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Sample records for c100-3 hcr43 ns5

  1. Orientifold dual for stuck NS5 branes

    CERN Document Server

    Feng, B; Karch, A; Uranga, Angel M; Feng, Bo; He, Yang-Hui; Karch, Andreas; Uranga, Angel

    2001-01-01

    We establish T-duality between NS5 branes stuck on an orientifold 8-plane in type I' and an orientifold construction in type IIB with D7 branes intersecting at angles. Two applications are discussed. For one we obtain new brane constructions, realizing field theories with gauge group a product of symplectic factors, giving rise to a large new class of conformal N=1 theories embedded in string theory. Second, by studying a D2 brane probe in the type I' background, we get some information on the still elusive (0,4) linear sigma model describing a perturbative heterotic string on an ADE singularity.

  2. Interaction study between HCV NS5A-D2 and NS5B using19F NMR.

    Science.gov (United States)

    Dujardin, Marie; Cantrelle, François-Xavier; Lippens, Guy; Hanoulle, Xavier

    2018-01-01

    The non structural protein 5A (NS5A) regulates the replication of the hepatitis C viral RNA through a direct molecular interaction of its domain 2 (NS5A-D2) with the RNA dependent RNA polymerase NS5B. Because of conflicting data in the literature, we study here this molecular interaction using fluorinated versions of the NS5A-D2 protein derived from the JFH1 Hepatitis C Virus strain. Two methods to prepare fluorine-labelled NS5A-D2 involving the biosynthetic incorporation of a 19 F-tryptophan using 5-fluoroindole and the posttranslational introduction of fluorine by chemical conjugation of 2-iodo-N-(trifluoromethyl)acetamide with the NS5A-D2 cysteine side chains are presented. The dissociation constants (K D ) between NS5A-D2 and NS5B obtained with these two methods are in good agreement, and yield values comparable to those derived previously from a surface plasmon resonance study. We compare benefits and limitations of both labeling methods to study the interaction between an intrinsically disordered protein and a large molecular target by 19 F NMR.

  3. Some considerations about NS5 and LST Hawking radiation

    Energy Technology Data Exchange (ETDEWEB)

    Lorente-Espin, Oscar, E-mail: oscar.lorente-espin@upc.edu [Departament de Fisica i Enginyeria Nuclear, Universitat Politecnica de Catalunya, Comte Urgell 187, E-08036 Barcelona (Spain)

    2011-09-26

    We have studied the Hawking radiation corresponding to the NS5 and Little String Theory (LST) black hole models using two semi-classical methods: the complex path method and a gravitational anomaly. After summarizing some known concepts about the thermodynamics of these theories, we have computed the emission rates for the two black hole models. The temperature calculated from, e.g. the well-known surface gravity expression, is shown to be identical to that obtained from both the computation of the gravitational anomaly and the complex path method. Moreover, the two semi-classical methods show that NS5 exhibits non-thermal behavior that contrasts with the thermal behavior of LST. We remark that energy conservation is the key factor leading to a non-thermal profile for NS5. In contrast, LST keeps a thermal profile even when energy conservation is considered because temperature in this model does not depend on energy.

  4. Discovery of potent macrocyclic HCV NS5A inhibitors.

    Science.gov (United States)

    Yu, Wensheng; Vibulbhan, Bancha; Rosenblum, Stuart B; Martin, Gregory S; Vellekoop, A Samuel; Holst, Christian L; Coburn, Craig A; Wong, Michael; Selyutin, Oleg; Ji, Tao; Zhong, Bin; Hu, Bin; Chen, Lei; Dwyer, Michael P; Jiang, Yueheng; Nair, Anilkumar G; Tong, Ling; Zeng, Qingbei; Agrawal, Sony; Carr, Donna; Rokosz, Laura; Liu, Rong; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Lahser, Fred; Asante-Appiah, Ernest; Fells, James; Kozlowski, Joseph A

    2016-08-01

    HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the treatment of HCV infection. Merck's effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  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...... growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. JFH1-based intergenotypic recombinants with genotype specific homotypic 5'UTR, or heterotypic 5'UTR (either of genotype 1a (strain H...

  7. Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B.

    Science.gov (United States)

    Wang, Yujing; Zhu, Zailing; Wang, Ping; Yu, Jialin; Wan, Lingzhu; Chen, Jun; Xiao, Ming

    2011-03-01

    The NS3-NS5B interaction of classical swine fever virus (CSFV) is important for viral replication. For characterisation of the interaction between the NS3 and NS5B, a series of NS5B mutants with deletion of N-, C-terminal amino acids and quadruple alanine substitution mutations were produced. GST pull-down assays and immunoprecipitation analyses showed that NS5B and some NS5B mutants have NS3 binding activity. Further experimental data indicated that CSFV NS5B might contain two NS3 binding sites, one covering amino acids 63-99 located at the N-terminal end, another covering amino acids 611-642 at the C-terminal end. Assays for RNA-dependent RNA polymerase (RdRp) activity revealed that CSFV NS3 is able to enhance the RdRp activity of NS5B and some NS5B mutants in vitro. The enhancement might be obtained by NS3 binding to the two terminal sequences of NS5B, which could be attractive targets for drug development against CSFV. Copyright © 2011. Published by Elsevier B.V.

  8. In Silico Approaches to the Design of NS5A Inhibitors.

    Science.gov (United States)

    Ivanenkov, Yan A; Veselov, Mark S; Aladinskiy, Vladimir A; Shakhbazyan, Artem G; Yartseva, Sofya M; Majouga, Alexander G; Aladinskaya, Anastasia V; Vantskul, Anton S; Leonov, Sergey V; Ivachtchenko, Alexandre V; Koteliansky, Victor E

    2016-01-01

    In recent years, nonstructural protein 5A (NS5A) has rapidly emerged as a promising therapeutic target for Hepatitis C (HCV) virus therapy. It is involved in both viral RNA replication and virus assembly and NS5A plays a critical role in the regulation of HCV life cycle. NS5A replication complex inhibitors (NS5A RCIs) have demonstrated strong antiviral activity in vitro and in vivo. However, wild-type resistance mutations and a wide range of genotypes significantly reduce their clinical efficacy. The exact mechanism of NS5A action still remains elusive, therefore several in silico models have been constructed to gain insight into the drug binding and subsequent structural optimization to overcome resistance. This paper provides a comprehensive overview of the computational studies towards NS5A mechanism of action and the design of novel small-molecule inhibitors.

  9. Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides

    Science.gov (United States)

    Chukkapalli, Vineela; Berger, Kristi L.; Kelly, Sean M.; Thomas, Meryl; Deiters, Alexander; Randall, Glenn

    2014-01-01

    Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies are HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation. PMID:25546252

  10. Prevalence of Resistance-Associated Substitutions in HCV NS5A, NS5B, or NS3 and Outcomes of Treatment With Ledipasvir and Sofosbuvir.

    Science.gov (United States)

    Sarrazin, Christoph; Dvory-Sobol, Hadas; Svarovskaia, Evguenia S; Doehle, Brian P; Pang, Phillip S; Chuang, Shu-Min; Ma, Julie; Ding, Xiao; Afdhal, Nezam H; Kowdley, Kris V; Gane, Edward J; Lawitz, Eric; Brainard, Diana M; McHutchison, John G; Miller, Michael D; Mo, Hongmei

    2016-09-01

    We evaluated the effects of baseline hepatitis C virus (HCV) NS5A, NS5B, and NS3 resistance-associated substitutions (RASs) on response to the combination of ledipasvir and sofosbuvir, with or without ribavirin, in patients with HCV genotype 1 infection. We analyzed data from 2144 participants in phase 2 and 3 studies of patients with HCV genotype 1a or b infection who received the combination of ledipasvir (90 mg) and sofosbuvir (400 mg) (ledipasvir/sofosbuvir) once daily, with or without ribavirin twice daily. Population and/or deep sequence analyses of the HCV NS3, NS5A, and NS5B genes were performed on blood samples collected at baseline. Overall, 16.0% of patients had detectable baseline RASs in NS5A. Among patients with HCV genotype 1b infection, there was no significant effect of baseline RASs in NS5A on sustained viral response 12 weeks after the end of treatment (SVR12) with ledipasvir/sofosbuvir and only a small effect in patients with HCV genotype 1a infection. RASs in NS5A that increased the half-maximal effective concentration to ledipasvir by more than 100-fold reduced the rate of SVR12 in treatment-naive patients given ledipasvir/sofosbuvir for 8 weeks (P = .011), but not for 12 weeks. These same baseline NS5A RASs reduced the percentage of treatment-experienced patients who achieved an SVR12 to 12 weeks (but not 24 weeks) ledipasvir/sofosbuvir (P ledipasvir/sofosbuvir in combination with ribavirin for 12 weeks. Overall, 2.5% of patients had baseline NS5B nucleotide inhibitor RASs (L159F, N142T, S282G, or L320S) and all achieved an SVR12. Of patients previously treated with protease inhibitors, 53.7% had RASs in NS3 and 96.5% achieved an SVR12. Baseline RASs in NS5A have minimal effects on patient responses to ledipasvir/sofosbuvir therapy. When these RASs do have effects, they could be largely overcome by extending treatment duration or through treatment intensification. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights

  11. Prevalence of natural polymorphisms at the HCV NS5A gene associated with resistance to daclatasvir, an NS5A inhibitor.

    Science.gov (United States)

    Plaza, Zulema; Soriano, Vincent; Vispo, Eugenia; del Mar Gonzalez, Maria; Barreiro, Pablo; Seclén, Eduardo; Poveda, Eva

    2012-01-01

    Daclatasvir (BMS-790052) is an investigational molecule that inhibits the HCV NS5A protein and shows potent antiviral activity apparently across all HCV genotypes. Selection of drug resistance mutations has been reported only for HCV genotype 1, and no information exists for other HCV variants and/or in HIV-HCV-coinfected individuals. All interferon-α-naive, HIV-HCV-coinfected patients newly attended at Hospital Carlos III (Madrid, Spain) in 2011 were identified. Changes reported to be associated with daclatasvir resistance in the in vitro replication system for HCV genotype/subtypes 1a/1b (M28T, Q30H/R, L31F/M/V, P32L and Y93C/H/N) were examined. A total of 78 HIV-HCV-coinfected individuals as well as 635 NS5A sequences deposited at Los Alamos HCV database were analysed. None of the NS5A sequences from HCV-1a or HCV-3 showed changes associated with daclatasvir resistance. By contrast, all NS5A sequences from HCV-4 harboured L31M. The double mutant L31M+Y93H was found in 7% of HCV-1b and 13% of HCV-4. Finally, all NS5A sequences from HCV-1b and HCV-4 harboured changes at codon 28 (M28L) and 30 (L30R), which are of unknown significance. The rate of all these NS5A polymorphisms did not differ significantly when comparing HIV-HCV-coinfected patients and sequences from HCV-monoinfected subjects deposited at Los Alamos HCV database. Primary resistance mutations to daclatasvir, an investigational HCV NS5A inhibitor, are not seen in HCV-1a or in HCV-3 as natural polymorphisms. By contrast, they can be recognized in most HCV-1b and HCV-4 strains, regardless HIV coinfection.

  12. 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

    ) from genotypes 1-6 and 2a(JFH1) NS5B-3' untranslated region, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants. METHODS: The HCV 5-5A recombinants with previously identified mutations in the NS3-helicase (F1464L), NS4A (A1672S), and NS5B (D2979G) were adapted and improved......, by incorporating additional recovered mutations that increased their propagation in Huh7.5 cells. Concentration-response profiles were determined for each DAA agent in replicate infected Huh7.5 cells. RESULTS: Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants did...... not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3 protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172 and by the NS5A inhibitor...

  13. Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A.

    Science.gov (United States)

    Sun, Jin-Hua; O'Boyle, Donald R; Fridell, Robert A; Langley, David R; Wang, Chunfu; Roberts, Susan B; Nower, Peter; Johnson, Benjamin M; Moulin, Frederic; Nophsker, Michelle J; Wang, Ying-Kai; Liu, Mengping; Rigat, Karen; Tu, Yong; Hewawasam, Piyasena; Kadow, John; Meanwell, Nicholas A; Cockett, Mark; Lemm, Julie A; Kramer, Melissa; Belema, Makonen; Gao, Min

    2015-11-12

    It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3-4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle.

  14. In vitro activity of daclatasvir on hepatitis C virus genotype 3 NS5A.

    Science.gov (United States)

    Wang, Chunfu; Valera, Lourdes; Jia, Lingling; Kirk, Melissa J; Gao, Min; Fridell, Robert A

    2013-01-01

    The NS5A replication complex inhibitor daclatasvir (DCV; BMS-790052) inhibits hybrid replicons containing hepatitis C virus (HCV) genotype 3a (HCV3a) NS5A genes with 50% effective concentrations (EC(50)s) ranging from 120 to 870 pM. Selection studies with a hybrid HCV3a replicon identified NS5A residues 31 and 93 as sites for DCV-selected resistance. Our results support the potential use of DCV as a component in combination therapies for HCV3a chronic infection.

  15. Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A.

    Science.gov (United States)

    Bujalowski, Paul J; Bujalowski, Wlodzimierz; Choi, Kyung H

    2017-06-01

    The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5' untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl 2 but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA 70 and SLA 80 , which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides. IMPORTANCE Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5' untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the

  16. 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.

  17. 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

  18. Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

    KAUST Repository

    Knodel, Markus

    2018-01-08

    Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

  19. Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

    Directory of Open Access Journals (Sweden)

    Markus M. Knodel

    2018-01-01

    Full Text Available Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

  20. Efficient hepatitis c virus genotype 1b core-NS5A recombinants permit efficacy testing of protease and NS5A inhibitors

    DEFF Research Database (Denmark)

    Pham, Long V.; Ramirez Almeida, Santseharay; Carlsen, Thomas H R

    2017-01-01

    Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants...... cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log10 focus-forming units (FFU)/ml. We subsequently succeeded...... in adapting a JFH1- based 5=UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all...

  1. In silico mutation analysis of non-structural protein-5 (NS5) dengue virus

    Science.gov (United States)

    Puspitasari, R. D.; Tambunan, U. S. F.

    2017-04-01

    Dengue fever is a world disease. It is endemic in more than 100 countries. Information about the effect of mutations in the virus is important in drug design and development. In this research, we studied the effect of mutation on NS5 dengue virus. NS5 is the large protein containing 67% amino acid similarity in DENV 1-4 and has multifunctional enzymatic activities. Dengue virus is an RNA virus that has very high mutation frequency with an average of 100 times higher than DNA mutations, and the accumulation of mutations will be possible to generate the new serotype. In this study, we report that mutation occurs in NS5 of DENV serotype 3, glutamine mutates into methionine at position 10 and threonine mutates into isoleucine at position 55. These residues are part of the domain named S-Adenosyl-L-Methionine-Dependent Methyltransferase (IPR029063).

  2. Alkyl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

    Science.gov (United States)

    Yu, Wensheng; Coburn, Craig A; Nair, Anilkumar G; Wong, Michael; Tong, Ling; Dwyer, Michael P; Hu, Bin; Zhong, Bin; Hao, Jinglai; Yang, De-Yi; Selyutin, Oleg; Jiang, Yueheng; Rosenblum, Stuart B; Kim, Seong Heon; Lavey, Brian J; Zhou, Guowei; Rizvi, Razia; Shankar, Bandarpalle B; Zeng, Qingbei; Chen, Lei; Agrawal, Sony; Carr, Donna; Rokosz, Laura; Liu, Rong; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Lahser, Fred; Asante-Appiah, Ernest; Nomeir, Amin; Kozlowski, Joseph A

    2016-08-01

    HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein we describe our continued research efforts around the alkyl "Z group" modification of the tetracyclic indole-based NS5A inhibitor MK-8742, which led to the discovery of a series of potent NS5A inhibitors. Compounds 10 and 19 are of particular interests since they are as potent as our previous leads and have much improved rat pharmacokinetic profiles. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

    Science.gov (United States)

    Yu, Wensheng; Coburn, Craig A; Nair, Anilkumar G; Wong, Michael; Rosenblum, Stuart B; Zhou, Guowei; Dwyer, Michael P; Tong, Ling; Hu, Bin; Zhong, Bin; Hao, Jinglai; Ji, Tao; Zan, Shuai; Kim, Seong Heon; Zeng, Qingbei; Selyutin, Oleg; Chen, Lei; Masse, Frederic; Agrawal, Sony; Liu, Rong; Xia, Ellen; Zhai, Ying; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Asante-Appiah, Ernest; Lin, Mingxiang; Kozlowski, Joseph A

    2016-08-01

    Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. 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....

  5. Discovery of fused tricyclic core containing HCV NS5A inhibitors with pan-genotype activity.

    Science.gov (United States)

    Yu, Wensheng; Coburn, Craig A; Yang, De-Yi; Meinke, Peter T; Wong, Michael; Rosenblum, Stuart B; Chen, Kevin X; Njoroge, George F; Chen, Lei; Dwyer, Michael P; Jiang, Yueheng; Nair, Anilkumar G; Selyutin, Oleg; Tong, Ling; Zeng, Qingbei; Zhong, Bin; Ji, Tao; Hu, Bin; Agrawal, Sony; Xia, Ellen; Zhai, Ying; Liu, Rong; Kong, Rong; Ingravallo, Paul; Asante-Appiah, Ernest; Nomeir, Amin; Fells, James; Kozlowski, Joseph A

    2016-07-01

    HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein, we describe research efforts that led to the discovery of a series of fused tricyclic core containing HCV NS5A inhibitors such as 24, 39, 40, 43, and 44 which have pan-genotype activity and are orally bioavailable in the rat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. 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.

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

    Science.gov (United States)

    Kwon, Hyock Joo; Xing, Weimei; Chan, Katie; Niedziela-Majka, Anita; Brendza, Katherine M; Kirschberg, Thorsten; Kato, Darryl; Link, John O; Cheng, Guofeng; Liu, Xiaohong; Sakowicz, Roman

    2015-01-01

    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.

  8. 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.

  9. Matched and mixed cap derivatives in the tetracyclic indole class of HCV NS5A inhibitors.

    Science.gov (United States)

    Dwyer, Michael P; Keertikar, Kerry M; Chen, Lei; Tong, Ling; Selyutin, Oleg; Nair, Anilkumar G; Yu, Wensheng; Zhou, Guowei; Lavey, Brian J; Yang, De-Yi; Wong, Michael; Kim, Seong Heon; Coburn, Craig A; Rosenblum, Stuart B; Zeng, Qingbei; Jiang, Yueheng; Shankar, Bandarpalle B; Rizvi, Razia; Nomeir, Amin A; Liu, Rong; Agrawal, Sony; Xia, Ellen; Kong, Rong; Zhai, Ying; Ingravallo, Paul; Asante-Appiah, Ernest; Kozlowski, Joseph A

    2016-08-15

    A matched and mixed capping SAR study was conducted on the tetracyclic indole class of HCV NS5A inhibitors to examine the influence of modifications of this region on the overall HCV virologic resistance profiles. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Alternative core development around the tetracyclic indole class of HCV NS5A inhibitors.

    Science.gov (United States)

    Tong, Ling; Yu, Wensheng; Coburn, Craig A; Meinke, Peter T; Nair, Anilkumar G; Dwyer, Michael P; Chen, Lei; Selyutin, Oleg; Rosenblum, Stuart B; Jiang, Yueheng; Fells, James; Hu, Bin; Zhong, Bin; Soll, Richard M; Liu, Rong; Agrawal, Sony; Xia, Ellen; Zhai, Ying; Kong, Rong; Ingravallo, Paul; Nomeir, Amin; Asante-Appiah, Ernest; Kozlowski, Joseph A

    2016-10-15

    Herein, we describe our research efforts to develop unique cores in molecules which function as HCV nonstructural protein 5A (NS5A) inhibitors. In particular, various fused tetracyclic cores were identified which showed genotype and mutant activities comparable to the indole-based tetracyclic core. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Holographic approach to deformations of NS5-brane distributions and exact CFTs

    CERN Document Server

    Fotopoulos, Angelos; Prezas, Nikolaos; Sfetsos, Konstadinos

    2008-01-01

    We consider general planar deformations of a circular distribution of NS5-branes. The near-horizon region of the latter admits, after a T-duality transformation, an exact conformal-field-theory description in terms of the coset model SU(2)/U(1) X SL(2,R)/U(1). We derive the exactly marginal operators corresponding to an infinitesimal planar deformation using the conjectured holography between the coset model and the little string theory that resides on the worldvolume of the NS5-branes. Subsequently, we perform a complementary analysis of the same deformations using the associated N=1 supersymmetric sigma model and verify the holographic correspondence. We explicitly demonstrate a precise match between the two approaches which rests upon a delicate interplay between exact conformal-field-theory operators and their semiclassical realizations in terms of target-space variables.

