclusion, VGX-6150 appears safe and a promising preventive vaccine candidate for HCV infection. PMID:28266565

  12. Rotating Shaft Tilt Angle Measurement Using an Inclinometer

    Directory of Open Access Journals (Sweden)

    Luo Jun

    2015-10-01

    Full Text Available This paper describes a novel measurement method to accurately measure the rotating shaft tilt angle of rotating machine for alignment or compensation using a dual-axis inclinometer. A model of the rotating shaft tilt angle measurement is established using a dual-axis inclinometer based on the designed mechanical structure, and the calculation equation between the rotating shaft tilt angle and the inclinometer axes outputs is derived under the condition that the inclinometer axes are perpendicular to the rotating shaft. The reversal measurement method is applied to decrease the effect of inclinometer drifts caused by temperature, to eliminate inclinometer and rotating shaft mechanical error and inclinometer systematic error to attain high measurement accuracy. The uncertainty estimation shows that the accuracy of rotating shaft tilt angle measurement depends mainly on the inclinometer uncertainty and its uncertainty is almost the same as the inclinometer uncertainty in the simulation. The experimental results indicate that measurement time is 4 seconds; the range of rotating shaft tilt angle is 0.002° and its standard deviation is 0.0006° using NS-5/P2 inclinometer, whose precision and resolution are ±0.01° and 0.0005°, respectively.

  13. Rotating Shaft Tilt Angle Measurement Using an Inclinometer

    Science.gov (United States)

    Luo, Jun; Wang, Zhiqian; Shen, Chengwu; Wen, Zhuoman; Liu, Shaojin; Cai, Sheng; Li, Jianrong

    2015-10-01

    This paper describes a novel measurement method to accurately measure the rotating shaft tilt angle of rotating machine for alignment or compensation using a dual-axis inclinometer. A model of the rotating shaft tilt angle measurement is established using a dual-axis inclinometer based on the designed mechanical structure, and the calculation equation between the rotating shaft tilt angle and the inclinometer axes outputs is derived under the condition that the inclinometer axes are perpendicular to the rotating shaft. The reversal measurement method is applied to decrease the effect of inclinometer drifts caused by temperature, to eliminate inclinometer and rotating shaft mechanical error and inclinometer systematic error to attain high measurement accuracy. The uncertainty estimation shows that the accuracy of rotating shaft tilt angle measurement depends mainly on the inclinometer uncertainty and its uncertainty is almost the same as the inclinometer uncertainty in the simulation. The experimental results indicate that measurement time is 4 seconds; the range of rotating shaft tilt angle is 0.002° and its standard deviation is 0.0006° using NS-5/P2 inclinometer, whose precision and resolution are ±0.01° and 0.0005°, respectively.

  14. PROTECTIVE EFFECTS OF PROPOLIS ON GAMMA- IRRADIATED NIGELLA SATIVA EXTRACT INDUCED BLOOD AND IMMUNE CHANGES IN WISTAR RATS

    Directory of Open Access Journals (Sweden)

    Osama Moseilhy Saleh

    2013-01-01

    Full Text Available The present study conducted to test the effect of Nigella Sativa (NS, 5 mg kg-1 of body weight, or γ-irradiated Nigella Sativa (GRNS on the changes of blood component profiles, liver, kidney functions and immune cytokines secretion in male Wistar rats. Moreover, the possible protection by propolis (200 mg kg-1 B. W. on the changes induced by NS and GRNS was examined. Results revealed that both NS and GRNS administration for two weeks induced changes in blood, GPT, GOT and urea levels and co-administration with propolis significantly ameliorated such changes. Also, liver histology showed numerous vacuolar degeneration and fatty changes in γ-irradiated groups which disappeared in presence of propolis. Kidney histology of NS administered rats showed less lymphocytic infiltration, while GRNS groups showed desquamation in the cytoplasm of the renal tubules, hemorrhage in the renal corpuscle and lymphocytic infiltration which disappeared when propolis given together with GRNS. Finally propolis induced protective effect on the changes induced in TNF-α and IL-10 secretion by either NS or GRNS in Wistar rats. In conclusion, the findings of present study clarified the protective effect of propolis on changes induced by γ-irradiated NS on blood, liver, kidney and cytokines changes in Wistar rats.