  12. Structure-activity relationships of proline modifications around the tetracyclic-indole class of NS5A inhibitors.

    Science.gov (United States)

    Tong, Ling; Yu, Wensheng; Coburn, Craig A; Chen, Lei; Selyutin, Oleg; Zeng, Qingbei; Dwyer, Michael P; Nair, Anilkumar G; Shankar, Bandarpalle B; Kim, Seong Heon; Yang, De-Yi; Rosenblum, Stuart B; Ruck, Rebecca T; Davies, Ian W; Hu, Bin; Zhong, Bin; Hao, Jinglai; Ji, Tao; Zan, Shuai; Liu, Rong; Agrawal, Sony; Carr, Donna; Curry, Stephanie; McMonagle, Patricia; Bystol, Karin; Lahser, Frederick; Ingravallo, Paul; Chen, Shiying; Asante-Appiah, Ernest; Kozlowski, Joseph A

    2016-11-01

    We describe the impact of proline modifications, in our tetracyclic-indole based series of nonstructural protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an improved and flattened resistance profiles. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. GPS2 is required for the association of NS5A with VAP-A and hepatitis C virus replication.

    Directory of Open Access Journals (Sweden)

    Guodong Xu

    Full Text Available Hepatitis C virus (HCV nonstructural protein 5A (NS5A is a component of the replication complex associated with various cellular proteins. It has been reported that G protein pathway suppressor 2 (GPS2 is a potential NS5A-binding factor, as identified in a yeast two-hybrid screens of human cDNA library using viral proteins as baits [1]. In this study, we demonstrated the interaction between GPS2 and NS5A in mammalian cells by coimmunoprecipitation analysis and found that both exogenously and endogenously expressed GPS2 interacted with NS5A of genotype 1b and 2a. Mutagenesis study demonstrated that Domain I of NS5A and coiled-coil domain of GPS2 are responsible for the interaction. Knockdown of GPS2 in hepatoma cell lines suppressed the replication of HCV RNA, which can be rescued by the expression of an RNAi-resistant GPS2. Furthermore, overexpression of GPS2 enhanced the association of NS5A with a proviral cellular factor, human vesicle-associated membrane protein-associated protein A (VAP-A, while knockdown of GPS2 disrupted interaction between VAP-A and NS5A. Taken together, our results suggest that GPS2 acts as a bridge between NS5A and VAP-A and is required for efficient HCV replication.

  14. 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

    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 (cLDs) or on the ......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...... JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A...... 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...

  15. Rotating NS5-brane solution and its exact string theoretical description

    OpenAIRE

    Sfetsos, K.

    1999-01-01

    We construct the most general solution in type-II string theory that represents N coincident non-extremal rotating NS5-branes and determine the relevant thermodynamic quantities. We show that in the field theory limit, it has an exact description. In particular, it can be obtained by an O(3,3) duality transformation on the exact string background for the coset model SL(2,R)_{-N}/U(1) \\times SU(2)_N. In the extreme supersymmetric limit we recover the multicenter solution, with a ring singulari...

  16. 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.

  17. 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.

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

    Science.gov (United States)

    Wang, Maolin; Wang, Kai; Yan, Aixia; Yu, Changyuan

    2012-01-01

    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. PMID:22605964

  19. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function.

    Science.gov (United States)

    Hebner, Christy M; Han, Bin; Brendza, Katherine M; Nash, Michelle; Sulfab, Maisoun; Tian, Yang; Hung, Magdeleine; Fung, Wanchi; Vivian, Randall W; Trenkle, James; Taylor, James; Bjornson, Kyla; Bondy, Steven; Liu, Xiaohong; Link, John; Neyts, Johan; Sakowicz, Roman; Zhong, Weidong; Tang, Hengli; Schmitz, Uli

    2012-01-01

    Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.

  20. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function.

    Directory of Open Access Journals (Sweden)

    Christy M Hebner

    Full Text Available Tegobuvir (TGV is a novel non-nucleoside inhibitor (NNI of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI of the viral polymerase.

  1. NS5A Sequence Heterogeneity and Mechanisms of Daclatasvir Resistance in Hepatitis C Virus Genotype 4 Infection.

    Science.gov (United States)

    Zhou, Nannan; Hernandez, Dennis; Ueland, Joseph; Yang, Xiaoyan; Yu, Fei; Sims, Karen; Yin, Philip D; McPhee, Fiona

    2016-01-15

    Daclatasvir is an NS5A inhibitor approved for treatment of infection due to hepatitis C virus (HCV) genotypes (GTs) 1-4. To support daclatasvir use in HCV genotype 4 infection, we examined a diverse genotype 4-infected population for HCV genotype 4 subtype prevalence, NS5A polymorphisms at residues associated with daclatasvir resistance (positions 28, 30, 31, or 93), and their effects on daclatasvir activity in vitro and clinically. We performed phylogenetic analysis of genotype 4 NS5A sequences from 186 clinical trial patients and 43 sequences from the European HCV database, and susceptibility analyses of NS5A polymorphisms and patient-derived NS5A sequences by using genotype 4 NS5A hybrid genotype 2a replicons. The clinical trial patients represented 14 genotype 4 subtypes; most prevalent were genotype 4a (55%) and genotype 4d (27%). Daclatasvir 50% effective concentrations for 10 patient-derived NS5A sequences representing diverse phylogenetic clusters were ≤0.080 nM. Most baseline sequences had ≥1 NS5A polymorphism at residues associated with daclatasvir resistance; however, only 3 patients (1.6%) had polymorphisms conferring ≥1000-fold daclatasvir resistance in vitro. Among 46 patients enrolled in daclatasvir trials, all 20 with baseline resistance polymorphisms achieved a sustained virologic response. Circulating genotype 4 subtypes are genetically diverse. Polymorphisms conferring high-level daclatasvir resistance in vitro are uncommon before therapy, and clinical data suggest that genotype 4 subtype and baseline polymorphisms have minimal impact on responses to daclatasvir-containing regimens. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.

  2. NS5A Sequence Heterogeneity and Mechanisms of Daclatasvir Resistance in Hepatitis C Virus Genotype 4 Infection

    OpenAIRE

    Zhou, Nannan; Hernandez, Dennis; Ueland, Joseph; Yang, Xiaoyan; Yu, Fei; Sims, Karen; Yin, Philip D.; McPhee, Fiona

    2015-01-01

    Background. ?Daclatasvir is an NS5A inhibitor approved for treatment of infection due to hepatitis C virus (HCV) genotypes (GTs) 1?4. To support daclatasvir use in HCV genotype 4 infection, we examined a diverse genotype 4?infected population for HCV genotype 4 subtype prevalence, NS5A polymorphisms at residues associated with daclatasvir resistance (positions 28, 30, 31, or 93), and their effects on daclatasvir activity in vitro and clinically. Methods. ?We performed phylogenetic analysis of...

  3. Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors.

    Science.gov (United States)

    McGivern, David R; Masaki, Takahiro; Williford, Sara; Ingravallo, Paul; Feng, Zongdi; Lahser, Frederick; Asante-Appiah, Ernest; Neddermann, Petra; De Francesco, Raffaele; Howe, Anita Y; Lemon, Stanley M

    2014-08-01

    All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  4. Discovery of silyl proline containing HCV NS5A inhibitors with pan-genotype activity: SAR development.

    Science.gov (United States)

    Nair, Anilkumar G; Zeng, Qingbei; Selyutin, Oleg; Rosenblum, Stuart B; Jiang, Yueheng; Yang, De-Yi; Keertikar, Kerry; Zhou, Guowei; Dwyer, Michael P; Kim, Seong Heon; Shankar, Bandarpalle; Yu, Wensheng; Tong, Ling; Chen, Lei; Mazzola, Robert; Caldwell, John; Tang, Haiqun; Allard, Melissa L; Buckle, Ronald N; Gauuan, Polivina Jolicia F; Holst, Christian L; Martin, Gregory S; Naicker, Kannan P; Vellekoop, Samuel; Agrawal, Sony; Liu, Rong; Kong, Rong; Ingravallo, Paul; Xia, Ellen; Zhai, Ying; Nomeir, Amin; Kozlowski, Joseph A

    2016-03-01

    HCV NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays thus making them attractive components for inclusion in an all oral fixed dose combination treatment regimen. Herein we describe the research efforts that led to the discovery of silyl proline containing HCV NS5A inhibitors such as 7e and 8a with pan-genotype activity profile and acceptable pharmacokinetic properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Substituted tetracyclic indole core derivatives of HCV NS5A inhibitor MK-8742.

    Science.gov (United States)

    Yu, Wensheng; Zhou, Guowei; Coburn, Craig A; Zeng, Qingbei; Tong, Ling; Dwyer, Michael P; Hu, Bin; Zhong, Bin; Hao, Jinglai; Ji, Tao; Zan, Shuai; Chen, Lei; Mazzola, Robert; Kim, Jae-Hun; Sha, Deyou; Selyutin, Oleg; Rosenblum, Stuart B; Lavey, Brian; Nair, Anilkumar G; Heon Kim, Seong; Keertikar, Kerry M; Rokosz, Laura; Agrawal, Sony; Liu, Rong; Xia, Ellen; Zhai, Ying; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Asante-Appiah, Ernest; Chen, Shiying; Kozlowski, Joseph A

    2016-10-01

    As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing substitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly. However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of the tetracyclic indole core had a positive impact on the potency against the resistance associated variants, such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation

    Science.gov (United States)

    Gawlik, Katarzyna; Baugh, James; Chatterji, Udayan; Lim, Precious J.; Bobardt, Michael D.; Gallay, Philippe A.

    2014-01-01

    Hepatitis C virus (HCV) infection is a major cause of liver disease. The molecular machinery of HCV assembly and particle release remains obscure. A better understanding of the assembly events might reveal new potential antiviral strategies. It was suggested that the nonstructural protein 5A (NS5A), an attractive recent drug target, participates in the production of infectious particles as a result of its interaction with the HCV core protein. However, prior to the present study, the NS5A-binding site in the viral core remained unknown. We found that the D1 domain of core contains the NS5A-binding site with the strongest interacting capacity in the basic P38-K74 cluster. We also demonstrated that the N-terminal basic residues of core at positions 50, 51, 59 and 62 were required for NS5A binding. Analysis of all substitution combinations of R50A, K51A, R59A, and R62A, in the context of the HCVcc system, showed that single, double, triple, and quadruple mutants were fully competent for viral RNA replication, but deficient in secretion of viral particles. Furthermore, we found that the extracellular and intracellular infectivity of all the mutants was abolished, suggesting a defect in the formation of infectious particles. Importantly, we showed that the interaction between the single and quadruple core mutants and NS5A was impaired in cells expressing full-length HCV genome. Interestingly, mutations of the four basic residues of core did not alter the association of core or NS5A with lipid droplets. This study showed for the first time that basic residues in the D1 domain of core that are critical for the formation of infectious extracellular and intracellular particles also play a role in core-NS5A interactions. PMID:24533158

  8. Ledipasvir plus sofosbuvir as salvage therapy for HCV genotype 1 failures to prior NS5A inhibitors regimens.

    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

    2017-07-01

    There is little information on retreatment efficacy and predictors of the combination of ledipasvir and sofosbuvir (ledipasvir/sofosbuvir) for patients who fail to respond to NS5A inhibitors. NS5A resistance variants are known to persist for long periods after such treatment. Here, we evaluated 54 patients with chronic HCV genotype 1b infection, free of decompensated cirrhosis, and hepatocellular carcinoma, for sustained virological response after 12 weeks (SVR12) of once-daily treatment with 90 mg ledipasvir and 400 mg sofosbuvir. Intention-to-treat analysis showed SVR12 of 70%. Using ultra-deep sequencing, non-responder to ledipasvir/sofosbuvir showed no change in the rates of detection of NS5A and NS5B resistant-variants at re-elevation of viral loads, relative to baseline. According to response to prior treatment, SVR12 rates were 18, 69, 94, and 100% in non response, viral breakthrough, relapse, and discontinuation due to adverse events, respectively. SVR12 rates in non response were significantly lower than those of the others. Multivariate analysis identified response to previous treatment (failure except for non response) and FIB4 index (ledipasvir/sofosbuvir is a potentially useful salvage treatment for patients who fail prior NS5A inhibitors-based therapy. Response to prior treatment was an important predictor of retreatment efficacy. © 2017 Wiley Periodicals, Inc.

  9. NS5A inhibitor, daclatasvir, for the treatment of chronic hepatitis C virus infection.

    Science.gov (United States)

    Herbst, D Alan; Reddy, K Rajender

    2013-10-01

    Chronic Hepatitis C virus (HCV) infection is a major pandemic. The current standard of care includes peginterferon and ribavirin plus one of two protease inhibitors, boceprevir and telaprevir, for Genotype 1 patients and peginterferon and ribavirin for all other genotypes. The treatment landscape is rapidly evolving as a number of direct-acting antivirals (DAA) are being developed in clinical trials. Daclatasvir, formerly labeled BMS-790052, is a first-in-class HCV NS5A inhibitor that has been demonstrated in Phase I and II trials to have a very potent antiviral effect across all genotypes and to have a potent clinical efficacy in both treatment naive and experienced cohorts. This review covers the whole spectrum of development of daclatasvir from Phase I to III programs. While daclatasvir has pangenotypic activity, it has a lower barrier to resistance in Genotype 1a but has been found to be very effective in Genotype 1b patients. However, Genotype 1a patients can be successfully treated with the addition of one or more DAAs alone or in combination with peginterferon and ribavirin. The future for daclatasvir and other DAAs is very encouraging in that all-oral therapies are likely to be effective and well-tolerated.

  10. Baseline NS5A resistance associated substitutions may impair DAA response in real-world hepatitis C patients.

    Science.gov (United States)

    Carrasco, Itzíar; Arias, Ana; Benítez-Gutiérrez, Laura; Lledó, Gemma; Requena, Silvia; Cuesta, Miriam; Cuervas-Mons, Valentín; de Mendoza, Carmen

    2017-10-09

    Oral DAA have demonstrated high efficacy as treatment of hepatitis C. However, the presence of resistance-associated substitutions (RAS) at baseline has occasionally been associated with impaired treatment response. Herein, we examined the impact of baseline RAS at the HCV NS5A gene region on treatment response in a real-life setting. All hepatitis C patients treated with DAA including NS5A inhibitors at our institution were retrospectively examined. The virus NS5A gene was analyzed using population sequencing at baseline and after 24 weeks of completing therapy in all patients that failed. All changes recorded at positions 28, 29, 30, 31, 32, 58, 62, 92, and 93 were considered. A total of 166 patients were analyzed. HCV genotypes were as follows: G1a (31.9%), G1b (48.2%), G3 (10.2%), and G4 (9.6%). Overall, 69 (41.6%) patients were coinfected with HIV and 46.7% had advanced liver fibrosis (Metavir F3-F4). Sixty (36.1%) patients had at least one RAS at baseline, including M28A/G/T (5), Q30X (12), L31I/F/M/V (6), T58P/S (25), Q/E62D (1), A92 K (7), and Y93C/H (15). Overall, 4.8% had two or more RAS, being more frequent in G4 (12.5%) followed by G1b (6.3%) and G1a (1.9%). Of 10 (6%) patients that failed DAA therapy, five had baseline NS5A RAS. No association was found for specific baseline RAS, although changes at position 30 were more frequent in failures than cures (22.2% vs 6.4%, P = 0.074). Moreover, the presence of two or more RAS at baseline was more frequent in failures (HR: 7.2; P = 0.029). Upon failure, six patients showed emerging RAS, including Q30C/H/R (3), L31M (1), and Y93C/H (2). Baseline NS5A RAS are frequently seen in DAA-naïve HCV patients. Two or more baseline NS5A RAS were found in nearly 5% and were significantly associated to DAA failure. Therefore, baseline NS5A testing should be considered when HCV treatment is planned with NS5A inhibitors. © 2017 Wiley Periodicals, Inc.

  11. Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

    Science.gov (United States)

    Hoffman, Brett; Li, Zhubing; Liu, Qiang

    2015-08-01

    Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

  12. Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1-7 and Escape Variants

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Pham, Long V; Mikkelsen, Lotte S

    2018-01-01

    NS5A inhibitors against HCV genotype 1-7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on HCV escape from treatment. METHODS: We measured the efficacy of different concentrations of daclatasvir, ledipasvir...

  13. Discovery of Chromane Containing Hepatitis C Virus (HCV) NS5A Inhibitors with Improved Potency against Resistance-Associated Variants.

    Science.gov (United States)

    Yu, Wensheng; Tong, Ling; Hu, Bin; Zhong, Bin; Hao, Jinglai; Ji, Tao; Zan, Shuai; Coburn, Craig A; Selyutin, Oleg; Chen, Lei; Rokosz, Laura; Agrawal, Sony; Liu, Rong; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Asante-Appiah, Ernest; Chen, Shiying; Kozlowski, Joseph A

    2016-11-23

    The discovery of potent and pan-genotypic HCV NS5A inhibitors faces many challenges including the significant diversity among genotypes, substantial potency shift conferred on some key resistance-associated variants, inconsistent SARs between different genotypes and mutants, and the lacking of models of inhibitor/protein complexes for rational inhibitor design. As part of ongoing efforts on HCV NS5A inhibition at Merck, we now describe the discovery of a novel series of chromane containing NS5A inhibitors. SAR studies around the "Z" group of the tetracyclic indole scaffold explored fused bicyclic rings as alternates to the phenyl group of elbasvir (1, MK-8742) and identified novel chromane and 2,3-dihydrobenzofuran derivatives as "Z" group replacements offered good potency across all genotypes. This effort, incorporating the C-1 fluoro substitution at the tetracyclic indole core, led to the discovery of a new series of NS5A inhibitors, such as compounds 14 and 25-28, with significantly improved potency against resistance-associated variants, such as GT2b, GT1a Y93H, and GT1a L31V. Compound 14 also showed reasonable PK exposures in preclinical species (rat and dog).

  14. Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.

    Science.gov (United States)

    Targett-Adams, Paul; Graham, Emily J S; Middleton, Jenny; Palmer, Amy; Shaw, Stephen M; Lavender, Helen; Brain, Philip; Tran, Thien Duc; Jones, Lyn H; Wakenhut, Florian; Stammen, Blanda; Pryde, David; Pickford, Chris; Westby, Mike

    2011-07-01

    The current standard of care for hepatitis C virus (HCV)-infected patients consists of lengthy treatment with interferon and ribavirin. To increase the effectiveness of HCV therapy, future regimens will incorporate multiple direct-acting antiviral (DAA) drugs. Recently, the HCV-encoded NS5A protein has emerged as a promising DAA target. Compounds targeting NS5A exhibit remarkable potency in vitro and demonstrate early clinical promise, suggesting that NS5A inhibitors could feature in future DAA combination therapies. Since the mechanisms through which these molecules operate are unknown, we have used NS5A inhibitors as tools to investigate their modes of action. Analysis of replicon-containing cells revealed dramatic phenotypic alterations in NS5A localization following treatment with NS5A inhibitors; NS5A was redistributed from the endoplasmic reticulum to lipid droplets. The NS5A relocalization did not occur in cells treated with other classes of HCV inhibitors, and NS5A-targeting molecules did not cause similar alterations in the localization of other HCV-encoded proteins. Time course analysis of the redistribution of NS5A revealed that the transfer of protein to lipid droplets was concomitant with the onset of inhibition, as judged by the kinetic profiles for these compounds. Furthermore, analysis of the kinetic profile of inhibition for a panel of test molecules permitted the separation of compounds into different kinetic classes based on their modes of action. Results from this approach suggested that NS5A inhibitors perturbed the function of new replication complexes, rather than acting on preformed complexes. Taken together, our data reveal novel biological consequences of NS5A inhibition, which may help enable the development of future assay platforms for the identification of new and/or different NS5A inhibitors.