  15. Universality of the topological string at large radius and NS-brane resurgence

    CERN Document Server

    Couso-Santamaria, Ricardo

    2015-01-01

    We show that there is a natural universal limit of the topological string free energies at the large radius point. The new free energies keep a nonholomorphic dependence on the complex structure moduli space and their functional form is the same for all Calabi-Yau geometries, compact and noncompact alike. The asymptotic nature of the free energy expansion changes in this limit due to a milder factorial growth of its coefficients, and this implies a transseries extension with instanton effects in $\\exp(- 1/g_s^2)$, of NS-brane type, rather than $\\exp(-1/g_s)$, of D-brane type. We show a relation between the instanton action of NS-brane type and the volume of the Calabi-Yau manifold which points to a possible interpretation in terms of NS5-branes. A similar rescaling limit has been considered recently leading to an Airy equation for the partition function which is here used to explain the resurgent properties of the rescaled transseries.

  16. Effects of Silymarin on Hepatitis C Virus and Heme Oxygenase-1 Gene Expression in Human Hepatoma Cells

    Science.gov (United States)

    Bonifaz, Vania; Shan, Ying; Lambrecht, Richard W.; Donohue, Susan E.; Moschenross, Darcy; Bonkovsky, Herbert L.

    2008-01-01

    Background/Aims Hepatitis C virus (HCV) infection is a global medical problem. The current standard treatment of chronic hepatitis C (CHC), pegylated interferon plus ribavirin, is prolonged, expensive, has serious side effects and, at best, is only 50% effective. Silymarin is a natural antioxidant often used by patients with CHC, although its efficacy for decreasing HCV levels or ameliorating CHC remains uncertain. HCV infection is associated with increased hepatic oxidative stress, and one of the antioxidant enzymes which protect cells against this stress is heme oxygenase-1 (HO-1). Methods We investigated effects of silymarin on HCV and HO-1 gene expression in Huh-7 cells, CNS3, and 9-13 cells (the latter two stably expressing HCV-proteins). Results Silymarin significantly down-regulated HCV core mRNA (by 20% - 36%) and protein (by 30%-60%) in CNS3 cells. In contrast, silymarin did not decrease HCV NS5A mRNA or protein expression in 9-13 cells. HO-1 mRNA was up-regulated (60%-400%) by silymarin in Huh-7, CNS3 and 9-13 cells, whereas Bach1 and Nrf2 mRNA levels were not affected. The effect of silymarin to down-regulate HCV core was not related to changes in the Jak-Stat signaling pathway. Conclusions Silymarin may be of benefit in CHC, although prospective, randomized, controlled trials are needed to be certain. PMID:18694403

  17. Large-density field theory, viscosity, and "$2k_F$" singularities from string duals

    CERN Document Server

    Polchinski, Joseph

    2012-01-01

    We analyze systems where an effective large-N expansion arises naturally in gauge theories without a large number of colors: a sufficiently large charge density alone can produce a perturbative string ('tHooft) expansion. One example is simply the well-known NS5/F1 system dual to $AdS_3\\times T^4\\times S^3$, here viewed as a 5+1 dimensional theory at finite density. This model is completely stable, and we find that the existing string-theoretic solution of this model yields two interesting results. First, it indicates that the shear viscosity is not corrected by $\\alpha'$ effects in this system. For flow perpendicular to the F1 strings the viscosity to entropy ratio take the usual value $1/4\\pi$, but for flow parallel to the F1's it vanishes as $T^2$ at low temperature. Secondly, it encodes singularities in correlation functions coming from low-frequency modes at a finite value of the momentum along the $T^4$ directions. This may provide a strong coupling analogue of finite density condensed matter systems fo...