  15. Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor.

    Science.gov (United States)

    Owens, Christopher M; Brasher, Bradley B; Polemeropoulos, Alex; Rhodin, Michael H J; McAllister, Nicole; Wong, Kelly A; Jones, Christopher T; Jiang, Lijuan; Lin, Kai; Or, Yat Sun

    2016-10-01

    EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Upadhyay, Anup K.; Cyr, Matthew; Longenecker, Kenton; Tripathi, Rakesh; Sun, Chaohong; Kempf, Dale J. (AbbVie)

    2017-02-21

    The rapid spread of the recentZika 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 ofZika 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 groupP21212 and containing two protein molecules in the asymmetric unit. The structure is similar to that reported for the NS5 protein fromJapanese encephalitis virusand suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain.

  17. Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication.

    Science.gov (United States)

    Berger, Carola; Romero-Brey, Inés; Radujkovic, Danijela; Terreux, Raphael; Zayas, Margarita; Paul, David; Harak, Christian; Hoppe, Simone; Gao, Min; Penin, Francois; Lohmann, Volker; Bartenschlager, Ralf

    2014-11-01

    Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  18. Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication.

    Science.gov (United States)

    Teramoto, Tadahisa; Balasubramanian, Anuradha; Choi, Kyung H; Padmanabhan, Radhakrishnan

    2017-06-09

    Four serotypes of mosquito-borne dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance for which there is no vaccine or antiviral drug available. The N-terminal domain of DENV NS5 has guanylyltransferase and methyltransferase (MTase), and the C-terminal region has the polymerase (POL), all of which are important for 5'-capping and RNA replication. The crystal structure of NS5 shows it as a dimer, but the functional evidence for NS5 dimer is lacking. Our studies showed that the substitution of DENV2 NS5 MTase or POL for DENV4 NS5 within DENV2 RNA resulted in a severe attenuation of replication in the transfected BHK-21 cells. A replication-competent species was evolved with the acquired mutations in the DENV2 and DENV4 NS5 MTase or POL domain or in the DENV2 NS3 helicase domain in the DENV2 chimera RNAs by repeated passaging of infected BHK-21 or mosquito cells. The linker region of seven residues in NS5, rich in serotype-specific residues, is important for the recovery of replication fitness in the chimera RNA. Our results, taken together, provide genetic evidence for a serotype-specific interaction between NS3 and NS5 as well as specific interdomain interaction within NS5 required for RNA replication. Genome-wide RNAseq analysis revealed the distribution of adaptive mutations in RNA quasispecies. Those within NS3 and NS5 are located at the surface and/or within the NS5 dimer interface, providing a functional significance to the crystal structure NS5 dimer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. QSAR study of HCV NS5B polymerase inhibitors using the genetic algorithm-multiple linear regression (GA-MLR)

    OpenAIRE

    Rafiei, Hamid; Khanzadeh, Marziyeh; Mozaffari, Shahla; Bostanifar, Mohammad Hassan; Avval, Zhila Mohajeri; Aalizadeh, Reza; Pourbasheer, Eslam

    2016-01-01

    Quantitative structure-activity relationship (QSAR) study has been employed for predicting the inhibitory activities of the Hepatitis C virus (HCV) NS5B polymerase inhibitors . A data set consisted of 72 compounds was selected, and then different types of molecular descriptors were calculated. The whole data set was split into a training set (80 % of the dataset) and a test set (20 % of the dataset) using principle component analysis. The stepwise (SW) and the genetic algorithm (GA) technique...

  20. Effect on hepatitis C virus replication of combinations of direct-acting antivirals, including NS5A inhibitor daclatasvir.

    Science.gov (United States)

    Pelosi, Lenore A; Voss, Stacey; Liu, Mengping; Gao, Min; Lemm, Julie A

    2012-10-01

    Three hepatitis C virus (HCV) inhibitors, asunaprevir (ASV; BMS-650032), daclatasvir (DCV; BMS-790052), and BMS-791325, each targeting a different nonstructural protein of the virus (NS3, NS5A, and NS5B, respectively), have independently demonstrated encouraging preclinical profiles and are currently undergoing clinical evaluation. Since drug-resistant variants have rapidly developed in response to monotherapy with almost all direct-acting antiviral agents (DAAs) for HCV, the need for combination therapies to effectively eradicate the virus from infected patients is clear. These studies demonstrated the additive-synergistic effects on replicon inhibition and clearance of combining NS3 protease or NS5B RNA polymerase inhibitors with the first-in-class, NS5A replication complex inhibitor daclatasvir (DCV) and reveal new resistance pathways for combinations of two small-molecule inhibitors that differ from those that develop during monotherapy. The results suggest that under a specific selective pressure, a balance must be reached in the fitness costs of substitutions in one target gene when substitutions are also present in another target gene. Further synergies and additional novel resistance substitutions were observed during triple-combination treatment relative to dual-drug therapy, indicating that, in combination, HCV inhibitors can exert cross-target influences on resistance development. Enhanced synergies in replicon inhibition and a reduced frequency of resistance together lend strong support to the utility of combinations of DAAs for the treatment of HCV, and the identification of altered resistance profiles during combination treatment provides useful information for monitoring resistance in the clinic.

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

    Directory of Open Access Journals (Sweden)

    Jinhua Xiang

    2008-07-01

    Full Text Available 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.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.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.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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

    Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed......, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced...

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  7. Susceptibilities of Genotype 1a, 1b, and 3 Hepatitis C Virus Variants to the NS5A Inhibitor Elbasvir

    Science.gov (United States)

    Curry, Stephanie; McMonagle, Patricia; Yeh, Wendy W.; Ludmerer, Steven W.; Jumes, Patricia A.; Marshall, William L.; Kong, Stephanie; Ingravallo, Paul; Black, Stuart; Pak, Irene; DiNubile, Mark J.

    2015-01-01

    Elbasvir is an investigational NS5A inhibitor with in vitro activity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistance-associated variants (RAVs) in vitro in a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier NCT01532973.) PMID:26303801

  8. Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing.

    Science.gov (United States)

    Bergfors, Assar; Leenheer, Daniël; Bergqvist, Anders; Ameur, Adam; Lennerstrand, Johan

    2016-02-01

    Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naïve-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naïve patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. 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

  10. Susceptibilities of genotype 1a, 1b, and 3 hepatitis C virus variants to the NS5A inhibitor elbasvir.

    Science.gov (United States)

    Liu, Rong; Curry, Stephanie; McMonagle, Patricia; Yeh, Wendy W; Ludmerer, Steven W; Jumes, Patricia A; Marshall, William L; Kong, Stephanie; Ingravallo, Paul; Black, Stuart; Pak, Irene; DiNubile, Mark J; Howe, Anita Y M

    2015-11-01

    Elbasvir is an investigational NS5A inhibitor with in vitro activity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistance-associated variants (RAVs) in vitro in a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier NCT01532973.). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. 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.

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    Moon Y F Tay

    2016-09-01

    Full Text Available 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.

  12. 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.

  13. Asymmetric binding to NS5A by daclatasvir (BMS-790052) and analogs suggests two novel modes of HCV inhibition.

    Science.gov (United States)

    Nettles, James H; Stanton, Richard A; Broyde, Joshua; Amblard, Franck; Zhang, Hongwang; Zhou, Longhu; Shi, Junxing; McBrayer, Tamara R; Whitaker, Tony; Coats, Steven J; Kohler, James J; Schinazi, Raymond F

    2014-12-11

    Symmetric, dimeric daclatasvir (BMS-790052) is the clinical lead for a class of picomolar inhibitors of HCV replication. While specific, resistance-bearing mutations at positions 31 and 93 of domain I strongly suggest the viral NS5A as target, structural mechanism(s) for the drugs' activities and resistance remains unclear. Several previous models suggested symmetric binding modes relative to the homodimeric target; however, none can fully explain SAR details for this class. We present semiautomated workflows to model potential receptor conformations for docking. Surprisingly, ranking docked hits with our library-derived 3D-pharmacophore revealed two distinct asymmetric binding modes, at a conserved poly-proline region between 31 and 93, consistent with SAR. Interfering with protein-protein interactions at this membrane interface can explain potent inhibition of replication-complex formation, resistance, effects on lipid droplet distribution, and virion release. These detailed interaction models and proposed mechanisms of action will allow structure-based design of new NS5A directed compounds with higher barriers to HCV resistance.

  14. Hepatitis C Virus NS5A Protein Down-regulates the Expression of Spindle Gene Aspm through PKR-p38 Signaling Pathway*S⃞

    Science.gov (United States)

    Wu, Shun-Chi; Chang, Shin C.; Wu, Hung-Yi; Liao, Pei-Ju; Chang, Ming-Fu

    2008-01-01

    Hepatitis C virus often causes persistent infection and hepatocellular carcinoma. Studies have demonstrated the roles of viral nonstructural protein 5A (NS5A) in the induction of chromosome aneuploidy, but the molecular mechanisms are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by laser capture microdissection. Gene expression profiles of the NS5A-expressing hepatocytes were examined by an Affymetrix oligonucleotide microarray system. Aspm (abnormal spindle-like, microcephaly associated), which encodes the mitotic spindle protein ASPM, was identified to be differentially expressed in the absence and the presence of NS5A. The down-regulation of Aspm mRNA and ASPM protein was confirmed by real time polymerase chain reaction and Western blot analysis, respectively, both in mouse model systems and in viral subgenomic replicon and in vitro transfection culturing systems. In addition, cultured cells that constitutively expressed NS5A protein showed G2/M cell cycle block and chromosome aneuploidy. Overexpression of ASPM relieved the G2/M cell cycle block. Furthermore, NS5A protein repressed the promoter activity of Aspm gene in a dose-dependent manner. The regulatory effect was abolished when amino acid substitutions P2209L, T2214A, and T2217G known to interrupt the NS5A-PKR interaction were introduced into the NS5A protein. This indicates that the down-regulation of Aspm expression is via the PKR-p38 signaling pathway. These results suggest that NS5A protein down-regulates the expression of the mitotic spindle protein ASPM and induces aberrant mitotic cell cycle associated with chromosome instability and hepatocellular carcinoma. PMID:18728014

  15. Hepatitis C virus NS5A protein down-regulates the expression of spindle gene Aspm through PKR-p38 signaling pathway.

    Science.gov (United States)

    Wu, Shun-Chi; Chang, Shin C; Wu, Hung-Yi; Liao, Pei-Ju; Chang, Ming-Fu

    2008-10-24

    Hepatitis C virus often causes persistent infection and hepatocellular carcinoma. Studies have demonstrated the roles of viral nonstructural protein 5A (NS5A) in the induction of chromosome aneuploidy, but the molecular mechanisms are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by laser capture microdissection. Gene expression profiles of the NS5A-expressing hepatocytes were examined by an Affymetrix oligonucleotide microarray system. Aspm (abnormal spindle-like, microcephaly associated), which encodes the mitotic spindle protein ASPM, was identified to be differentially expressed in the absence and the presence of NS5A. The down-regulation of Aspm mRNA and ASPM protein was confirmed by real time polymerase chain reaction and Western blot analysis, respectively, both in mouse model systems and in viral subgenomic replicon and in vitro transfection culturing systems. In addition, cultured cells that constitutively expressed NS5A protein showed G(2)/M cell cycle block and chromosome aneuploidy. Overexpression of ASPM relieved the G(2)/M cell cycle block. Furthermore, NS5A protein repressed the promoter activity of Aspm gene in a dose-dependent manner. The regulatory effect was abolished when amino acid substitutions P2209L, T2214A, and T2217G known to interrupt the NS5A-PKR interaction were introduced into the NS5A protein. This indicates that the down-regulation of Aspm expression is via the PKR-p38 signaling pathway. These results suggest that NS5A protein down-regulates the expression of the mitotic spindle protein ASPM and induces aberrant mitotic cell cycle associated with chromosome instability and hepatocellular carcinoma.

  16. A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A.

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    C T Ranjith-Kumar

    Full Text Available RNA synthesis by the genotype 1b hepatitis C virus (HCV polymerase (NS5B transiently expressed in Human embryonic kidney 293T cells or liver hepatocytes was found to robustly stimulate RIG-I-dependent luciferase production from the interferon β promoter in the absence of exogenously provided ligand. This cell-based assay, henceforth named the 5BR assay, could be used to examine HCV polymerase activity in the absence of other HCV proteins. Mutations that decreased de novo initiated RNA synthesis in biochemical assays decreased activation of RIG-I signaling. In addition, NS5B that lacks the C-terminal transmembrane helix but remains competent for RNA synthesis could activate RIG-I signaling. The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling. Furthermore, non-nucleoside inhibitor benzothiadiazines (BTDs that bind within the template channel of the 1b NS5B were found to inhibit the readout from the 5BR assay. Mutation M414T in NS5B that rendered the HCV replicon resistant to BTD was also resistant to BTDs in the 5BR assay. Co-expression of the HCV NS5A protein along with NS5B and RIG-I was found to inhibit the readout from the 5BR assay. The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B. Lastly, NS5B from all six major HCV genotypes showed robust activation of RIG-I in the 5BR assay. In summary, the 5BR assay could be used to validate inhibitors of the HCV polymerase as well as to elucidate requirements for HCV-dependent RNA synthesis.

  17. Discovery of Ruzasvir (MK-8408): A Potent, Pan-Genotype HCV NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Polymorphisms.

    Science.gov (United States)

    Tong, Ling; Yu, Wensheng; Chen, Lei; Selyutin, Oleg; Dwyer, Michael P; Nair, Anilkumar G; Mazzola, Robert; Kim, Jae-Hun; Sha, Deyou; Yin, Jingjun; Ruck, Rebecca T; Davies, Ian W; Hu, Bin; Zhong, Bin; Hao, Jinglai; Ji, Tao; Zan, Shuai; Liu, Rong; Agrawal, Sony; Xia, Ellen; Curry, Stephanie; McMonagle, Patricia; Bystol, Karin; Lahser, Frederick; Carr, Donna; Rokosz, Laura; Ingravallo, Paul; Chen, Shiying; Feng, Kung-I; Cartwright, Mark; Asante-Appiah, Ernest; Kozlowski, Joseph A

    2017-01-12

    We describe the research that led to the discovery of compound 40 (ruzasvir, MK-8408), a pan-genotypic HCV nonstructural protein 5A (NS5A) inhibitor with a "flat" GT1 mutant profile. This NS5A inhibitor contains a unique tetracyclic indole core while maintaining the imidazole-proline-valine Moc motifs of our previous NS5A inhibitors. Compound 40 is currently in early clinical trials and is under evaluation as part of an all-oral DAA regimen for the treatment of chronic HCV infection.

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

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    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

  19. Characterization of NS5A polymorphisms and their impact on response rates in patients with HCV genotype 2 treated with daclatasvir-based regimens.

    Science.gov (United States)

    Zhou, Nannan; Han, Zhou; Hartman-Neumann, Sandra; DeGray, Brenda; Ueland, Joseph; Vellucci, Vincent; Hernandez, Dennis; McPhee, Fiona

    2016-12-01

    Daclatasvir (DCV) is a pan-genotypic non-structural protein 5A (NS5A) inhibitor that is approved for treatment of hepatitis C virus (HCV) genotype (GT)1 and GT3 in the USA and GT1, GT3 and GT4 in Europe. We set out to examine the impact of daclatasvir-based regimens on the sustained virologic response (SVR) in patients with GT2 infection with respect to GT2 subtype and NS5A polymorphisms at amino acid positions associated with daclatasvir resistance. Analyses were performed on 283 GT2 NS5A sequences from five daclatasvir regimen-based clinical trials (ClinicalTrials.gov: NCT-01257204, NCT-01359644, NCT-02032875, NCT-02032888 and NCT-01616524) and 143 NS5A sequences from the Los Alamos HCV database. Susceptibility analyses of substitutions at amino acid positions associated with daclatasvir resistance and patient-derived NS5A sequences were performed using an in vitro HCV replication assay. Of 13 GT2 subtypes identified from 426 NS5A sequences, the most prevalent were GT2a (32%), GT2b (48%) and GT2c (10%). The most prevalent NS5A polymorphism was L31M (GT2a = 88%; GT2b = 59%; GT2c = 10%). Substitutions identified in 96% of GT2 NS5A sequences exhibited daclatasvir EC50 values ranging from 0.005 to 20 nM when tested in vitro. A similar range in daclatasvir EC50 values was observed for 16 diverse GT2 patient-derived NS5A sequences (EC50 = 0.005-60 nM). Depending on the daclatasvir-based regimen studied (daclatasvir/interferon-based or daclatasvir/sofosbuvir-based), SVR rates ranged from 90% to 100% in GT2 patients with the most prevalent baseline NS5A-L31M polymorphism, compared with from 96% to 100% without this polymorphism. High SVR rates were achieved in patients infected with GT2 treated with daclatasvir-based regimens irrespective of GT2 subtype or baseline NS5A polymorphisms. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e

  20. Small-molecule inhibitors of hepatitis C virus (HCV) non-structural protein 5A (NS5A): a patent review (2010-2015).

    Science.gov (United States)

    Ivanenkov, Yan A; Aladinskiy, Vladimir A; Bushkov, Nikolay A; Ayginin, Andrey A; Majouga, Alexander G; Ivachtchenko, Alexandre V

    2017-04-01

    Non-structural 5A (NS5A) protein has achieved a considerable attention as an attractive target for the treatment of hepatitis C (HCV). A number of novel NS5A inhibitors have been reported to date. Several drugs having favorable ADME properties and mild side effects were launched into the pharmaceutical market. For instance, daclatasvir was launched in 2014, elbasvir is currently undergoing registration, ledipasvir was launched in 2014 as a fixed-dose combination with sofosbuvir (NS5B inhibitor). Areas covered: Thomson integrity database and SciFinder database were used as a valuable source to collect the patents on small-molecule NS5A inhibitors. All the structures were ranked by the date of priority. Patent holder and antiviral activity for each scaffold claimed were summarized and presented in a convenient manner. A particular focus was placed on the best-in-class bis-pyrrolidine-containing NS5A inhibitors. Expert opinion: Several first generation NS5A inhibitors have recently progressed into advanced clinical trials and showed superior efficacy in reducing viral load in infected subjects. Therapy schemes of using these agents in combination with other established antiviral drugs with complementary mechanisms of action can address the emergence of resistance and poor therapeutic outcome frequently attributed to antiviral drugs.

  1. Hepatitis C Virus NS5A Protein Triggers Oxidative Stress by Inducing NADPH Oxidases 1 and 4 and Cytochrome P450 2E1

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    Olga A. Smirnova

    2016-01-01

    Full Text Available Replication of hepatitis C virus (HCV is associated with the induction of oxidative stress, which is thought to play a major role in various liver pathologies associated with chronic hepatitis C. NS5A protein of the virus is one of the two key viral proteins that are known to trigger production of reactive oxygen species (ROS. To date it has been considered that NS5A induces oxidative stress by altering calcium homeostasis. Herein we show that NS5A-induced oxidative stress was only moderately inhibited by the intracellular calcium chelator BAPTA-AM and not at all inhibited by the drug that blocks the Ca2+ flux from ER to mitochondria. Furthermore, ROS production was not accompanied by induction of ER oxidoreductins (Ero1, H2O2-producing enzymes that are implicated in the regulation of calcium fluxes. Instead, we found that NS5A contributes to ROS production by activating expression of NADPH oxidases 1 and 4 as well as cytochrome P450 2E1. These effects were mediated by domain I of NS5A protein. NOX1 and NOX4 induction was mediated by enhanced production of transforming growth factor β1 (TGFβ1. Thus, our data show that NS5A protein induces oxidative stress by several multistep mechanisms.