  18. Ribavirin: Past, present and future

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    Before the advent of direct acting antiviral agents (DAAs)ribavirin, associated to pegylated-interferon playeda crucial role in the treatment of chronic hepatitis C,preventing relapses and breakthroughs. In the presentera of new potent DAAs, a place is still devoted tothe drug. Ribavirin associated with sofosbuvir alone isefficient in the treatment of most cases of G2 infectedpatients. All options currently available for the lastdifficult-to-treat cirrhotic G3 patients contain ribavirin.Reducing treatment duration to 12 wk in G1 or G4cirrhotic compensated patients is feasible thanks toribavirin. Retreating patients with acquired anti NS5Aresistance-associated variants using ribavirin-basedstrategies could be useful. The addition of ribavirin withDAAs combinations however, leads to more frequentbut mild adverse events especially in cirrhotic patients.Preliminary data with interferon-free second generationDAAs combinations without ribavirin suggest thatfuture of the drug is jeopardized even in difficult-totreatpatients The optimization of ribavirin dosageaccording to an early monitoring of blood levels hasbeen suggested to be relevant in double therapy withpeginterferon or sofosbuvir but not with very potentcombinations of more than two DAAs.

  19. 新型抗丙型肝炎病毒药索非布韦的研究进展%Advances in research of sofosbuvir-the new drug against hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    屈慧新; 王玉泽; 王彩霞; 史慧静; 王玲玲

    2016-01-01

    目的 评价新型抗丙型肝炎病毒(hepatitis C virus.HCV)药索非布韦的药理作用、安全药理学、药代动力学、药物相互作用、临床研究、不良反应、耐药性.方法 查阅相关文献26篇,对索非布韦基础与临床的研究进展进行了归纳与总结.结果与结论 索非布韦(sofosbuvir)是经美国食品药品管理局(FDA)批准的首个NS5B HCV聚合酶抑制剂,对所有HCV基因型均有效,本药安全性及耐受性良好,与其它药物间相互作用少,具有优良的药代动力学特性和强大的抗病毒能力,其强大的治疗效果给丙型肝炎患者带来了新希望.

  20. Overall efficacy and safety results of sofosbuvir-based therapies in phase II and III studies.

    Science.gov (United States)

    Mangia, Alessandra; Piazzolla, Valeria

    2014-12-15

    The uridine nucleotide analogue sofosbuvir is a selective hepatitis C virus NS5B polymerase inhibitor, active regardless of genotype. We analyzed data on efficacy and safety of sofosbuvir, either in combination with pegylated interferon alfa-2a and ribavirin, or in combination with ribavirin alone as part of an interferon free regimen in more than 1300 patients. Treatment with sofosbuvir for 12 weeks in combination with P/R, in naïve genotype 1 patients was mainly studied in Neutrino. The efficacy of sofosbuvir as part of an all-oral combination including ribavirin alone, was explored in 555 naïve, ineligible and previous treatment failure genotype 2/3 patients. Rates of Sustained Viral Response in genotype 1 and 2 were higher than 85%. For genotype 3 and 4, a European study, Valence, and a US study on patients of Egyptian origin showed that naïve patients are cured at high rates by the all-oral combination given for 24 weeks. The efficacy of sofosbuvir plus P/R for 12 weeks in previous treatment failure genotype 3 has also been demonstrated. Sofosbuvir-based combinations are safe and well tolerated without side effects directly related to the drug. A large body of evidence suggests that sofosbuvir marks a revolution in HCV treatment.

  1. 全球首个抗HCV全口服新药索非布韦%The World's First Oral Anti-hepatitis C Virus Drug Sofosbuvir

    Institute of Scientific and Technical Information of China (English)

    刘正和

    2014-01-01

    丙型肝炎病毒感染极大地威胁着公众健康,目前的标准治疗方案是聚乙二醇修饰的干扰素-α(PegIFN)与广谱抗病毒药物利巴韦林(RBV)联合,然而这种疗法的持续病毒学应答相对较低,新的研究如直接作用的抗病毒药(DAAs)提供了一种新的治疗策略,以提高疗效和耐受性.索非布韦是首个全口服抗丙型肝炎病毒药物,通过抑制NS5B聚合酶来有效抑制HCV的复制,从而达到治疗效果.本文主要就其化学合成、作用机理、药代动力学特征及临床疗效和安全性做一综述.