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

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    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

  3. Daclatasvir: the first of a new class of drugs targeted against hepatitis C virus NS5A.

    Science.gov (United States)

    Gentile, I; Borgia, F; Coppola, N; Buonomo, A R; Castaldo, G; Borgia, G

    2014-01-01

    Hepatitis C virus (HCV) infection affects about 160 million people worldwide. It is treated with pegylatedinterferon (peg-IFN) and ribavirin, and in the case of patients affected by genotype 1, also with a protease inhibitor (telaprevir or boceprevir). Despite a good success rate, IFN-based combinations are contraindicated in several patients (e.g. decompensated cirrhosis, patients with psychiatric disorders, severe heart diseases or autoimmune disorders) and are associated with frequent adverse events that ultimately reduce their use. Numerous oral drugs are in an advanced phase of clinical development, and in some cases, in IFN-free combinations. This review focuses on preclinical and clinical data regarding daclatasvir (BMS-790052), which is a highly selective HCV NS5A replication complex inhibitor effective against HCV genotypes 1, 2, 3 and 4. In vitro data show that daclatasvir exerts a very potent antiviral effect against several HCV genotypes. Its pharmacokinetics is optimal and allows once-a-day oral administration. Its adverse event profile is good. Clinical data regarding its efficacy in combination with peg-IFN, ribavirin or other direct antiviral agents are impressive (rates of sustained virological response range between 60% and 100% in treatment-naïve patients). The only drawback of this drug appears to be a relatively low genetic barrier to resistance. In conclusion, daclatasvir, especially in combinations with other antiviral agents, is a very promising drug for the treatment of chronic hepatitis C.

  4. A 96-well based analysis of replicon elimination with the HCV NS5A replication complex inhibitor daclatasvir.

    Science.gov (United States)

    O'Boyle, Donald R; Nower, Peter T; Sun, Jin-Hua; Fridell, Robert; Wang, Chunfu; Valera, Lourdes; Gao, Min

    2013-10-01

    A 96-well based replicon elimination and colony formation assay is presented for comparing the resistance barrier of the hepatitis C virus (HCV) NS5A replication complex inhibitor daclatasvir (DCV, BMS-790052) on three HCV genotypes (gts) in a proof of concept experimental protocol. The 96-well assay format provides both individual colony as well as population characterization and is readily applicable to other HCV direct-acting antiviral agents (DAAs). The assay provides an assessment of HCV replication levels over a 5log10 range by measuring a luciferase reporter resident in the HCV replicons. Individual colony status can be measured with a separate and compatible resazurin assay to assess relative host cell fitness following inhibitor treatments. The methods employed are non-toxic and leave intact isolatable colonies that can be used for phenotyping and genotyping. The utility of the assay is demonstrated by the identification and isolation of resistant variants as well as in the ranking of the relative resistance barrier for the replication complex inhibitor DCV for gts 1a, 1b and 2a. The format provides a quantitative ranking based upon luciferase activity and has the ability to monitor DAA resistance development over time for large numbers of compounds. Copyright © 2013. Published by Elsevier B.V.

  5. QSAR study of HCV NS5B polymerase inhibitors using the genetic algorithm-multiple linear regression (GA-MLR).

    Science.gov (United States)

    Rafiei, Hamid; Khanzadeh, Marziyeh; Mozaffari, Shahla; Bostanifar, Mohammad Hassan; Avval, Zhila Mohajeri; Aalizadeh, Reza; Pourbasheer, Eslam

    2016-01-01

    Quantitative structure-activity relationship (QSAR) study has been employed for predicting the inhibitory activities of the Hepatitis C virus (HCV) NS5B polymerase inhibitors . A data set consisted of 72 compounds was selected, and then different types of molecular descriptors were calculated. The whole data set was split into a training set (80 % of the dataset) and a test set (20 % of the dataset) using principle component analysis. The stepwise (SW) and the genetic algorithm (GA) techniques were used as variable selection tools. Multiple linear regression method was then used to linearly correlate the selected descriptors with inhibitory activities. Several validation technique including leave-one-out and leave-group-out cross-validation, Y-randomization method were used to evaluate the internal capability of the derived models. The external prediction ability of the derived models was further analyzed using modified r(2), concordance correlation coefficient values and Golbraikh and Tropsha acceptable model criteria's. Based on the derived results (GA-MLR), some new insights toward molecular structural requirements for obtaining better inhibitory activity were obtained.

  6. Label Free Inhibitor Screening of Hepatitis C Virus (HCV NS5B Viral Protein Using RNA Oligonucleotide

    Directory of Open Access Journals (Sweden)

    Sang Eun Kim

    2011-06-01

    Full Text Available Globally, over 170 million people (ca. 3% of the World’s population are infected with the hepatitis C virus (HCV, which can cause serious liver diseases such as chronic hepatitis, evolving into subsequent health problems. Driven by the need to detect the presence of HCV, as an essential factor in diagnostic medicine, the monitoring of viral protein has been of great interest in developing simple and reliable HCV detection methods. Despite considerable advances in viral protein detection as an HCV disease marker, the current enzyme linked immunosorbent assay (ELISA based detection methods using antibody treatment have several drawbacks. To overcome this bottleneck, an RNA aptamer become to be emerged as an antibody substitute in the application of biosensor for detection of viral protein. In this study, we demonstrated a streptavidin-biotin conjugation method, namely, the RNA aptamer sensor system that can quantify viral protein with detection level of 700 pg mL−1 using a biotinylated RNA oligonucleotide on an Octet optical biosensor. Also, we showed this method can be used to screen inhibitors of viral protein rapidly and simply on a biotinylated RNA oligonucleotide biosensor. Among the inhibitors screened, (−-Epigallocatechin gallate showed high binding inhibition effect on HCV NS5B viral protein. The proposed method can be considered a real-time monitoring method for inhibitor screening of HCV viral protein and is expected to be applicable to other types of diseases.

  7. Discovery of novel dengue virus NS5 methyltransferase non-nucleoside inhibitors by fragment-based drug design.

    Science.gov (United States)

    Benmansour, Fatiha; Trist, Iuni; Coutard, Bruno; Decroly, Etienne; Querat, Gilles; Brancale, Andrea; Barral, Karine

    2017-01-05

    With the aim to help drug discovery against dengue virus (DENV), a fragment-based drug design approach was applied to identify ligands targeting a main component of DENV replication complex: the NS5 AdoMet-dependent mRNA methyltransferase (MTase) domain, playing an essential role in the RNA capping process. Herein, we describe the identification of new inhibitors developed using fragment-based, structure-guided linking and optimization techniques. Thermal-shift assay followed by a fragment-based X-ray crystallographic screening lead to the identification of three fragment hits binding DENV MTase. We considered linking two of them, which bind to proximal sites of the AdoMet binding pocket, in order to improve their potency. X-ray crystallographic structures and computational docking were used to guide the fragment linking, ultimately leading to novel series of non-nucleoside inhibitors of flavivirus MTase, respectively N-phenyl-[(phenylcarbamoyl)amino]benzene-1-sulfonamide and phenyl [(phenylcarbamoyl)amino]benzene-1-sulfonate derivatives, that show a 10-100-fold stronger inhibition of 2'-O-MTase activity compared to the initial fragments. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. Preclinical Profile and Clinical Efficacy of a Novel Hepatitis C Virus NS5A Inhibitor, EDP-239.

    Science.gov (United States)

    Owens, Christopher M; Brasher, Bradley B; Polemeropoulos, Alex; Rhodin, Michael H J; McAllister, Nicole; Peng, Xiaowen; Wang, Ce; Ying, Lu; Cao, Hui; Lawitz, Eric; Poordad, Fred; Rondon, Juan; Box, Terry D; Zeuzem, Stefan; Buggisch, Peter; Lin, Kai; Qiu, Yao-Ling; Jiang, Lijuan; Colvin, Richard; Or, Yat Sun

    2016-10-01

    EDP-239, a novel hepatitis C virus (HCV) inhibitor targeting nonstructural protein 5A (NS5A), has been investigated in vitro and in vivo EDP-239 is a potent, selective inhibitor with potency at picomolar to nanomolar concentrations against HCV genotypes 1 through 6. In the presence of human serum, the potency of EDP-239 was reduced by less than 4-fold. EDP-239 is additive to synergistic with other direct-acting antivirals (DAAs) or host-targeted antivirals (HTAs) in blocking HCV replication and suppresses the selection of resistance in vitro Furthermore, EDP-239 retains potency against known DAA- or HTA-resistant variants, with half-maximal effective concentrations (EC50s) equivalent to those for the wild type. In a phase I, single-ascending-dose, placebo-controlled clinical trial, EDP-239 demonstrated excellent pharmacokinetic properties that supported once daily dosing. A single 100-mg dose of EDP-239 resulted in reductions in HCV genotype 1a viral RNA of >3 log10 IU/ml within the first 48 h after dosing and reductions in genotype 1b viral RNA of >4-log10 IU/ml within 96 h. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Long term persistence of NS5A inhibitor-resistant hepatitis C virus in patients who failed daclatasvir and asunaprevir therapy.

    Science.gov (United States)

    Yoshimi, Satoshi; Imamura, Michio; Murakami, Eisuke; Hiraga, Nobuhiko; Tsuge, Masataka; Kawakami, Yoshiiku; Aikata, Hiroshi; Abe, Hiromi; Hayes, C Nelson; Sasaki, Tamito; Ochi, Hidenori; Chayama, Kazuaki

    2015-11-01

    Although interferon-free antiviral treatment is expected to improve treatment of hepatitis C, it is unclear to what extent pre-existing drug-resistant amino acid substitutions influence response to therapy. The impact of pre-existing drug-resistant substitutions on virological response to daclatasvir and asunaprevir combination therapy was studied in genotype 1b hepatitis C virus (HCV)-infected patients. Thirty-one patients were treated with daclatasvir and asunaprevir for 24 weeks. Twenty-six patients achieved sustained virological response (SVR), three patients experienced viral breakthrough, and two patients relapsed. Direct sequencing analysis of HCV showed the existence of daclatasvir-resistant NS5A-L31M or -Y93H/F variants in nine out of 30 patients (30%) prior to treatment, while asunaprevir-resistant NS3-D168 mutations were not detected in any patient. All 21 patients with wild-type NS5A-L31 and -Y93 achieved SVR, whereas only four out of nine patients (44%) with L31M or Y93F/H substitutions achieved SVR (P = 0.001). Ultra-deep sequencing analysis showed that treatment failure was associated with the emergence of both NS5A-L31/Y93 and NS3-D168 variants. NS5A-L31/Y93 variants remained at high frequency through post-treatment weeks 103 through 170, while NS3-D168 variants were replaced by wild-type in all patients. In conclusion, pre-existence of NS5A inhibitor-resistant substitutions compromised the response to daclatasvir and asunaprevir combination therapy, and treatment failure was associated with the emergence of both NS5A-L31/Y93 and NS3-D168 variants. While asunaprevir-resistant variants that emerged during therapy returned to wild-type, daclatasvir-resistant variants tended to persist in the absence of the drug. © 2015 Wiley Periodicals, Inc.

  10. NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome.

    Science.gov (United States)

    Zeuzem, Stefan; Mizokami, Masashi; Pianko, Stephen; Mangia, Alessandra; Han, Kwang-Hyub; Martin, Ross; Svarovskaia, Evguenia; Dvory-Sobol, Hadas; Doehle, Brian; Hedskog, Charlotte; Yun, Chohee; Brainard, Diana M; Knox, Steven; McHutchison, John G; Miller, Michael D; Mo, Hongmei; Chuang, Wan-Long; Jacobson, Ira; Dore, Gregory J; Sulkowski, Mark

    2017-05-01

    The efficacy of NS5A inhibitors for the treatment of patients chronically infected with hepatitis C virus (HCV) can be affected by the presence of NS5A resistance-associated substitutions (RASs). We analyzed data from 35 phase I, II, and III studies in 22 countries to determine the pretreatment prevalence of various NS5A RASs, and their effect on outcomes of treatment with ledipasvir-sofosbuvir in patients with genotype 1 HCV. NS5A gene deep sequencing analysis was performed on samples from 5397 patients in Gilead clinical trials. The effect of baseline RASs on sustained virologic response (SVR) rates was assessed in the 1765 patients treated with regimens containing ledipasvir-sofosbuvir. Using a 15% cut-off, pretreatment NS5A and ledipasvir-specific RASs were detected in 13% and 8% of genotype 1a patients, respectively, and in 18% and 16% of patients with genotype 1b. Among genotype 1a treatment-naïve patients, SVR rates were 91% (42/46) vs. 99% (539/546) for those with and without ledipasvir-specific RASs, respectively. Among treatment-experienced genotype 1a patients, SVR rates were 76% (22/29) vs. 97% (409/420) for those with and without ledipasvir-specific RASs, respectively. Among treatment-naïve genotype 1b patients, SVR rates were 99% for both those with and without ledipasvir-specific RASs (71/72 vs. 331/334), and among treatment-experienced genotype 1b patients, SVR rates were 89% (41/46) vs. 98% (267/272) for those with and without ledipasvir-specific RASs, respectively. Pretreatment ledipasvir-specific RASs that were present in 8-16% of patients have an impact on treatment outcome in some patient groups, particularly treatment-experienced patients with genotype 1a HCV. The efficacy of treatments using NS5A inhibitors for patients with chronic hepatitis C virus (HCV) infection can be affected by the presence of NS5A resistance-associated substitutions (RASs). We reviewed results from 35 clinical trials where patients with genotype 1 HCV infection

  11. High-Resolution Hepatitis C Virus Subtyping Using NS5B Deep Sequencing and Phylogeny, an Alternative to Current Methods

    Science.gov (United States)

    Gregori, Josep; Rodríguez-Frias, Francisco; Buti, Maria; Madejon, Antonio; Perez-del-Pulgar, Sofia; Garcia-Cehic, Damir; Casillas, Rosario; Blasi, Maria; Homs, Maria; Tabernero, David; Alvarez-Tejado, Miguel; Muñoz, Jose Manuel; Cubero, Maria; Caballero, Andrea; delCampo, Jose Antonio; Domingo, Esteban; Belmonte, Irene; Nieto, Leonardo; Lens, Sabela; Muñoz-de-Rueda, Paloma; Sanz-Cameno, Paloma; Sauleda, Silvia; Bes, Marta; Gomez, Jordi; Briones, Carlos; Perales, Celia; Sheldon, Julie; Castells, Lluis; Viladomiu, Lluis; Salmeron, Javier; Ruiz-Extremera, Angela; Quiles-Pérez, Rosa; Moreno-Otero, Ricardo; López-Rodríguez, Rosario; Allende, Helena; Romero-Gómez, Manuel; Guardia, Jaume; Esteban, Rafael; Garcia-Samaniego, Javier; Forns, Xavier

    2014-01-01

    Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes. PMID:25378574

  12. Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus.

    Science.gov (United States)

    Bollati, Michela; Milani, Mario; Mastrangelo, Eloise; Ricagno, Stefano; Tedeschi, Gabriella; Nonnis, Simona; Decroly, Etienne; Selisko, Barbara; de Lamballerie, Xavier; Coutard, Bruno; Canard, Bruno; Bolognesi, Martino

    2009-01-09

    The mRNA-capping process starts with the conversion of a 5'-triphosphate end into a 5'-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5'-5' phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2'OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2'O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, (N7Me)GpppG, and (N7Me)GpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2'O-methyltransferase activities.

  13. Addition of ribavirin to daclatasvir plus asunaprevir for chronic hepatitis C 1b patients with baseline NS5A resistance-associated variants improved response

    Directory of Open Access Journals (Sweden)

    Chun-Ming Hong

    2017-04-01

    Conclusion: In genotype 1b chronic hepatitis C patients with NS5A-Y93H polymorphism, the addition of ribavirin to daclatasvir/asunaprevir may increase the SVR12 rate with minimal side effects, and thus deserves more comprehensive trials in resource-limited areas.

  14. 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.

  15. The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

    Science.gov (United States)

    Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

    2017-12-07

    Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Discovery of Novel Hepatitis C Virus NS5B Polymerase Inhibitors by Combining Random Forest, Multiple e-Pharmacophore Modeling and Docking.

    Directory of Open Access Journals (Sweden)

    Yu Wei

    Full Text Available The NS5B polymerase is one of the most attractive targets for developing new drugs to block Hepatitis C virus (HCV infection. We describe the discovery of novel potent HCV NS5B polymerase inhibitors by employing a virtual screening (VS approach, which is based on random forest (RB-VS, e-pharmacophore (PB-VS, and docking (DB-VS methods. In the RB-VS stage, after feature selection, a model with 16 descriptors was used. In the PB-VS stage, six energy-based pharmacophore (e-pharmacophore models from different crystal structures of the NS5B polymerase with ligands binding at the palm I, thumb I and thumb II regions were used. In the DB-VS stage, the Glide SP and XP docking protocols with default parameters were employed. In the virtual screening approach, the RB-VS, PB-VS and DB-VS methods were applied in increasing order of complexity to screen the InterBioScreen database. From the final hits, we selected 5 compounds for further anti-HCV activity and cellular cytotoxicity assay. All 5 compounds were found to inhibit NS5B polymerase with IC50 values of 2.01-23.84 μM and displayed anti-HCV activities with EC50 values ranging from 1.61 to 21.88 μM, and all compounds displayed no cellular cytotoxicity (CC50 > 100 μM except compound N2, which displayed weak cytotoxicity with a CC50 value of 51.3 μM. The hit compound N2 had the best antiviral activity against HCV, with a selective index of 32.1. The 5 hit compounds with new scaffolds could potentially serve as NS5B polymerase inhibitors through further optimization and development.

  17. Structural and functional analysis of methylation and 5'-RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5.

    Science.gov (United States)

    Egloff, Marie-Pierre; Decroly, Etienne; Malet, Hélène; Selisko, Barbara; Benarroch, Delphine; Ferron, François; Canard, Bruno

    2007-09-21

    The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase(DV), is involved in two of four steps required for the formation of the viral mRNA cap (7Me)GpppA(2'OMe), the guanine-N7 and the adenosine-2'O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2'O-methyltransferase (MTase) activity has been shown on capped (7Me+/-)GpppAC(n) RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 A to 2.8 A resolution of NS5MTase(DV) in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of (7Me)GpppAC(4) and (7Me)GpppAC(5) RNAs is inhibited in the presence of GTP, (7Me)GTP and (7Me)GpppA but not by ATP. This particular position of the cap is in accordance with the 2'O-methylation step. A model was generated of a ternary 2'O-methylation complex of NS5MTase(DV), (7Me)GpppA and AdoMet. RNA-binding increased when (7Me+/-)GpppAGC(n-1) starting with the consensus sequence GpppAG, was used instead of (7Me+/-)GpppAC(n). In the NS5MTase(DV)-GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5' end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2'O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.

  18. A Refined Model of the HCV NS5A protein bound to daclatasvir explains drug-resistant mutations and activity against divergent genotypes.

    Science.gov (United States)

    Barakat, Khaled H; Anwar-Mohamed, Anwar; Tuszynski, Jack A; Robins, Morris J; Tyrrell, D Lorne; Houghton, Michael

    2015-02-23

    Many direct-acting antiviral agents (DAAs) that selectively block hepatitis C virus (HCV) replication are currently under development. Among these agents is Daclatasvir, a first-in-class inhibitor targeting the NS5A viral protein. Although Daclatasvir is the most potent HCV antiviral molecule yet developed, its binding location and mode of binding remain unknown. The drug exhibits a low barrier to resistance mutations, particularly in genotype 1 viruses, but its efficacy against other genotypes is unclear. Using state-of-the-art modeling techniques combined with the massive computational power of Blue Gene/Q, we identified the atomic interactions of Daclatasvir within NS5A for different HCV genotypes and for several reported resistant mutations. The proposed model is the first to reveal the detailed binding mode of Daclatasvir. It also provides a tool to facilitate design of second generation drugs, which may confer less resistance and/or broader activity against HCV.