  2. Sofosbuvir: a review of its use in patients with chronic hepatitis C.

    Science.gov (United States)

    Keating, Gillian M

    2014-07-01

    Sofosbuvir (Sovaldi(®)) is a nucleotide hepatitis C virus (HCV) NS5B polymerase inhibitor that has pangenotypic antiviral activity and a high genetic barrier to resistance. This article reviews the clinical efficacy and tolerability of sofosbuvir in patients with chronic hepatitis C and summarizes its pharmacological properties. Interferon-free treatment with sofosbuvir plus ribavirin achieved high sustained virological response (SVR) rates in treatment-naïve and treatment-experienced patients with HCV genotype 2 or 3 infection, and also had efficacy in patients with HCV genotype 1 infection. Sofosbuvir plus ribavirin was also effective in patients co-infected with HCV and HIV, and sofosbuvir plus ribavirin administered prior to liver transplantation prevented recurrent HCV infection in the majority of patients who had HCV RNA levels below the limit of quantification at the time of transplantation. Sofosbuvir plus peginterferon-α-2a and ribavirin achieved high SVR rates in patients with HCV genotype 1 infection, and also appeared effective in patients with HCV genotype 4, 5 or 6 infection. Oral sofosbuvir was generally well tolerated in patients with chronic hepatitis C. The most commonly reported adverse events and laboratory abnormalities were consistent with those expected with ribavirin and peginterferon-α. In conclusion, sofosbuvir represents an important advance in the treatment of chronic hepatitis C.

  3. A new anti-hepatitis C virus drug: sofosbuvir%口服抗丙型肝炎病毒新药索福布韦

    Institute of Scientific and Technical Information of China (English)

    曾婧娉; 张弋

    2014-01-01

    面对全球约1.7亿慢性丙肝患者,现今的标准治疗方案,即聚乙二醇干扰素(Peg-IFN)联合利巴韦林(RBV),其总治愈率不足50%.因此,近年来旨在提高治愈率的直接抗病毒药物(DAAs)不断问世,其中,丙型肝炎病毒(HCV)聚合酶抑制剂索福布韦作用于病毒RNA复制的NS5B聚合酶位点,中止HCV复制.相较于标准治疗方案,基于索福布韦的联合治疗方案治愈率更高.本文就索福布韦的药理作用、药动学、临床研究及安全性等作一综述.

  4. 丙型肝炎无干扰素全口服治疗临床Ⅲ期试验进展%Progress of interferon-free oral drugs in phase Ⅲ clinical trials for hepatitis C treatment

    Institute of Scientific and Technical Information of China (English)

    唐紫薇; 任浩; 戚中田

    2015-01-01

    Hepatitis C virus is a single stranded RNA virus categorized to the genus Flavirus.HCV infection is transmitted via blood and characterized by high chronic rate.Hepatitis C poses a major health threat worldwide with approximately 160 million chronically infected individuals.The standard therapy for HCV infection in the past decade is using pegylated IFN-α plus ribavirin.However,IFN-α treatment showed low response rate,high averse effect,and would be eventually replaced by emerging new drugs.The combination of ribavirin therapy may prevent relapse,as well as increase sustained response rate,while its usage remains to be further investigated.In 2011,FDA approved the NS3 protease and NS5B inhibitor telaprevir and boceprevir as the first generation of direct-acting antiviral agents.In 2013,the approval of sofosbuvir in hepatitis C treatment opened a new chapter for all-oral but non-interferon therapy.During the 2014 International Liver Congress,the annual meeting held by the European Association for the Study of the Liver (EASL),several promising therapies against hepatitis C infection concluded from phase Ⅲ trials were reported.The patients included treatment naive and treatment-experienced,with or without cirrhosis,as well as patients infected with type 1 HCV.The sustained virologic response rates reached over 90 %.This review summarizes the recent progresses from phase Ⅲ trials on all-oral,interferon-free anti-HCV virus treatments.

  5. 抗丙型肝炎病毒药:索氟布韦(sofosbuvir)

    Institute of Scientific and Technical Information of China (English)

    陈本川

    2014-01-01

    索氟布韦(sofosbuvir)为美国FDA首次批准上市的抗丙型肝炎病毒新药,是一种口服有效的HCV NS5B聚合酶抑制剂,对HCV基因2和3型引起的慢性丙型肝炎( CHC)可全口服治疗,对基因1和4型CHC需与聚乙二醇干扰素α和利巴韦林联用。该文对索氟布韦适应证、剂量与用法、用药注意事项、非临床药理毒理学、临床药理毒理学、临床研究、不良反应及知识产权状态和国内外研究进展等进行介绍。

  6. Safety, Efficacy, and Tolerability of Sofosbuvir and Ribavirin in Management of Recurrent Hepatitis C Virus Genotype 4 After Living Donor Liver Transplant in Egypt: What Have We Learned so far?