  19. Aza follow-ups to BI 207524, a thumb pocket 1 HCV NS5B polymerase inhibitor. Part 1: Mitigating the genotoxic liability of an aniline metabolite.

    Science.gov (United States)

    Beaulieu, Pierre L; Bolger, Gordon; Duplessis, Martin; Gagnon, Alexandre; Garneau, Michel; Stammers, Timothy; Kukolj, George; Duan, Jianmin

    2015-03-01

    A series of heterocyclic aza-analogs of BI 207524 (2), a potent HCV NS5B polymerase thumb pocket 1 inhibitor, was investigated with the goal to reduce the liability associated with the release of a genotoxic aniline metabolite in vivo. Analog 4, containing a 2-aminopyridine aniline isostere that is negative in the Ames test was identified, and was found to provide comparable GT1a/1b potency to 2. Although the cross-species PK profile, poor predicted human liver distribution of analog 4 and allometry principles projected high doses to achieve a strong antiviral response in patients, this work has provided a path forward toward the design of novel thumb pocket 1 NS5B polymerase inhibitors with improved safety profiles. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. 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

    mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy...... 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......With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi...

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

    Directory of Open Access Journals (Sweden)

    Anirban Mandal

    2016-01-01

    Full Text Available 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.

  2. Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life.

    Science.gov (United States)

    Guedj, Jeremie; Dahari, Harel; Rong, Libin; Sansone, Natasha D; Nettles, Richard E; Cotler, Scott J; Layden, Thomas J; Uprichard, Susan L; Perelson, Alan S

    2013-03-05

    The nonstructural 5A (NS5A) protein is a target for drug development against hepatitis C virus (HCV). Interestingly, the NS5A inhibitor daclatasvir (BMS-790052) caused a decrease in serum HCV RNA levels by about two orders of magnitude within 6 h of administration. However, NS5A has no known enzymatic functions, making it difficult to understand daclatasvir's mode of action (MOA) and to estimate its antiviral effectiveness. Modeling viral kinetics during therapy has provided important insights into the MOA and effectiveness of a variety of anti-HCV agents. Here, we show that understanding the effects of daclatasvir in vivo requires a multiscale model that incorporates drug effects on the HCV intracellular lifecycle, and we validated this approach with in vitro HCV infection experiments. The model predicts that daclatasvir efficiently blocks two distinct stages of the viral lifecycle, namely viral RNA synthesis and virion assembly/secretion with mean effectiveness of 99% and 99.8%, respectively, and yields a more precise estimate of the serum HCV half-life, 45 min, i.e., around four times shorter than previous estimates. Intracellular HCV RNA in HCV-infected cells treated with daclatasvir and the HCV polymerase inhibitor NM107 showed a similar pattern of decline. However, daclatasvir treatment led to an immediate and rapid decline of extracellular HCV titers compared to a delayed (6-9 h) and slower decline with NM107, confirming an effect of daclatasvir on both viral replication and assembly/secretion. The multiscale modeling approach, validated with in vitro kinetic experiments, brings a unique conceptual framework for understanding the mechanism of action of a variety of agents in development for the treatment of HCV.

  3. Hepatitis C virus (HCV evades NKG2D-dependent NK cell responses through NS5A-mediated imbalance of inflammatory cytokines.

    Directory of Open Access Journals (Sweden)

    Damien Sène

    Full Text Available Understanding how hepatitis C virus (HCV induces and circumvents the host's natural killer (NK cell-mediated immunity is of critical importance in efforts to design effective therapeutics. We report here the decreased expression of the NKG2D activating receptor as a novel strategy adopted by HCV to evade NK-cell mediated responses. We show that chronic HCV infection is associated with expression of ligands for NKG2D, the MHC class I-related Chain (MIC molecules, on hepatocytes. However, NKG2D expression is downmodulated on circulating NK cells, and consequently NK cell-mediated cytotoxic capacity and interferon-γ production are impaired. Using an endotoxin-free recombinant NS5A protein, we show that NS5A stimulation of monocytes through Toll-like Receptor 4 (TLR4 promotes p38- and PI3 kinase-dependent IL-10 production, while inhibiting IL-12 production. In turn, IL-10 triggers secretion of TGFβ which downmodulates NKG2D expression on NK cells, leading to their impaired effector functions. Moreover, culture supernatants of HCV JFH1 replicating Huh-7.5.1 cells reproduce the effect of recombinant NS5A on NKG2D downmodulation. Exogenous IL-15 can antagonize the TGFβ effect and restore normal NKG2D expression on NK cells. We conclude that NKG2D-dependent NK cell functions are modulated during chronic HCV infection, and demonstrate that this alteration can be prevented by exogenous IL-15, which could represent a meaningful adjuvant for therapeutic intervention.

  4. Natural non-homologous recombination led to the emergence of a duplicated V3-NS5A region in HCV-1b strains associated with hepatocellular carcinoma.

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    Hélène Le Guillou-Guillemette

    Full Text Available The emergence of new strains in RNA viruses is mainly due to mutations or intra and inter-genotype homologous recombination. Non-homologous recombinations may be deleterious and are rarely detected. In previous studies, we identified HCV-1b strains bearing two tandemly repeated V3 regions in the NS5A gene without ORF disruption. This polymorphism may be associated with an unfavorable course of liver disease and possibly involved in liver carcinogenesis. Here we aimed at characterizing the origin of these mutant strains and identifying the evolutionary mechanism on which the V3 duplication relies.Direct sequencing of the entire NS5A and E1 genes was performed on 27 mutant strains. Quasispecies analyses in consecutive samples were also performed by cloning and sequencing the NS5A gene for all mutant and wild strains. We analyzed the mutant and wild-type sequence polymorphisms using Bayesian methods to infer the evolutionary history of and the molecular mechanism leading to the duplication-like event.Quasispecies were entirely composed of exclusively mutant or wild-type strains respectively. Mutant quasispecies were found to have been present since contamination and had persisted for at least 10 years. This V3 duplication-like event appears to have resulted from non-homologous recombination between HCV-1b wild-type strains around 100 years ago. The association between increased liver disease severity and these HCV-1b mutants may explain their persistence in chronically infected patients.These results emphasize the possible consequences of non-homologous recombination in the emergence and severity of new viral diseases.

  5. Evaluación de un péptido quimérico NS4/NS5 para la detección de anticuerpos contra VHC

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    lvonne Gómez; Milenen Hernández; Carlos Martínez; Marleby García; Antonio Melchor

    2001-01-01

    Para el diagnóstico del virus de la hepatitis C, se utilizan las pruebas de Elisa, debido a su sensibilidad y especificidad. Gran parte de estas pruebas se basa en el empleo de péptidos sintéticos y, en la actualidad, de péptidos quiméricos. En este estudio se sintetizó un péptido quimérico que comprende secuencias inmunodominantes de las regiones no estructurales NS4 y NS5 del virus de la hepatitis C. El péptido representativo de las dos secuencias está separado por un brazo espaciador de do...

  6. Addition of ribavirin to daclatasvir plus asunaprevir for chronic hepatitis C 1b patients with baseline NS5A resistance-associated variants improved response.

    Science.gov (United States)

    Hong, Chun-Ming; Liu, Chun-Jen; Yeh, Shiou-Hwei; Chen, Pei-Jer

    2017-04-01

    Daclatasvir is a nonstructural protein 5A inhibitor with potent activity against hepatitis C virus genotypes 1-6 in vitro, and asunaprevir is a nonstructural protein 3 protease inhibitor with activity against genotypes 1, 4, 5, and 6. Despite a 90% sustained virologic response (SVR) rate, the SVR rate in patients with baseline NS5A-L31/Y93H polymorphisms decreased to around 40%. Therefore, an alternative regimen under the consideration of cost-effectiveness would be important. Whether the addition of ribavirin could improve the SVR rate among this group of patients remains unknown and hence our case series was reported. For six adult chronic hepatitis C 1b patients with a pre-existing NS5A-Y93H (>20%) polymorphism, we added ribavirin (800 mg/d) to daclatasvir/asunaprevir for 24 weeks and followed through 12-weeks post-treatment. Four of these patients received interferon/ribavirin treatment before but relapsed, while the other two were naïve cases. Two of them had liver cirrhosis and one had hepatocellular carcinoma postcurative therapy. The primary efficacy end-point was undetectable hepatitis C virus RNA (hepatitis C virus RNA level ofdaclatasvir/asunaprevir may increase the SVR12 rate with minimal side effects, and thus deserves more comprehensive trials in resource-limited areas. Copyright © 2016. Published by Elsevier B.V.

  7. Discovery of a Hepatitis C Virus NS5B Replicase Palm Site Allosteric Inhibitor (BMS-929075) Advanced to Phase 1 Clinical Studies

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    Yeung, Kap-Sun; Beno, Brett R.; Parcella, Kyle; Bender, John A.; Grant-Young, Katherine A.; Nickel, Andrew; Gunaga, Prashantha; Anjanappa, Prakash; Bora, Rajesh Onkardas; Selvakumar, Kumaravel; Rigat, Karen; Wang, Ying-Kai; Liu, Mengping; Lemm, Julie; Mosure, Kathy; Sheriff, Steven; Wan, Changhong; Witmer, Mark; Kish, Kevin; Hanumegowda, Umesh; Zhuo, Xiaoliang; Shu, Yue-Zhong; Parker, Dawn; Haskell, Roy; Ng, Alicia; Gao, Qi; Colston, Elizabeth; Raybon, Joseph; Grasela, Dennis M.; Santone, Kenneth; Gao, Min; Meanwell, Nicholas A.; Sinz, Michael; Soars, Matthew G.; Knipe, Jay O.; Roberts, Susan B.; Kadow, John F.

    2017-05-04

    The hepatitis C virus (HCV) NS5B replicase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Inspired by the overlay of bound structures of three structurally distinct NS5B palm site allosteric inhibitors, the high-throughput screening hit anthranilic acid 4, the known benzofuran analogue 5, and the benzothiadiazine derivative 6, an optimization process utilizing the simple benzofuran template 7 as a starting point for a fragment growing approach was pursued. A delicate balance of molecular properties achieved via disciplined lipophilicity changes was essential to achieve both high affinity binding and a stringent targeted absorption, distribution, metabolism, and excretion profile. These efforts led to the discovery of BMS-929075 (37), which maintained ligand efficiency relative to early leads, demonstrated efficacy in a triple combination regimen in HCV replicon cells, and exhibited consistently high oral bioavailability and pharmacokinetic parameters across preclinical animal species. The human PK properties from the Phase I clinical studies of 37 were better than anticipated and suggest promising potential for QD administration.

  8. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH interaction with 3' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein

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    Chou Shih-Jie

    2009-04-01

    Full Text Available Abstract Replication of the Japanese encephalitis virus (JEV genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5 in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.

  9. Emergence of hepatitis C virus NS5A L31V plus Y93H variant upon treatment failure of daclatasvir and asunaprevir is relatively resistant to ledipasvir and NS5B polymerase nucleotide inhibitor GS-558093 in human hepatocyte chimeric mice.

    Science.gov (United States)

    Kai, Yugo; Hikita, Hayato; Tatsumi, Tomohide; Nakabori, Tasuku; Saito, Yoshinobu; Morishita, Naoki; Tanaka, Satoshi; Nawa, Takatoshi; Oze, Tsugiko; Sakamori, Ryotaro; Yakushijin, Takayuki; Hiramatsu, Naoki; Suemizu, Hiroshi; Takehara, Tetsuo

    2015-11-01

    Resistance-associated variants (RAVs) emerge at multiple positions spanning hepatitis C virus (HCV) NS3/4A and NS5A regions upon failure of asunaprevir/daclatasvir combination therapy. It has not been determined whether the emergence of such RAVs have an impact on re-treatment by a combination of ledipasvir and sofosbuvir, a potent regimen for HCV genotype 1 infection. TK-NOG human hepatocyte chimeric mice were inoculated with sera from a patient with treatment failure of asunaprevir/daclatasvir therapy. They developed persistent HCV infection with triple variants of NS3/4A D168V, NS5A L31V plus Y93H. Administration of ledipasvir/GS-558093 (a NS5B nucleotide analog) in these mice failed to achieve end-of-treatment response or sustained virologic response, which was in sharp contrast to the results in mice with wild-type virus infection. The administration of telaprevir/GS-558093 successfully achieved it in those mice. Treatment failure with asunaprevir/daclatasvir may limit further treatment options. This population may represent a growing unmet medical need.

  10. Comparison of daclatasvir resistance barriers on NS5A from hepatitis C virus genotypes 1 to 6: implications for cross-genotype activity.

    Science.gov (United States)

    Wang, Chunfu; Jia, Lingling; O'Boyle, Donald R; Sun, Jin-Hua; Rigat, Karen; Valera, Lourdes; Nower, Peter; Huang, Xin; Kienzle, Bernadette; Roberts, Susan; Gao, Min; Fridell, Robert A

    2014-09-01

    A comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b>4a≥5a>6a≅1a>2a JFH>3a>2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC50s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC50s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC50s ranging from 2 to 250 nM). The in vitro data suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Identification and Analysis of Novel Inhibitors against NS3 Helicase and NS5B RNA-Dependent RNA Polymerase from Hepatitis C Virus 1b (Con1

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    Na Yang

    2017-11-01

    Full Text Available Hepatitis C virus (HCV leads to severe liver diseases, including liver fibrosis, cirrhosis and hepatocellular carcinoma. Non-structural protein 3 helicase (NS3h and non-structural protein 5B RNA-dependent RNA polymerase (NS5B are involved in the replication of HCV RNA genome, and have been proved to be excellent targets for discovery of direct-acting antivirals. In this study, two high-throughput screening systems, fluorescence polarization (FP-based ssDNA binding assay and fluorescence intensity (FI-based dsRNA formation assay, were constructed to identify candidate NS3h and NS5B inhibitors, respectively. A library of approximately 800 small molecules and crude extracts, derived from marine microorganisms or purchased from the National Compound Resource Center, China, were screened, with three hits selected for further study. Natural compound No.3A5, isolated from marine fungi, inhibited NS3h activity with an IC50 value of 2.8 μM. We further demonstrated that compound No.3A5 inhibited the abilities of NS3h to bind ssDNA in electrophoretic mobility shift assay and to hydrolyze ATP. The NS3h-inhibitory activity of compound No.3A5 was reversible in our dilution assay, which indicated there was no stable NS3h-No.3A5 complex formed. Additionally, compound No.3A5 exhibited no binding selectivity on NS3h or single strand binding protein of Escherichia coli. In NS5B assays, commercial compounds No.39 and No.94 previously reported as kinase inhibitors were found to disrupt dsRNA formation, and their IC50 values were 62.9 and 18.8 μM, respectively. These results highlight how identifying new uses for existing drugs is an effective method for discovering novel HCV inhibitors. To our knowledge, all inhibitors reported in this study were originally discovered with HCV anti-non-structural protein activities in vitro.

  12. Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

    Science.gov (United States)

    Asante-Appiah, Ernest; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Chase, Robert; Nickle, David; Qiu, Ping; Howe, Anita; Lahser, Frederick C

    2017-07-01

    Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC50) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The

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

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    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.

  14. A unique phosphorylation-dependent eIF4E assembly on 40S ribosomes co-ordinated by hepatitis C virus protein NS5A that activates internal ribosome entry site translation.

    Science.gov (United States)

    Panda, Swarupa; Vedagiri, Dhiviya; Viveka, Thangaraj Soundara; Harshan, Krishnan Harinivas

    2014-09-01

    We previously reported that the HCV (hepatitis C virus) protein NS5A up-regulated mRNA cap binding eIF4F (eukaryotic initiation factor 4F) complex assembly through mTOR (mechanistic target of rapamycin)-4EBP1 (eIF4E-binding protein 1) pathway and that NS5A (non-structural protein 5A) physically interacted with translation apparatus. In the present study, we demonstrate that NS5A co-ordinates a unique assembly of the cap binding protein eIF4E and 40S ribosome to form a complex that we call ENR (eIF4E-NS5A-ribosome). Recruitment of NS5A and eIF4E to 40S ribosome was confirmed by polysome fractionation, subcellular fractionation and high-salt-wash immunoprecipitation. These observations were also confirmed in HCV-infected cells, validating its biological significance. eIF4E phosphorylation was critical for ENR assembly. 80S ribosome dissociation and RNase integrity assays revealed that, once associated, the ENR complex is stable and RNA interaction is dispensable. Both the N- and C-terminal regions of NS5A domain 1 were indispensable for this assembly and for the NS5A-induced HCV IRES (internal ribosome entry site) activation. The present study demonstrates that NS5A initially associates with phosphorylated eIF4E of eIF4F complex and subsequently recruits it to 40S ribosomes. This is the first time the interaction of viral protein with both eIF4E and ribosomes has been reported. We propose that this assembly would determine the outcome of HCV infection and pathogenesis through regulation of viral and host translation.

  15. Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H Strain Frequency in Genotype 1b HCV by Invader Assay.

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    Satoshi Yoshimi

    Full Text Available Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001. The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694. Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4, higher serum HCV RNA levels (p=4.3 x 10-7, and lower HCC risk (p=6.9 x 10-3 than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

  16. A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals novel NS5 mutation

    Science.gov (United States)

    Schmidt, DJ; Pickett, BE; Camacho, D; Comach, G; Xhaja, K; Lennon, NJ; Rizzolo, K; de Bosch, N; Becerra, A; Nogueira, ML; Mondini, A; da Silva, EV; Vasconcelos, PF; Muñoz-Jordán, JL; Santiago, GA; Ocazionez, R; Gehrke, L; Lefkowitz, EJ; Birren, BW; Henn, MR; Bosch, I

    2013-01-01

    Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3(DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008 respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of 7 amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may

  17. Persistence of resistant variants in hepatitis C virus-infected patients treated with the NS5A replication complex inhibitor daclatasvir.

    Science.gov (United States)

    Wang, Chunfu; Sun, Jin-Hua; O'Boyle, Donald R; Nower, Peter; Valera, Lourdes; Roberts, Susan; Fridell, Robert A; Gao, Min

    2013-05-01

    Daclatasvir (DCV; BMS-790052) is a hepatitis C virus (HCV) NS5A replication complex inhibitor (RCI) with picomolar to low nanomolar potency and broad genotypic coverage in vitro. Viral RNA declines have been observed in the clinic for both alpha interferon-ribavirin (IFN-α-RBV) and IFN-RBV-free regimens that include DCV. Follow-up specimens (up to 6 months) from selected subjects treated with DCV in 14-day monotherapy studies were analyzed for genotype and phenotype. Variants were detected by clonal sequencing in specimens from baseline and were readily detected by population sequencing following viral RNA breakthrough and posttreatment. The major amino acid substitutions generating resistance in vivo were at residues M28, Q30, L31, and Y93 for genotype 1a (GT-1a) and L31 and Y93 for GT-1b, similar to the resistance substitutions observed with the in vitro replicon system. The primary difference in the resistance patterns observed in vitro and in vivo was the increased complexity of linked variant combinations observed in clinical specimens. Changes in the percentage of individual variants were observed during follow-up; however, the overall percentage of variants in the total population persisted up to 6 months. Our results suggest that during the 14-day monotherapy, most wild-type virus was eradicated by DCV. After the end of DCV treatment, viral fitness, rather than DCV resistance, probably determines which viral variants emerge as dominant in populations.

  18. Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

    Science.gov (United States)

    Boyce, Sarah E.; Tirunagari, Neeraj; Niedziela-Majka, Anita; Perry, Jason; Wong, Melanie; Kan, Elaine; Lagpacan, Leanna; Barauskas, Ona; Hung, Magdeleine; Fenaux, Martijn; Appleby, Todd; Watkins, William J.; Schmitz, Uli; Sakowicz, Roman

    2014-01-01

    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. PMID:24416288

  19. Impact of Pre-existing NS5A-L31 or -Y93H Minor Variants on Response Rates in Patients Infected with HCV Genotype-1b Treated with Daclatasvir/Asunaprevir.