    Directory of Open Access Journals (Sweden)

    Dabbous

    2016-04-01

    Full Text Available Background Recurrence of HCV after living donor liver transplant (LDLT is nearly universal, with almost one third of recipients developing cirrhosis and graft failure within 5 years after LDLT. Different studies have been published on the effect of sofosbuvir after liver transplantation on recurrent HCV with different genotypes. Objectives The aim of this study was to evaluate the efficacy, safety, and tolerability of sofosbuvir and ribavirin in LDLT recipients with recurrent HCV genotype 4. Patients and Methods Thirty-nine Egyptian LDLT recipients were treated for recurrent HCV after LDLT with nucleos(tide analog NS5B polymerase inhibitor, sofosbuvir, and ribavirin without pegylated interferon for 6 months (November 2014 to June 2015 in this intention-to-treat analysis. Results One recipient died 1 week after starting the treatment, but the remaining 38 patients completed 24 weeks of treatment and were then followed for 12 weeks after end of treatment (EOT. The sustained virological response (SVR at week 12 after EOT was achieved in 76% (29/38 of recipients. SVR was significantly higher in treatment-naïve patients and in recipients with a low stage of fibrosis. Only 2 (5% recipients developed severe pancytopenia and acute kidney injury. Conclusions We recommend initiating treatment as soon as possible after liver transplantation with newer combinations, such as ledipasvir/sofosbuvir or sofosbuvir/simeprevir, rather than sofosbuvir with Ribavirin, to achieve higher rates of SVR.

  7. 治疗泛基因型丙型肝炎复方药--Epclusa®

    Institute of Scientific and Technical Information of China (English)

    陈本川

    2016-01-01

    Epclusa®由美国吉利德科学公司( Gilead)研发,为2种抗丙型肝炎病毒( HCV)药:核苷类似物HCV RNA聚合酶抑制药sofosbuvir(索非布韦)和HCV蛋白酶NS5A抑制药velpatasvir(维帕他韦)组成。对所有成人HCV 6种基因型慢性感染,伴或不伴有肝硬化患者都有治疗作用;对中度至重度失代偿性肝硬化患者,联用利巴韦林有治疗作用。获得美国食品药品管理局( FDA)优先审评地位,于2016年6月28日批准上市。该文对Epclusa®的非临床毒理学、临床药理毒理学、临床试验、适应证、剂量与服药方法、用药注意事项与警示、不良反应及知识产权状态与国内外研究进展等进行介绍。

  8. 治疗基因1型慢性丙型肝炎的复方制剂--HARVONITM

    Institute of Scientific and Technical Information of China (English)

    陈本川

    2015-01-01

    HarvoniTM是2种抗丙型肝炎病毒( HCV)药固定剂量的复方制剂,由HCVNS5B 聚合酶抑制药索非布韦(sofosbuvir)400 mg 和HCV蛋白酶NS5A抑制药ledipasvir(暂译名:雷迪帕韦)90 mg组合而成。是美国食品药品管理局( FDA)批准的第一个用于治疗基因1型丙型肝炎的全口服复方制剂,无需联用注射干扰素,可单药使用,也可与其他抗HCV口服药物,如利巴韦林联合使用。该文对HarvoniTM非临床药理毒理学、临床药理毒理学、临床研究、适应证、剂量与用法、用药注意事项、不良反应及知识产权状态和国内外研究进展等进行介绍。

  9. Sofosbuvir: A novel treatment option for chronic hepatitis C infection

    Directory of Open Access Journals (Sweden)

    Harmeet Kaur Bhatia

    2014-01-01

    Full Text Available Hepatitis C currently infects more than 170 million people around the world, leading to significant morbidity and mortality. The current standard of care for HCV infection, including one of the two protease inhibitors, telaprevir or boceprevir, for 12-32 weeks, along with pegylated interferon alfa-2a (PEG-IFN-α and ribavirin for up to 48 weeks, is unsatisfactory in many cases, either because of lack of efficacy or because of treatment-related adverse effects. There is an urgent need of new drugs with improved efficacy as well as a safety profile. Sofosbuvir, a recently approved nucleotide analog, is a highly potent inhibitor of the NS5B polymerase in the Hepatitis C virus (HCV, and has shown high efficacy in combination with several other drugs, with and without PEG-INF, against HCV. It offers many advantages due to its high potency, low side effects, oral administration, and high barrier to resistance. The efficacy and safety were demonstrated in many large and well-designed phase 2 and phase 3 clinical trials like NEUTRINO, PROTON, ELECTRON, ATOMIC, COSMOS, FUSION, FISSION, NUCLEAR, POSITRON, and the like. It is generally well-tolerated. Adverse events that occurred include: Headache, insomnia, fatigue, nausea, dizziness, pruritis, upper respiratory tract infections, rash, back pain, grade 1 anemia, and grade 4 lymphopenia; however, the exact safety profile can only be judged when this drug is actually used on a large scale.

  10. 治疗丙肝药物索非布韦的研究进展%Research progress in sofosbuvir for the treatment of hepatitis C

    Institute of Scientific and Technical Information of China (English)

    叶腾飞; 卞晓岚; 朱利明

    2015-01-01

    索非布韦(sofosbuvir,SOF)是全球首个无需同时使用干扰素即可实现全口服治疗丙肝的药物,为丙型肝炎病毒(HCV)特异性NS5B聚合酶的核苷抑制剂.临床试验表明,SOF和利巴韦林二联全口服方案可用于治疗基因2型、3型HCV的感染,用药12周总体持续病毒学应答率(SVR)为50%~78%.SOF联合聚乙二醇干扰素和利巴韦林可用于基因1型和4型HCV的感染,用药12周的SVR达90%.SOF因其服用简便,治愈率高,不良反应发生率低等特点,使其极有可能成为"重磅炸弹"型药物.

  11. 治疗慢性丙型肝炎新药Sofosbuvir%A New Drug for Chronic Hepatitis: C-Sofosbuvir

    Institute of Scientific and Technical Information of China (English)

    吴叶红; 刘海净; 刘欢

    2014-01-01

    鉴于丙型肝炎病毒(HCV)感染全球高发趋势,目前治疗方案因禁忌、严重不良反应等导致其使用的局限性,因此需要一种更有效、安全、简便、不适宜干扰素治疗的治疗药物.Sofosbuvir是具有这些特性的直接抗病毒药物,为HCV NS5 B聚合酶的尿苷核苷酸类似物抑制药,可有效对抗多种基因型HCV感染,具有良好的安全性和耐受性.本文综述Sofosbuvir的作用机制、药动学、不良反应、药物相互作用及临床试验.

  12. Development of sofosbuvir for the treatment of hepatitis C virus infection.

    Science.gov (United States)

    Lawitz, Eric; Jacobson, Ira M; Nelson, David R; Zeuzem, Stefan; Sulkowski, Mark S; Esteban, Rafael; Brainard, Diana; McNally, John; Symonds, William T; McHutchison, John G; Dieterich, Douglas; Gane, Edward

    2015-11-01

    The nucleotide analog NS5B polymerase inhibitor sofosbuvir was approved by the U.S. Food and Drug Administration (FDA) in December 2013 for the treatment of chronic hepatitis C virus (HCV) infection in combination with ribavirin or peginterferon and ribavirin. Sofosbuvir was developed to meet an urgent medical need for shorter, safer, simplified, more effective HCV treatment regimens and to reduce or eliminate the need for peginterferon. New treatment regimens were especially required for patient populations with limited treatment options, including patients who had failed prior HCV therapy, those with compensated and decompensated cirrhosis, and those who were either intolerant of or had contraindications to interferon. Sofosbuvir plus ribavirin for patients with genotype 2 or 3 HCV infection was the first approved all-oral treatment option. Sofosbuvir is also the backbone of the first regimen available for patients awaiting liver transplantation to prevent HCV recurrence, as well as the first oral interferon-free regimen for patients coinfected with HCV and HIV. This paper describes the development of sofosbuvir up to its original FDA approval.