    Science.gov (United States)

    Hernandez, Dennis; Yu, Fei; Huang, Xin; Kirov, Stefan; Pant, Saumya; McPhee, Fiona

    2016-07-01

    The combination of daclatasvir (DCV, pan-genotypic NS5A inhibitor) plus asunaprevir (ASV; NS3 protease inhibitor) is approved in Japan, Korea and other countries for the treatment of chronic hepatitis C virus (HCV) genotype (GT)-1. A high (~90 to 100%) sustained virologic response (SVR) with DCV/ASV therapy has been achieved by excluding patients infected with HCV GT-1b with baseline NS5A resistance-associated variants (RAVs) at L31 or Y93H detected by direct sequencing (DS). We set out to determine whether patients with minor variants at NS5A-L31 or -Y93H, detected by next-generation sequencing (NGS), impacted SVR rates with DCV/ASV therapy. Baseline samples from 222 interferon (IFN)-ineligible/intolerant (N = 135) and prior non-responder (N = 87) patients infected with GT-1b who were treated with DCV/ASV for 24 weeks in the Phase 3 clinical study AI447026 were prepared for NGS (Ion-Torrent platform). The prevalence of baseline NS5A RAVs and their impact on SVR when observed at ≥1% by NGS in a patient's virus population were examined. NGS and DS (sensitivity ≥20%) data were compared. The prevalence of baseline NS5A RAVs at L31 or Y93H was 29% (63/219) and 18% (39/214) by NGS and DS, respectively. SVR24 rates were comparable in patients without observed baseline L31 or Y93H polymorphisms whether assessed by NGS (96%; 148/154) or by the less sensitive DS platform (95%; 164/173). Optimal SVR rates (≥95%) to DCV/ASV treatment were achieved using DS to exclude patients infected with GT-1b with NS5A RAVs at L31 or Y93H representing ≥20% of their virus population. Exclusion by NGS of patients with minor variants in NS5A (<20%) did not enhance SVR rates. These results suggest that the presence of minor variants in NS5A does not appear to impact the overall SVR rate in patients with GT-1b treated with DCV/ASV. This study was sponsored by Bristol-Myers Squibb. ClinicalTrials.gov identifier: NCT01497834.

  20. Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides (7Me)GpppAC(n) and GpppAC(n).

    Science.gov (United States)

    Selisko, Barbara; Peyrane, Frédéric F; Canard, Bruno; Alvarez, Karine; Decroly, Etienne

    2010-01-01

    The flavivirus RNA genome contains a conserved cap-1 structure, (7Me)GpppA(2'OMe)G, at the 5' end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2'O)-MTases (2'O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2'O-MTase activity of NS5MTase of dengue virus (NS5MTase(DV)) using purified, short, capped RNA substrates ((7Me)GpppAC(n) or GpppAC(n)). NS5MTase(DV) methylated both types of substrate exclusively at the 2'O position. The efficiency of 2'O-methylation did not depend on the methylation of the N7 position. Using (7Me)GpppAC(n) and GpppAC(n) substrates of increasing chain lengths, it was found that both NS5MTase(DV) 2'O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K(m) values for (7Me)GpppAC(5) and the co-substrate S-adenosyl-L-methionine (AdoMet) were determined (0.39 and 3.26 microM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2'O-MTase activity of NS5MTase(DV) showed a low turnover of 3.25x10(-4) s(-1). Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTase(DV), which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC(50) 0.34 microM) and sinefungin (IC(50) 0.63 microM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.

  1. 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.

    Science.gov (United States)

    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

    2008-07-15

    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(50) values. The most potent compound exhibited IC(50) less than 10nM against the genotype 1b HCV polymerase and EC(50) of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.

  2. Assessment of pharmacokinetic interactions of the HCV NS5A replication complex inhibitor daclatasvir with antiretroviral agents: ritonavir-boosted atazanavir, efavirenz and tenofovir.

    Science.gov (United States)

    Bifano, Marc; Hwang, Carey; Oosterhuis, Berend; Hartstra, Jan; Grasela, Dennis; Tiessen, Renger; Velinova-Donga, Maria; Kandoussi, Hamza; Sevinsky, Heather; Bertz, Richard

    2013-01-01

    Approximately one-third of all HIV-infected individuals are coinfected with HCV, many of whom will receive concomitant treatment for both infections. With the advent of direct-acting antivirals (DAAs) for HCV, potential drug interactions between antiretrovirals and DAAs require evaluation prior to co-therapy. Three open-label studies were conducted in healthy subjects to assess potential interactions between the investigational first-in-class HCV NS5A replication complex inhibitor daclatasvir and representative antiretrovirals atazanavir/ritonavir, efavirenz and tenofovir disoproxil fumarate. Target exposure was that of 60 mg daclatasvir alone. Dose-normalized (60 mg) geometric mean ratios of daclatasvir AUCτ for 20 mg ± atazanavir/ritonavir (2.10 [90% CI 1.95, 2.26]) and 120 mg ± efavirenz (0.68 [0.60, 0.78]) showed less than the three-fold elevation and two-fold reduction, respectively, in systemic exposure predicted by prior interaction studies with potent inhibitors/inducers of CYP3A4. Daclatasvir dose adjustment to 30 mg once daily with atazanavir/ritonavir and 90 mg once daily with efavirenz is predicted to normalize AUCτ relative to the target exposure (geometric mean ratios 1.05 [0.98, 1.13] and 1.03 [0.90, 1.16], respectively). Atazanavir exposure (Cmax, AUCτ and C24 trough) and efavirenz Ctrough under coadministration were similar to historical data without daclatasvir. No clinically relevant interactions between daclatasvir and tenofovir disoproxil fumarate were observed for either drug, and no dosing adjustments were indicated. Daclatasvir was well tolerated in all three studies. The pharmacokinetic data support coadministration of daclatasvir with atazanavir/ritonavir, efavirenz and/or tenofovir. A Phase III study in HIV-HCV coinfection has commenced using the described dose modifications.

  3. Immunogenicity of Multi-Epitope DNA and Peptide Vaccine Candidates Based on Core, E2, NS3 and NS5B HCV Epitopes in BALB/c Mice.

    Science.gov (United States)

    Pishraft Sabet, Leila; Taheri, Tahereh; Memarnejadian, Arash; Mokhtari Azad, Talat; Asgari, Fatemeh; Rahimnia, Ramin; Alavian, Seyed Moayed; Rafati, Sima; Samimi Rad, Katayoun

    2014-10-01

    Hypervariability of HCV proteins is an important obstacle to design an efficient vaccine for HCV infection. Multi-epitope vaccines containing conserved epitopes of the virus could be a promising approach for protection against HCV. Cellular and humoral immune responses against multi-epitope DNA and peptide vaccines were evaluated in BALB/c mice. In this experimental study, multi-epitope DNA- and peptide-based vaccines for HCV infection harboring immunodominant CD8+ T cell epitopes (HLA-A2 and H2-Dd) from Core (132-142), NS3 (1073-1081) and NS5B (2727-2735), a Th CD4+ epitope from NS3 (1248-1262) and a B-cell epitope from E2 (412-426) were designed. Multi-epitope DNA and peptide vaccines were tested in two regimens as heterologous DNA/peptide (group 1) and homologous peptide/peptide (group 2) prime/boost vaccine in BALB/c mice model. Electroporation was used for delivery of the DNA vaccine. Peptide vaccine was formulated with Montanide ISA 720 (M720) as adjuvant. Cytokine assay and antibody detection were performed to analyze the immune responses. Mice immunized with multi-epitope peptide formulated with M720 developed higher HCV-specific levels of total IgG, IgG1 and IgG2a than those immunized with multi-epitope DNA vaccine. IFN-γ levels in group 2 were significantly higher than group 1 (i.e. 3 weeks after the last immunization; 37.61 ± 2.39 vs. 14.43 ± 0.43, P epitope DNA and peptide-vaccines confirmed their specific immunogenicity in BALB/c mice. However, lower Th1 immune responses in mice immunized with DNA vaccine suggests further investigations to improve the immunogenicity of the multi-epitope DNA vaccine through immune enhancers.

  4. Prevalence of relevant NS5A resistance-associated substitutions to elbasvir in genotype 1a hepatitis C virus patients in Spain.

    Science.gov (United States)

    Palladino, Claudia; Esteban-Cartelle, Beatriz; Mate-Cano, Irene; Sánchez-Carrillo, Marta; Resino, Salvador; Briz, Verónica

    2017-05-15

    Resistance-associated substitutions (RASs) to the new HCV NS5A inhibitor elbasvir may limit its efficacy and lead to virological failure in HCV-GT1a-infected patients. There are no data outside clinical trials evaluating their prevalence and impact in grazoprevir/elbasvir in GT1a-infected patients in Spain. A multicentre cross-sectional study of 632 initial patients was conducted. In 13 of these patients, the sample could not be amplified or a consensus sequence by Sanger sequencing could not be performed. Ultimately, 617 HCV-G1a-infected individuals treated at 84 Spanish hospitals from the 17 autonomous communities plus the 2 autonomous cities of Spain were analysed. HCV population sequencing was used to identify RAS to elbasvir and the mutational pattern and drug sensitivity were confirmed by geno2pheno[HCV]. Viruses bearing RASs to elbasvir were present in 6.2% of HCV-G1a infected patients. The most common RASs were the Y93C/H/N and Q30E/H/R (2.4% and 2.3%, respectively). Only 3.4% of the identified RASs to elbasvir conferred reduced susceptibility to elbasvir by geno2pheno[HCV], which exclusively identified the positions Q30H/R (n=7) and Y93C/H/N (n=8) as single mutations and Q30H+Y93H (n=4) and Q30R+Y93H (n=2) as double mutations as the major RASs to elbasvir. A lower prevalence of RASs to elbasvir was observed in our HCV-G1a Spanish cohort than reported previously in clinical trials evaluating patients from the USA. This information may be essential to guide the implementation of grazoprevir/elbasvir in Spain and to manage G1a-infected patients. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  5. Combined 3D-QSAR, molecular docking, molecular dynamics simulation, and binding free energy calculation studies on the 5-hydroxy-2H-pyridazin-3-one derivatives as HCV NS5B polymerase inhibitors.

    Science.gov (United States)

    Yu, Haijing; Fang, Yu; Lu, Xia; Liu, Yongjuan; Zhang, Huabei

    2014-01-01

    The NS5B RNA-dependent RNA polymerase (RdRP) is a promising therapeutic target for developing novel anti-hepatitis C virus (HCV) drugs. In this work, a combined molecular modeling study was performed on a series of 193 5-hydroxy-2H-pyridazin-3-one derivatives as inhibitors of HCV NS5B Polymerase. The best 3D-QSAR models, including CoMFA and CoMSIA, are based on receptor (or docking). Furthermore, a 40-ns molecular dynamics (MD) simulation and binding free energy calculations using docked structures of NS5B with ten compounds, which have diverse structures and pIC50 values, were employed to determine the detailed binding process and to compare the binding modes of the inhibitors with different activities. On one side, the stability and rationality of molecular docking and 3D-QSAR results were validated by MD simulation. The binding free energies calculated by the MM-PBSA method gave a good correlation with the experimental biological activity. On the other side, by analyzing some differences between the molecular docking and the MD simulation results, we can find that the MD simulation could also remedy the defects of molecular docking. The analyses of the combined molecular modeling results have identified that Tyr448, Ser556, and Asp318 are the key amino acid residues in the NS5B binding pocket. The results from this study can provide some insights into the development of novel potent NS5B inhibitors. © 2013 John Wiley & Sons A/S.

  6. 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

  7. Analysis of sequences of hepatitis C virus NS5A genotype 1 in HIV-coinfected patients with a null response to nitazoxanide or peg-interferon plus ribavirin.

    Science.gov (United States)

    Sede, M; Laufer, N; Ojeda, D; Gun, A; Cahn, P; Quarleri, J

    2013-09-01

    Even though new drugs have been approved for treatment of hepatitis C virus (HCV) infection, the risk of drug-drug interactions and concern about overlapping toxicities has hindered the development of studies in HIV/HCV-coinfected individuals. Traditional treatment with pegylated interferon plus ribavirin (peg-IFN + RBV) is very expensive and has a low rate of sustained virological response in coinfected patients, especially if they are infected with HCV genotype 1. Nitazoxanide (NTZ) is a drug that is being evaluated for the treatment of chronic HCV infection, both in HCV-monoinfected and HIV/HCV-coinfected patients. Understanding the NTZ resistance mechanism could allow the development of resistance to be minimized and would expand the treatment options, mainly in special populations such as HIV/HCV-coinfected patients. Similarly to IFN, NTZ increases the activity of the cellular protein kinase activated by double-stranded RNA (PKR), a key kinase in the innate antiviral response. In order to elucidate whether sequence heterogeneity in the PKR-binding domain of HCV NS5A genotype 1 could influence the antiviral activity of either NTZ monotherapy or peg-IFN + RBV, baseline and end-of-therapy plasma samples from two groups of eleven non-responder HIV/HCV-coinfected patients that had received NTZ or peg-IFN + RBV were studied. Most of the HCV NS5A sequences examined at the end of therapy did not change from the baseline, even after 30 days course of antiviral therapy. An extensive comparison of HCV NS5A genotype 1 and 4 sequences from the database with reported IFN therapy outcome was performed in order to infer their phylogenetic relationships. The HCV genotype 1 NS5A nucleotide sequences from therapy-non-responder patients were intermingled amongst those from the database, irrespective of their IFN-therapy outcome. When comparing NS5A-PKRBD amino acid sequences, significant differences were observed in genotype 4, but not in genotype 1 (p  0

  8. 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.

  9. NS5A resistance leading to failure of 24-week therapy with sofosbuvir/ledipasvir and ribavirin for the treatment of hepatitis C genotype 1a infection in a HIV-1 co-infected patient.

    Science.gov (United States)

    Sevastianova, Ksenia; Dean, Jonathan; Bannan, Ciaran; Coghlan, Miriam; Farrell, Gillian; Murray, Catherine; De Gascun, Cillian F; Bergin, Colm

    2016-09-01

    Herein we report a previously undescribed case of treatment-emergent non-structural protein 5A (NS5A) resistance mutations, Q30H and Y93C, leading to a failure of 24-week course of sofosbuvir/ledipasvir+ribavirin therapy for the treatment of hepatitis C virus (HCV) genotype 1a in interferon-experienced, human immunodeficiency virus type 1 (HIV-1) co-infected patient with cirrhosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. A novel simple assay system to quantify the percent HCV-RNA levels of NS5A Y93H mutant strains and Y93 wild-type strains relative to the total HCV-RNA levels to determine the indication for antiviral therapy with NS5A inhibitors.

    Directory of Open Access Journals (Sweden)

    Yoshihito Uchida

    Full Text Available Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.Both the wild-type and mutant strains were quantified, with a threshold of 4.0 Log copies/mL, in 295 of the 332 patients (88.9%, and the percentage of the mutant strains relative to the total HCV-RNA level in the serum was calculated. The percentage was 0% in 237 patients (80.3% and 100% in 23 patients (7.8%, identical to the results of direct sequencing. Both wild-type and mutant strains were detected in the remaining 35 patients (11.9%, at levels between 1% and 99%, despite the mutant strains having been undetectable by direct sequencing in 11 patients with percentages of these strains of less than 25%.A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established. This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.

  11. Identification of the NS5B S282T resistant variant and two novel amino acid substitutions that affect replication capacity in hepatitis C virus-infected patients treated with mericitabine and danoprevir.

    Science.gov (United States)

    Tong, Xiao; Li, Lewyn; Haines, Kristin; Najera, Isabel

    2014-06-01

    Baseline and posttreatment samples from hepatitis C virus (HCV) genotype (GT) 1-infected patients who received a combination of danoprevir and mericitabine from a phase II clinical study (INFORM-SVR) were analyzed. In addition to resistance monitoring, sequencing and phenotypic assays were combined with statistical analysis to identify potential novel amino acid substitutions associated with treatment outcome. The NS5B S282T substitution associated with mericitabine resistance was identified in 2/30 viral breakthrough patients and was replaced by wild-type viruses after cessation of drug treatment (during follow-up). The NS3 R155K substitution associated with danoprevir resistance was also observed in these 2 patients. All 69 GT 1a-infected patients who experienced viral breakthrough on treatment or relapsed during follow-up (relapsers) developed NS3 R155K. Among GT 1b-infected patients, substitutions at the danoprevir resistance locus NS3 D168 were observed in 15/20 subjects, whereas substitutions at the danoprevir resistance locus NS3 R155 were observed in 5/20 subjects. Interestingly, the baseline polymorphism NS5B Q47H was more prevalent in GT 1a-infected patients who achieved a sustained virologic response at follow-up week 24 (SVR24) than in non-SVR24 patients (2/13 versus 0/72), and a postbaseline NS3 S122G substitution was more prevalent in GT 1a-infected patients with viral breakthrough than in relapsers (4/22 versus 0/47). Neither substitution conferred resistance to danoprevir or mericitabine, but the substitutions reduced (NS5B Q47H) or improved (NS3 S122G) replication capacity by 2- to 4-fold. The NS5B S282T mericitabine-resistant variant was rare and did not persist once drug was discontinued. NS5B Q47H and NS3 S122G are two newly identified substitutions that affected replication capacity and were enriched in distinct treatment response groups. (This study has been registered at ClinicalTrials.gov under registration no. NCT01278134.). Copyright

  12. Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

    Science.gov (United States)

    Daniel, Hubert D-J; David, Joel; Raghuraman, Sukanya; Gnanamony, Manu; Chandy, George M; Sridharan, Gopalan; Abraham, Priya

    2017-05-01

    Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing. © 2016 Wiley Periodicals, Inc.

  13. 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

    Science.gov (United States)

    2012-01-01

    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 lichen planus. The adjusted odds ratios for these three factors were 6.58, 3.53 and 2.58, respectively, and each was statistically significant. No significant differences in viral factors, such as aa substitutions in the core region and ISDR of NS5A, were detected between the two groups (groups 2-A and -B). 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. PMID:22490000

  14. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

    Science.gov (United States)

    Lahser, Frederick C; Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y M; Asante-Appiah, Ernest

    2016-05-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  15. Previous failure of interferon-based therapy does not alter the frequency of HCV NS3 protease or NS5B polymerase inhibitor resistance-associated variants: longitudinal analysis in HCV/HIV co-infected patients.

    Science.gov (United States)

    Sede, Mariano M; Laufer, Natalia L; Quarleri, Jorge

    2015-08-01

    Since 2011, treatment of chronic hepatitis C virus (HCV) includes direct-acting antivirals (DAAs) in addition to pegylated interferon-α (peg-IFN) and ribavirin (RBV). IFN-based treatment induces strong cytotoxic T-lymphocyte activity directed to the protease- and polymerase-derived epitopes. This enhanced immunological pressure could favour the emergence of viral epitope variants able to evade immune surveillance and, when resistance-associated variants (RAVs) are implicated, could also be co-selected as a hitchhiking effect. This study analysed the dynamics of the frequency of protease and polymerase inhibitor RAVs that could affect future HCV treatment in human immunodeficiency virus (HIV) co-infected patients on stable antiretroviral therapy with previous IFN-based treatment failure. HCV genotype 1a RNA was extracted from plasma samples of 18 patients prior to and during (24h and 4, 12, 24 and 48 weeks) therapy with peg-IFN+RBV. Next-generation sequencing was performed on HCV-RNA populations using NS3 and NS5B PCR-amplified coding regions. Two measures of genetic diversity were used to compare virus populations: average pairwise nucleotide diversity (π) and Tajima's D statistic. Several protease and polymerase RAVs were detected in all subjects at very low frequencies (<5%), and in most cases their presence was not constant during follow-up. Only samples from two patients for each region exhibited Q80R/K/L and A421V as highly predominant variants. No significant differences were observed among sampling times for either π or D values. In conclusion, previous therapy and failure of peg-IFN+RBV were not associated with an increase in DAA-targeting NS3 or NS5B RAVs that naturally exist in HIV co-infected subjects. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  16. Effectiveness and safety of sofosbuvir-based regimens plus an NS5A inhibitor for patients with HCV genotype 3 infection and cirrhosis. Results of a multicenter real-life cohort.