  13. Brane/antibrane Configurations in Type IIA and M-Theory

    Science.gov (United States)

    Marsano, Joseph

    We investigate the relation between large N duality applied to systems of D5's and /lineD5's wrapping vanishing cycles of local CY in type IIB and M-theory lifts of the NS5/D4//lineD4 systems in type IIA to which they are related by T-duality. Through a simple example based on a local CY constructed using an A2 singularity, we review this well-known correspondence in the supersymmetric setting and describe the manner in which it generalizes when antibranes are added. Agreement between the IIB and IIA pictures, which supports the assertion that {N}=2 supersymmetry is spontaneously broken in these systems at string tree level, is demonstrated when gs ≪ 1. Novel nonholomorphic features can arise away from this regime and their physical origin is discussed. This note is based on talks given at KITP, Harvard University, TIFR, the University of Tokyo at Hongo, the 2007 Les Houches Summer School, and the 2007 Simons Workshop, is based on work done in collaboration with K. Papadodimas and M. Shigemori, and contains some previously unpublished results.

  14. In-silico analysis of putative HCV epitopes against Pakistani human leukocyte antigen background: An approach towards development of future vaccines for Pakistani population.

    Science.gov (United States)

    Ashraf, Naeem Mahmood; Bilal, Muhammad; Mahmood, Malik Siddique; Hussain, Aadil; Mehboob, Muhammad Zubair

    2016-09-01

    Mounting burden of HCV-infected individuals and soaring cost of treatment is a serious source of unease for developing countries. Numbers of various approaches have been anticipated to develop a vaccine against HCV but the majority of them proved ineffective. Development of vaccine by considering geographical distribution of HCV genotypes and host genetics shows potential. In this research article, we have tried to predict most putative HCV epitopes which are efficiently restricted by most common HLA alleles in Pakistani population through different computational algorithms. Thirteen selected, experimentally identified epitopes sequences were used to derived consensus sequences in all genotypes of HCV. Obtained consensus sequences were used to predict their binding affinities with most prevalent HLA alleles in Pakistani population. Two Class-I epitopes from NS4B region, one from Class-I epitope from NS5A and one Class-II epitope from NS3 region showed effective binding and proved to be highly putative to boost immune response. A cocktail of these four have been checked for population coverage and they gave 75.53% for Pakistani Asian and 70.77% for Pakistani Mixed populations with no allergenic response. Computational algorithms are robust way to shortlist potential candidate epitopes for vaccine development but further, in vivo and in-vitro studies are required to confirm their immunogenic properties.

  15. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

    Science.gov (United States)

    Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

    2015-01-01

    The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

  16. Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Naïve Patients

    Science.gov (United States)

    Marascio, Nadia; Pavia, Grazia; Strazzulla, Alessio; Dierckx, Tim; Cuypers, Lize; Vrancken, Bram; Barreca, Giorgio Settimo; Mirante, Teresa; Malanga, Donatella; Oliveira, Duarte Mendes; Vandamme, Anne-Mieke; Torti, Carlo; Liberto, Maria Carla; Focà, Alfredo

    2016-01-01

    Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure. PMID:27618896

  17. The Cascade is a MMS Instanton

    CERN Document Server

    Evslin, J

    2004-01-01

    Wrap m D5-branes around the 2-cycle of a conifold, place n D3-branes at a point and watch the system relax. The D5-branes source m units of RR 3-form flux on the 3-cycle, which cause dielectric NS5-branes to nucleate and repeatedly sweep out the 3-cycle, each time gaining m units of D3-charge while the stack of D5-branes loses m units of D3-charge. A similar description of the Klebanov-Strassler cascade has been proposed by Kachru, et al. when m>>m-n. Using the T-dual MQCD we argue that the above process occurs for any m and n and in particular may continue for more than one step. The nonbaryonic roots of the SQCD vacua lead to new cascades because, for example, the 3-cycle swept does not link all of the D5's. This decay is the S-dual of a MMS instanton, which is the decay into flux of a brane that is trivial in twisted K-theory. This provides the first evidence for the S-dual of the K-theory classification that does not itself rely upon any strong/weak duality.

  18. The IR obstruction to UV completion for Dante's Inferno model with higher-dimensional gauge theory origin

    Science.gov (United States)

    Furuuchi, Kazuyuki; Koyama, Yoji

    2016-06-01

    We continue our investigation of large field inflation models obtained from higher-dimensional gauge theories, initiated in our previous study [1]. We focus on Dante's Inferno model which was the most preferred model in our previous analysis. We point out the relevance of the IR obstruction to UV completion, which co