    Science.gov (United States)

    Alonso, S; Riveiro-Barciela, M; Fernandez, I; Rincón, D; Real, Y; Llerena, S; Gea, F; Olveira, A; Fernandez-Carrillo, C; Polo, B; Carrión, J A; Gómez, A; Devesa, M J; Baliellas, C; Castro, Á; Ampuero, J; Granados, R; Pascasio, J M; Rubín, A; Salmeron, J; Badia, E; Planas, J M M; Lens, S; Turnes, J; Montero, J L; Buti, M; Esteban, R; Fernández-Rodríguez, C M

    2017-04-01

    Patients with HCV genotype 3 (GT3) infection and cirrhosis are currently the most difficult to cure. We report our experience with sofosbuvir+daclatasvir (SOF+DCV) or sofosbuvir/ledipasvir (SOF/LDV), with or without ribavirin (RBV) in clinical practice in this population. This was a multicenter observational study including cirrhotic patients infected by HCV GT3, treated with sofosbuvir plus an NS5A inhibitor (May 2014-October 2015). In total, 208 patients were included: 98 (47%) treatment-experienced, 42 (20%) decompensated and 55 (27%) MELD score >10. In 131 (63%), treatment was SOF+DCV and in 77 (37%), SOF/LDV. Overall, 86% received RBV. RBV addition and extension to 24 weeks was higher in the SOF/LDV group (95% vs 80%, P=.002 and 83% vs 72%, P=.044, respectively). A higher percentage of decompensated patients were treated with DCV than LDV (25% vs 12%, P=.013). Overall, SVR12 was 93.8% (195/208): 94% with SOF+DCV and 93.5% with SOF/LDV. SVR12 was achieved in 90.5% of decompensated patients. Eleven treatment failures: 10 relapses and one breakthrough. RBV addition did not improve SVR (RR: 1.08; P=.919). The single factor associated with failure to achieve SVR was platelet count <75×10E9/mL (RR: 3.50, P=.019). In patients with MELD <10, type of NS5A inhibitor did not impact on SVR12 (94% vs 97%; adjusted RR: 0.49). Thirteen patients (6.3%) had serious adverse events, including three deaths (1.4%) and one therapy discontinuation (0.5%), higher in decompensated patients (16.7% vs 3.6%, P<.006). In patients with GT3 infection and cirrhosis, SVR12 rates were high with both SOF+DCV and SOF/LDV, with few serious adverse events. © 2016 John Wiley & Sons Ltd.

  17. GB virus C (GBV-C) evolutionary patterns revealed by analyses of reference genomes, E2 and NS5B sequences amplified from viral strains circulating in the Lisbon area (Portugal).

    Science.gov (United States)

    Parreira, Ricardo; Branco, Cristina; Piedade, João; Esteves, Aida

    2012-01-01

    GBV-C is a non-pathogenic virus that is largely dispersed in different human populations. The phylogenetic analysis of the 5'-untranslated region (5'UTR) of the GBV-C genome has led to the segregation of viral strains into six genotypes, but incongruent results are frequently obtained depending on the genome region analyzed. In this report, different phylogenetic approaches and multivariate statistics were combined to disclose evolutionary patterns that contribute to shape GBV-C evolution. The data here presented indicate: (i) that the phylogenetic noise was mostly determined by the size of the analyzed sequence, rather than by its position on the viral genome; (ii) that most genomic segments in the coding sequence seemed to evolve under a similar evolution model, which was different from that which best fits the 5'UTR, with overall large heterogeneity of rate change across the sequence; (iii) that due to saturation of transversions occurring in the 5'UTR at genetic distances GBV-C evolution extensively, this being shown for both reference genomes and NS5B GBV-C sequences amplified from Portuguese residents. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. 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.

  20. 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.

  1. Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 3: Further optimization of the 2-, 6-, and 7'-substituents and initial pharmacokinetic assessments.

    Science.gov (United States)

    Li, Lian-Sheng; Zhou, Yuefen; Murphy, Douglas E; Stankovic, Nebojsa; Zhao, Jingjing; Dragovich, Peter S; Bertolini, Thomas; Sun, Zhongxiang; Ayida, Benjamin; Tran, Chinh V; Ruebsam, Frank; Webber, Stephen E; Shah, Amit M; Tsan, Mei; Showalter, Richard E; Patel, Rupal; Lebrun, Laurie A; Bartkowski, Darian M; Nolan, Thomas G; Norris, Daniel A; Kamran, Ruhi; Brooks, Jennifer; Sergeeva, Maria V; Kirkovsky, Leo; Zhao, Qiang; Kissinger, Charles R

    2008-06-01

    5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.

  2. The antiviral protein viperin inhibits HCV replication via interaction with NS5A

    OpenAIRE

    Helbig, Karla J.; Nicholas S Eyre; Yip, Evelyn; Narayana, Sumudu; Li, Kui; Fiches, Guillaume; McCartney, Erin M; Jangra, Rohit K.; Lemon, Stanley M.; Beard, Michael R.

    2011-01-01

    The interferon-stimulated gene viperin has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here we demonstrate that viperin plays an integral part in the ability of interferon to limit replication of cell culture derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and Fluorescence Resonance Energy Transfer (FRET) ana...

  3. Daclatasvir: A NS5A Replication Complex Inhibitor for Hepatitis C Infection.

    Science.gov (United States)

    Smith, Michael A; Regal, Randolph E; Mohammad, Rima A

    2016-01-01

    To review the pharmacology, efficacy, and safety of daclatasvir in the treatment of patients with chronic hepatitis C virus (HCV) infection. A literature search through EMBASE and PubMed was conducted (January 1966 to August 2015) using the terms BMS-790052, daclatasvir, and hepatitis C. References from retrieved articles were reviewed for any additional material. Additionally, the new drug application and prescribing information were retrieved. The literature search was limited to human studies published in English. Phase 1, 2, and 3 studies describing the pharmacology, pharmacokinetics, efficacy, and safety of daclatasvir for HCV were identified. Daclatasvir, a nonstructural 5A protein inhibitor, combined with sofosbuvir, is indicated for adult patients with chronic HCV genotype 3 regardless of treatment or cirrhosis status. The phase III ALLY-3 trial (n = 152) demonstrated that daclatasvir taken once daily with sofosbuvir for 12 weeks was effective at achieving sustained virological response (SVR) rates in treatment-naïve (97%) and treatment-experienced (94%) patients without cirrhosis. Patients with cirrhosis had significantly lower SVR rates (58 and 69%, respectively). The most common adverse drug events associated with daclatasvir and sofosbuvir in ALLY-3 were headache (20%), fatigue (19%), and nausea (12%). Daclatasvir, when combined with sofosbuvir, is an effective agent to treat HCV genotype 3, with SVR rates above 90% for patients without cirrhosis who are treatment naïve or experienced. SVR rates for treatment-naïve or -experienced patients with cirrhosis are not as robust (58%-69%). © The Author(s) 2015.

  4. Grazoprevir, Ruzasvir, and Uprifosbuvir for HCV After NS5A Treatment Failure

    DEFF Research Database (Denmark)

    Wyles, David; Wedemeyer, Heiner; Ben-Ari, Ziv

    2017-01-01

    a single dose of grazoprevir, ruzasvir, and uprifosbuvir plus ribavirin due to serious adverse events of vomiting and tachycardia. The presence of baseline resistance-associated substitutions had no impact on SVR12. No participant who completed treatment in either study experienced virologic failure...

  5. Zika virus NS5 protein potential inhibitors: an enhanced in silico approach in drug discovery.

    Science.gov (United States)

    Ramharack, Pritika; Soliman, Mahmoud E S

    2017-04-17

    The re-emerging Zika virus (ZIKV) is an arthropod-borne virus that has been described to have explosive potential as a worldwide pandemic. The initial transmission of the virus was through a mosquito vector, however, evolving modes of transmission has allowed the spread of the disease over continents. The virus has already been linked to irreversible chronic central nervous system conditions. The concerns of the scientific and clinical community are the consequences of Zika viral mutations, thus suggesting the urgent need for viral inhibitors. There have been large strides in vaccine development against the virus but there are still no FDA approved drugs available. Rapid rational drug design and discovery research is fundamental in the production of potent inhibitors against the virus that will not just mask the virus, but destroy it completely. In silico drug design allows for this prompt screening of potential leads, thus decreasing the consumption of precious time and resources. This study demonstrates an optimized and proven screening technique in the discovery of two potential small molecule inhibitors of ZIKV Methyltransferase and RNA dependent RNA polymerase. This in silico 'per-residue energy decomposition pharmacophore' virtual screening approach will be critical in aiding scientists in the discovery of not only effective inhibitors of Zika viral targets, but also a wide range of anti-viral agents.

  6. Pharmacokinetics of a Cholesterol-conjugated Aptamer Against the Hepatitis C Virus (HCV) NS5B Protein

    Science.gov (United States)

    Lee, Chang Ho; Lee, Soo-Han; Kim, Ji Hyun; Noh, Yook-Hwan; Noh, Gyu-Jeong; Lee, Seong-Wook

    2015-01-01

    Hepatitis C virus (HCV) is the major cause of progressive liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Previously, we reported that a 29 nucleotide-long 2'-F pyrimidine modified RNA aptamer against the HCV nonstructural protein 5B efficiently inhibited HCV replication and suppressed HCV infectious virus particle formation in a cell culture system. In this study, we modified this aptamer through conjugation of cholesterol for in vivo availability. This cholesterol-conjugated aptamer (chol-aptamer) efficiently entered the cell and inhibited HCV RNA replication, without any alteration in gene expression profiling including innate immune response-related genes. Moreover, systemic administration of the chol-aptamer was well tolerated without any abnormalities in mice. To evaluate the pharmacokinetics of the chol-aptamer in vivo, dose proportionality, bioavailability, and pharmacokinetic parameters were evaluated by noncompartmental analyses in normal BALB/c mice. Population analysis was performed using nonlinear mixed effects modeling. Moreover, the pharmacokinetics of two different routes (intravenous, IV, versus intraperitoneal, IP) were compared. Cholesterol conjugation showed dose proportionality, extended the time that the aptamer was in the plasma, and enhanced aptamer exposure to the body. Noticeably, the IV route was more suitable than the IP route due to the chol-aptamer remaining in the plasma for a longer period of time. PMID:26440598

  7. Escape of Tick-Borne Flavivirus from 2'-C-Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness

    Czech Academy of Sciences Publication Activity Database

    Eyer, Luděk; Kondo, H.; Zouharová, D.; Hirano, M.; Valdés, James J.; Muto, M.; Kastl, T.; Kobayashi, S.; Haviernik, J.; Igarashi, K.; Kariwa, H.; Vaculovicova, M.; Černý, Jiří; Kizek, R.; Kroeger, A.; Lienenklaus, S.; Dejmek, Milan; Nencka, Radim; Palus, Martin; Salát, J.; De Clercq, E.; Yoshii, K.; Růžek, Daniel

    2017-01-01

    Roč. 91, č. 21 (2017), č. článku e01028-17. ISSN 0022-538X R&D Projects: GA MZd(CZ) NV16-34238A Institutional support: RVO:60077344 Keywords : antiviral agents * antiviral therapy * escape mutant * tick-borne * encephalitis virus * tick-borne pathogens Subject RIV: EE - Microbiology, Virology Impact factor: 4.663, year: 2016

  8. 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

    of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa...

  9. Drug: D10165 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available B inhibitor ... HCV NS5B is RNA-dependent RNA polymerase(RdRP). Treatment of hepatitis C NS5B polymerase ... CAS: 1071517-39-9 PubChem: 135626883 ChEMBL: CHEMBL1076263 LigandBox: D10165 ...

  10. Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Scheel, Troels K H; Jensen, Tanja B

    2009-01-01

    -6 viruses had similar spread kinetics, intracellular Core, NS5A, and lipid amounts, and colocalization of Core and NS5A with lipids. Treatment with interferon-alpha2b but not ribavirin or amantadine showed a significant antiviral effect. Infection with all genotypes could be blocked by specific antibodies...

  11. Regulation of hepatitis C virus replication by nuclear translocation of nonstructural 5A protein and transcriptional activation of host genes.

    Science.gov (United States)

    Maqbool, Muhammad Ahmad; Imache, Mohamed R; Higgs, Martin R; Carmouse, Sophie; Pawlotsky, Jean-Michel; Lerat, Hervé

    2013-05-01

    Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is involved in regulating viral replication through its direct interaction with the HCV RNA-dependent RNA polymerase. NS5A also alters infected cell metabolism through complex interactions with numerous host cell proteins. NS5A has furthermore been suggested to act as a transcriptional activator, although the impact on viral replication is unclear. To study this, HCV NS5A variants were amplified from hepatic tissue from an HCV-infected patient, and their abilities to activate gene transcription were analyzed in a single-hybrid yeast (Saccharomyces cerevisiae) model. Different variants isolated from the same patient displayed different transactivational activities. When these variants were inserted into the HCV subgenomic replicon system, they demonstrated various levels of RNA replication, which correlated with their transactivational activities. We showed that the C-terminal fragment of NS5A was localized to the nucleus and that a functional NS5A nuclear localization signal and cellular caspase activity were required for this process. Furthermore, nuclear localization of NS5A was necessary for viral replication. Finally, we demonstrate that nuclear NS5A binds to host cell promoters of several genes previously identified as important for efficient HCV RNA replication, inducing their transcription. Taken together, these results demonstrate a new mechanism by which HCV modulates its cellular environment, thereby enhancing viral replication.

  12. Screening of cellular proteins that interact with the classical swine ...

    Indian Academy of Sciences (India)

    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 ...

  13. The rearrangement of motif F in the flavivirus RNA-directed RNA polymerase.

    Science.gov (United States)

    Potapova, Ulyana; Feranchuk, Sergey; Leonova, Galina; Belikov, Sergei

    2017-11-04

    In the flavivirus genus, the non-structural protein NS5 plays a central role in RNA viral replication and constitutes a major target for drug discovery. One of the prime challenges in the study of NS5 protein is to investigate the interplay between the two protein domains, namely, the RNA-dependent RNA polymerase (RdRp) domain and the methyltransferase (MTase) domain. These investigations could clarify the multiple roles of NS5 protein in the virus life cycle. Here we present the results of sequence analyses and structural bioinformatics studies of NS5 protein, which suggest that the conserved motif F in the NS5 protein could act as a lock which controls the rearrangement of the domains and as a switch in the protein enzymatic activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. 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.

  15. 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.

    2012-01-01

    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. PMID:22345449

  16. 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.

  17. 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.

  18. 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.

  19. Daclatasvir: potential role in hepatitis C

    National Research Council Canada - National Science Library

    Lee, Choongho

    2013-01-01

    .... 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...

  20. Drug: D10477 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available Arabinofuranosyl type antiviral ... DG02000 ... NS5B inhibitor Chemical group: DG02119 ... Treatment of hepatitis C ... CAS: 940908-79-2 PubChem: 172232570 ChEMBL: CHEMBL562967 ...

  1. Drug: D10442 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available inhibitor Chemical group: DG01818 ... Treatment of chronic hepatitis C infection HCV NS5A [CPD:C18292] ... CAS: 1256388-51-8 PubChem: 172232535 ChEBI: 85089 ChEMBL: CHEMBL2374220 ...

  2. Daclatasvir

    Science.gov (United States)

    ... C virus (HCV) NS5A inhibitors. It works by stopping the virus that causes hepatitis C from spreading ... Be sure to mention any of the following: amiodarone (Cordarone, Nexterone, Pacerone); certain antibiotics such as clarithromycin ( ...

  3. Sofosbuvir, Velpatasvir, and Voxilaprevir

    Science.gov (United States)

    ... C virus (HCV) NS5A inhibitor. It works by stopping the virus that causes hepatitis C from spreading ... Be sure to mention any of the following: amiodarone (Nexterone, Pacerone); cholesterol-lowering medications (statins) such as ...

  4. Ombitasvir, Paritaprevir, Ritonavir, and Dasabuvir

    Science.gov (United States)

    ... C virus (HCV) NS5A inhibitor. It works by stopping the virus that causes hepatitis C from spreading ... Nizoral, Xolegel); medications for irregular heartbeat such as amiodarone (Cordarone, Nexterone, Pacerone), bepridil (no longer available in ...

  5. Dasabuvir, Ombitasvir, Paritaprevir, and Ritonavir

    Science.gov (United States)

    ... C virus (HCV) NS5A inhibitor. It works by stopping the virus that causes hepatitis C from spreading ... Nizoral, Xolegel); medications for irregular heartbeat such as amiodarone (Cordarone, Nexterone, Pacerone), bepridil (no longer available in ...

  6. Ombitasvir, Paritaprevir, and Ritonavir

    Science.gov (United States)

    ... C virus (HCV) NS5A inhibitor. It works by stopping the virus that causes hepatitis C from spreading ... Nizoral, Xolegel); medications for irregular heartbeat such as amiodarone (Cordarone, Nexterone, Pacerone), bepridil (no longer available in ...

  7. Ledipasvir and Sofosbuvir

    Science.gov (United States)

    ... medications called HCV NS5A inhibitors. It works by stopping the virus that causes hepatitis C from spreading ... Be sure to mention any of the following: amiodarone (Cordarone, Nexterone, Pacerone), certain medications for seizures such ...

  8. DEVELOPMENT OF ASSAY METHOD AND FORCED DEGRADATION STUDY OF LEDIPASVIR AND SOFOSBUVIR BY RP-HPLC IN TABLET FORMULATION

    OpenAIRE

    S. Naazneen*, A. Sridevi

    2017-01-01

    Chronic hepatitis C virus (HCV) infection is one of the most common etiologies of liver-related mortality throughout the world. Sofosbuvir and ledipasvir are inhobits HCV NS5B and HCV NS5A polymerase respectively. No published LC-MS/MS and HPLC based methods for simultaneous estimation of ledipasvir and sofosbuvir. Therefore, A stability indicating high performance liquid Chromatographic (HPLC) method was developed and validated for estimation of both drugs. Chromatographic separation was ach...

  9. 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

  10. Analysis of Ribonucleotide 5'-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays.

    Science.gov (United States)

    Lu, Gaofei; Bluemling, Gregory R; Collop, Paul; Hager, Michael; Kuiper, Damien; Gurale, Bharat P; Painter, George R; De La Rosa, Abel; Kolykhalov, Alexander A

    2017-03-01

    Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5'-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2'-C-methyl- and 2'-C-ethynyl-substituted analog 5'-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors. Copyright © 2017 American Society for Microbiology.

  11. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A.

    Directory of Open Access Journals (Sweden)

    Margarita Zayas

    2016-01-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein (NS5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI and two intrinsically disordered domains (DII and DIII interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2. We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core-RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i SC-dependent recruitment of replication complexes to core protein and (ii BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.

  12. Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of viral nonstructural proteins.

    Directory of Open Access Journals (Sweden)

    Teymur Kazakov

    2015-04-01

    Full Text Available Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV, an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3-5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3-5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.

  13. Successful retreatment with sofosbuvir plus ledipasvir for cirrhotic patients with hepatitis C virus genotype 1b, who discontinued the prior treatment with asunaprevir plus daclatasvir: A case series and review of the literature.

    Science.gov (United States)

    Haga, Yuki; Kanda, Tatsuo; Yasui, Shin; Nakamura, Masato; Ooka, Yoshihiko; Takahashi, Koji; Wu, Shuang; Nakamoto, Shingo; Arai, Makoto; Chiba, Tetsuhiro; Maruyama, Hitoshi; Yokosuka, Osamu; Takada, Nobuo; Moriyama, Mitsuhiko; Imazeki, Fumio; Kato, Naoya

    2018-01-12

    Interferon-free treatment results in higher sustained virologic response (SVR) rates, with no serious adverse events in hepatitis C virus (HCV)-infected patients. However, in some patients with treatment-failure in HCV NS5A inhibitor-including interferon-free regimens, the treatment-emergent HCV NS5A resistance-associated variants (RAVs), which are resistant to interferon-free retreatment including HCV NS5A inhibitors, are observed. In HCV-infected Japanese patients with daclatasvir and asunaprevir treatment failure, retreatment with sofosbuvir and ledipasvir could lead to only ∼70% SVR rates. Three HCV genotype (GT)-1b-infected cirrhotic patients who discontinued the combination of daclatasvir and asunaprevir due to adverse drug reactions within 4 weeks; retreatment with sofosbuvir and ledipasvir combination could result in SVR in these patients without RAVs. One HCV GT-1b-infected cirrhotic patient who discontinued the combination of daclatasvir and asunaprevir due to viral breakthrough at week 10; retreatment with sofosbuvir and ledipasvir combination for this patient with the treatment-emergent HCV NS5A RAV-Y93H resulted in viral relapse at week 4 after the end of the treatment. Retreatment with sofosbuvir and ledipasvir is effective for HCV GT-1b patients who discontinue the combination of daclatasvir and asunaprevir within 4 weeks. The treatment response should be related to the existence of treatment-emergent HCV NS5A RAVs, but may not be related to the short duration of treatment.

  14. Emergence of drug resistance-associated variants and changes in serum lipid profiles in sofosbuvir plus ledipasvir-treated chronic hepatitis C patients.

    Science.gov (United States)

    Kan, Hiromi; Imamura, Michio; Kawakami, Yoshiiku; Daijo, Kana; Teraoka, Yuji; Honda, Fumi; Nakamura, Yuki; Morio, Kei; Kobayashi, Tomoki; Nakahara, Takashi; Nagaoki, Yuko; Kawaoka, Tomokazu; Tsuge, Masataka; Aikata, Hiroshi; Hayes, Clair Nelson; Miki, Daiki; Ochi, Hidenori; Honda, Yoji; Mori, Nami; Takaki, Shintaro; Tsuji, Keiji; Chayama, Kazuaki

    2017-11-01

    Combination of sofosbuvir plus ledipasvir therapy has been expected to enhance sustained virological response (SVR) rates in hepatitis C virus (HCV) genotype 1 chronic infected patients. We analyzed the emergence of drug resistance-associated variants (RAVs) in treatment failure and changes in lipid profiles in sofosbuvir/ledipasvir-treated patients. A total of 176 patients with chronic HCV genotype 1 infection without decompensated liver cirrhosis were treated with sofosbuvir/ledipasvir for 12 weeks. NS5A and NS5B RAVs were determined by either Invader assay or direct sequencing. Serum lipid-related markers were measured at the start of treatment and at week 4 in patients who received sofosbuvir/ledipasvir and ombitasvir/paritaprevir/ritonavir therapies. SVR was achieved in 94.9% (167 out of 176) of patients. SVR12 rate was 97.1% for patietns with low frequncy (75%) of NS5A RAVs. In multivariate regression analysis, higher albumin (odds ratio [OR] = 0.020 for presence; P = 0.007), and NS5A-L31/Y93 RAVs with a population frequency ledipasvir-treated patients. These elevations were greater than in ombitasvir/paritaprevir/ritonavir-treated patients. In conclusion, NS5A multi-RAVs are likely to develop in patients who fail to respond to sofosbuvir/ledipasvir therapy. Inhibition of HCV replication with sofosbuvir might affect lipid metabolism. © 2017 Wiley Periodicals, Inc.

  15. 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

    2016-01-01

    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. PMID:27021315

  16. Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template

    Science.gov (United States)

    2015-01-01

    The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D 1H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3′(−)SL by 1H NMR, the base pair at the stem’s edge was identified as the most stable component of the structure. 1H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3′(−)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process. PMID:25310724

  17. Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Gottwein, Judith M

    2012-01-01

    Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant...... in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive...... mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation...

  18. 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

    to sofosbuvir—the only nucleotide analog approved for treatment of chronic HCV infection. Methods The developed HCV genotype 3a full-length genome (DBN3a), with a strain-DBN coding sequence, modified NS5B consensus sequence, pS52 untranslated regions, and coding mutations from a culture-efficient JFH1-based......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....... Sofosbuvir, MK-3682, dasabuvir, or combinations of sofosbuvir and ledipasvir or sofosbuvir and velpatasvir had decreased efficacy against infection with the DBN3a sofosbuvir escape variant. Conclusions We developed a system for highly efficient culture of HCV genotype 3a. Genotype 1a has a high genetic...

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

    Directory of Open Access Journals (Sweden)

    Takeya Tsutsumi

    Full Text Available 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.

  20. Long-Term Follow-Up of Resistance-Associated Substitutions in Hepatitis C Virus in Patients in Which Direct Acting Antiviral-Based Therapy Failed

    Directory of Open Access Journals (Sweden)

    Kanako Yoshida

    2017-05-01

    Full Text Available We evaluated the transition of dominant resistance-associated substitutions (RASs in hepatitis C virus during long-term follow-up after the failure of DAAs (direct acting antivirals-based therapy. RASs in non-structure (NS3/4A, NS5A, NS5B, and deletions in NS5A from 20 patients who failed simeprevir/pegylated-interferon/ribavirin (SMV/PEG-IFN/RBV and 25 patients who failed daclatasvir/asunaprevir (DCV/ASV treatment were examined by direct sequencing. With respect to SMV/PEG-IFN/RBV treatment, RAS was detected at D168 in NS3/4A but not detected in NS5A and NS5B at treatment failure in 16 of 20 patients. During the median follow-up period of 64 weeks, the RAS at D168 became less dominant in 9 of 16 patients. Among 25 DCV/ASV failures, RASs at D168, L31, and Y93 were found in 57.1%, 72.2%, and 76.9%, respectively. NS5A deletions were detected in 3 of 10 patients treated previously with SMV/PEG-IFN/RBV. The number of RASs in the breakthrough patients exceeded that in relapsers (mean 3.9 vs. 2.7, p < 0.05. RAS at D168 in NS3/4A became less dominant in 6 of 15 patients within 80 weeks. Y93H emerged at the time of relapse, then decreased gradually by 99% at 130 weeks post-treatment. Emerged RASs were associated with the clinical course of treatment and could not be detected during longer follow-up.

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

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-03-01

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

  2. An update on the treatment of genotype-1 chronic hepatitis C infection: lessons from recent clinical trials.

    Science.gov (United States)

    Wendt, Astrid; Bourlière, Marc

    2013-12-01

    The launch of first-generation protease inhibitors (PIs) was a major step forward in hepatitis C virus (HCV) treatment. However, this major advance is, up to now, restricted to genotype-1 (GT-1) patients. However, the ongoing development of new direct-acting antiviral agents (DAAs) allows new hope for the future. 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 (NS5B.I) include nucleoside/nucleotide inhibitors (NIs) and nonnucleotide inhibitors (NNIs). NIs have high efficacy across all genotypes. Sofosbuvir has highly potent antiviral activity across all genotypes in association with pegylated interferon (IFN) 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 PI can cure GT-1b null-responder patients in an IFN-free regimen. In addition, several studies demonstrate that IFN-free regimens with DAA combinations are able to cure a large number of either naïve or treatment-experienced GT-1 patients. Moreover, a quadruple regimen with PR is able to cure almost all GT-1 null-responders. The development of pan-genotypic DAAs (NIs or NS5A.I) allows new combinations with or without PR that increase the rate of sustained virological response (SVR) 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 in GT-1 patients.

  3. Virological escape in HCV genotype-1-infected patients receiving daclatasvir plus ribavirin and peginterferon alfa-2a or alfa-2b.

    Science.gov (United States)

    McPhee, Fiona; Hernandez, Dennis; Zhou, Nannan; Yu, Fei; Ueland, Joseph; Monikowski, Aaron; Chayama, Kazuaki; Toyota, Joji; Izumi, Namiki; Yokosuka, Osamu; Kawada, Norifumi; Osaki, Yukio; Hughes, Eric A; Watanabe, Hideaki; Ishikawa, Hiroki; Kumada, Hiromitsu

    2014-01-01

    Daclatasvir (DCV; BMS-790052) is a picomolar inhibitor of HCV non-structural protein 5A (NS5A) and has demonstrated efficacy in patients chronically infected with HCV. In the double-blind, randomized studies AI444021 and AI444022, 71 Japanese patients chronically infected with HCV genotype 1 (predominantly genotype 1b) received DCV (10 mg or 60 mg) plus peginterferon alfa-2b or alfa-2a and ribavirin. Virological failure occurred in 14% (5/36) of treatment-naive patients and 54% (19/35) of prior alfa/ribavirin non-responders. Resistance testing was performed on baseline samples and samples with HCV RNA≥1,000 IU/ml at week 1 through post-treatment week 24. Baseline NS5A resistance-associated polymorphisms had less impact on virological response rates than IL28B genotype. All patients with virological failure had NS5A DCV-resistant variants at the time of failure. The predominant NS5A variants were L31V/M/I plus Y93H; this combination was detected in 100% (5/5) of treatment-naive patients and 74% (14/19) of non-responders with failure. Emergent resistance variants in prior non-responders (four viral breakthroughs, one relapse) were more varied with novel combinations such as L31F-ΔP32 and L28M-R30Q-A92K detected. Significant loss in DCV antiviral activity was generally only seen with ≥ two resistance-associated NS5A substitutions. All DCV-resistant variants were still detected at end of study. Virological failure in HCV genotype 1b treatment-naive Japanese patients receiving DCV plus alfa-2a/ribavirin or alfa-2b/ribavirin was associated with enrichment of NS5A resistance variants L31V/M-Y93H. In prior non-responders, emergent variants associated with failure also included NS5A-A92K or NS5A-ΔP32. As with L31-Y93 variants, these variants persisted.

  4. 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.

  5. HCV非结构蛋白5A对HIV长末端重复序列影响的初步探讨

    Directory of Open Access Journals (Sweden)

    PENG Milin

    2014-02-01

    Full Text Available ObjectiveTo investigate the effect of nonstructural protein 5A (NS5A encoded by the human hepatitis C virus (HCV RNA genome on human immunodeficiency virus (HIV long terminal repeat (LTR and to provide an experimental basis for the study on the effect of HCV on HIV. MethodsHepatocellular carcinoma Huh7 cells were divided into blank group, control group, and experimental group to be transfected with plasmid pGL3-LTR-Luc (containing luciferase reporter gene driven by the LTR promoter, plasmid pRc/CMV plus plasmid pGL3-LTR-Luc, and plasmid pCNS5A (containing HCV NS5A gene plus plasmid pGL3-LTR-Luc, respectively; Huh7 cells were collected 48 h later. The protein and mRNA expression levels of HCV NS5A were measured by immunocytochemistry, Western blot, and RT-PCR. The relative luciferase activity was measured to evaluate the HIV LTR activity and the effect of HCV NS5A on HIV LTR. The activity values were expressed as mean±SD, and Levene′s test of homogeneity of variance was used; comparison between all groups was made by one-way analysis of variance (ANOVA, and comparison between two groups was made by least significant difference (LSD test. ResultsThe mRNA and protein expression of HCV NS5A was detected in the cytoplasm of Huh7 cells in experimental group. The one-way ANOVA showed that there were significant differences in LTR luciferase activity between the three groups (F=7.876, P=0002. The LSD test showed that the experimental group had a significantly higher relative luciferase activity than the blank group and control group (22476±4471 vs 15887±3039, P=0.002; 22476±4471 vs 16321±4162, P=0.008. ConclusionHuh7 cells can be transfected with the HCV NS5A expression plasmid (pCNS5A. HCV NS5A can activate HIV LTR, which suggests that HCV NS5A may be one of the molecular mechanisms of HCV promoting HIV replication.

  6. Selection of regimens for patients with hepatitis C virus genotype/serotype discrepancy.

    Science.gov (United States)

    Katsushima, Shinji; Kobata, Tatsuro; Komeda, Toshiki; Hamada, Seiko; Chikugo, Kouki; Nakano, Shigeharu; Shimogama, Tsubasa; Kumagai, Ken; Ohta, Yoshiyuki; Endoh, Bunji; Esaka, Naoki; Iwamoto, Satoru; Kasahara, Katsuhiro; Shima, Nobuko; Mizumoto, Yoshinori

    2017-01-01

    In the present study, the usefulness of the resistance-associated variant (RAV) analysis to select direct acting antiviral (DAA) drugs for patients with hepatitis C virus (HCV) genotype/serotype discrepancy was evaluated. The core-genotype and serotype were determined in the 559 patients recruited in the study. The RAV analysis and NS5B-genotype determination were performed in the eight patients who exhibited a genotype/serotype discrepancy. One of these patients exhibited a core-genotype 1b/serotype 2, and detection by RAV analysis was possible in this patient. The other seven patients demonstrated a core-genotype 2/serotype 1, and detection using the RAV analysis was possible in four of them. The NS5B-genotype was 1b in all patients in whom detection using the RAV analysis was possible and was other than 1b in patients in whom detection using the RAV analysis was impossible. The RAV analysis could detect RNA sequences specific to genotype 1b in the NS5A region. Therefore, in patients with genotype/serotype discrepancy in whom detection using the RAV analysis is possible, the treatment regimens should be selected based on the assumption that HCV with genome that is highly homologous to genotype 1b is present in the NS5A region.

  7. Regulatory mechanisms of viral hepatitis B and C

    Indian Academy of Sciences (India)

    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 ...

  8. Structural basis of Zika virus methyltransferase inhibition by sinefungin

    Czech Academy of Sciences Publication Activity Database

    Hercík, Kamil; Brynda, Jiří; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 162, č. 7 (2017), s. 2091-2096 ISSN 0304-8608 R&D Projects: GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : crystal structure * flavivirus RNA * NS5 Subject RIV: CE - Biochemistry Impact factor: 2.058, year: 2016

  9. Nuclear import inhibitor N-(4-hydroxyphenyl) retinamide targets Zika virus (ZIKV) nonstructural protein 5 to inhibit ZIKV infection.

    Science.gov (United States)

    Wang, Chunxiao; Yang, Sundy N Y; Smith, Kate; Forwood, Jade K; Jans, David A

    2017-12-02

    In the absence of approved therapeutics, Zika virus (ZIKV)'s recent prolific outbreaks in the Americas, together with impacts on unborn fetuses of infected mothers, make it a pressing human health concern worldwide. Although a key player in viral replication in the infected host cell cytoplasm, ZIKV non-structural protein 5 (NS5) appears to contribute integrally to pathogenesis by localising in the host cell nucleus, in similar fashion to NS5 from Dengue virus (DENV). We show here for the first time that ZIKV NS5 is recognized with high nanomolar affinity by the host cell importin α/β1 heterodimer, and that this interaction can be blocked by the novel DENV NS5 targeting inhibitor N-(4-hydroxyphenyl) retinamide (4-HPR). Importantly, we show that 4-HPR has potent anti-ZIKV activity at low μM concentrations. With an established safety profile for human use, 4-HPR represents an exciting possibility as an anti-ZIKV agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Download this PDF file

    African Journals Online (AJOL)

    Dr Olaleye

    activity. MATERIALS AND METHODS. General. The 1H and 13C NMR spectra were recorded in CDCl3- d6 at 400 MHz. The HRESIMS data were obtained using an Agilent MS TOF ... Phenomenex C18 column (10.0 × 250 mm) and an. 1100 Series multiple ... Two probes were complementary to the NS5A region of the HCV ...

  11. Real-World Experiences with the Combination Treatment of Ledipasvir plus Sofosbuvir for 12 Weeks in HCV Genotype 1-Infected Japanese Patients: Achievement of a Sustained Virological Response in Previous Users of Peginterferon plus Ribavirin with HCV NS3/4A Inhibitors.

    Science.gov (United States)

    Kanda, Tatsuo; Yasui, Shin; Nakamura, Masato; Suzuki, Eiichiro; Arai, Makoto; Ooka, Yoshihiko; Ogasawara, Sadahisa; Chiba, Tetsuhiro; Saito, Tomoko; Haga, Yuki; Takahashi, Koji; Sasaki, Reina; Wu, Shuang; Nakamoto, Shingo; Tawada, Akinobu; Maruyama, Hitoshi; Imazeki, Fumio; Kato, Naoya; Yokosuka, Osamu

    2017-04-25

    The aim of this study was to characterize the treatment response and serious adverse events of ledipasvir plus sofosbuvir therapies in Japanese patients infected with hepatitis C virus (HCV) genotype 1 (GT1). This retrospective study analyzed 240 Japanese HCV GT1 patients treated for 12 weeks with 90 mg of ledipasvir plus 400 mg of sofosbuvir daily. Sustained virological response at 12 weeks post-treatment (SVR12) was achieved in 236 of 240 (98.3%) patients. Among treatment-naïve patients, SVR12 was achieved in 136 of 138 (98.6%) patients, and among treatment-experienced patients, SVR12 was achieved in 100 of 102 (98.0%) patients. In patients previously treated with peginterferon plus ribavirin with various HCV NS3/4A inhibitors, 100% SVR rates (25/25) were achieved. Two relapsers had HCV NS5A resistance-associated variants (RAVs), but no HCV NS5B-S282 was observed after they relapsed. We experienced two patients with cardiac events during treatment. In conclusion, combination of ledipasvir plus sofosbuvir for 12 weeks is a potential therapy for HCV GT1 patients. Caution is needed for HCV NS5A RAVs, which were selected by HCV NS5A inhibitors and cardiac adverse events.

  12. 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 (ledipasvir 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. © 2016 Wiley Periodicals, Inc.

  13. Spread of hepatitis C virus among European injection drug users infected with HIV: A phylogenetic analysis

    NARCIS (Netherlands)

    van Asten, Liselotte; Verhaest, Inge; Lamzira, Saida; Hernandez-Aguado, Ildefonso; Zangerle, Robert; Boufassa, Faroudy; Rezza, Giovanni; Broers, Barbara; Robertson, J. Roy; Brettle, Raymond P.; McMenamin, Jim; Prins, Maria; Cochrane, Alexandra; Simmonds, Peter; Coutinho, Roel A.; Bruisten, Sylvia

    2004-01-01

    To describe the spread of hepatitis C virus (HCV) among HCV/human immunodeficiency virus (HIV)-coinfected injection drug users (IDUs), the molecular epidemiology of HCV was studied among 108 IDUs from 7 European countries. Phylogenetic analysis based on the NS5B region showed great sequence

  14. Geometric Tachyon and Warm Inflation

    Science.gov (United States)

    Bhattacharjee, Anindita; Deshamukhya, Atri

    2013-03-01

    The inflationary models developed in presence of a background radiation can be a solution to the reheating problem faced by common cold (isentropic) inflationary scenario. A D-brane system comprising of k Neuvo-Schwarz (NS) 5-branes with a transverse circle and BPS D3-branes with world volume parallel to the NS 5-branes, placed at a point on the transverse circle diametrically to NS 5-brane has a point of unstable equilibrium and the D3-brane has a geometric tachyonic mode associated with displacement of the brane along the circle. Cold inflationary scenario has been studied in connection with this geometric tachyon [S. Panda, M. Sami and S. Tsujikawa, Phys. Rev. D73, 023515 (2006)] where it was found that one needs a background of minimum 104 branes to realize a viable inflationary model. In this piece of work, we have tried to study a model of inflation driven by this geometric tachyon in presence of radiation. We have found that compared to the isentropic scenario, to satisfy the observational bounds, the number of background branes required in this case reduces drastically and a viable model can be obtained with even six to seven NS 5-branes in the background. In this context, we have also analyzed the non-gaussianity associated with the model and observed that the concerned parameter lies well within the observation limit.

  15. Catalyst-free aldol reaction of N-substituted rhodanines on aqueous ...

    Indian Academy of Sciences (India)

    N S DEVI

    2018-02-07

    Feb 7, 2018 ... treatment of diabetic neuropathy.7 Additionally, they are known to inhibit numerous targets such as RNA polymerase,8 HIV-1 integrase,9 PMT1 mannosyl trans- ferase,10 JSP-1 phosphatases,11 hepatitis C virus NS5B polymerase12 and cathepsin D.13 Due to high potential of rhodanine derivatives in drug ...

  16. Rol de las proteínas no estructurales en los eventos de replicación del ARN del virus dengue: propuesta de un modelo de replicación del ARN

    Directory of Open Access Journals (Sweden)

    Carlos Yábar V

    2003-03-01

    Full Text Available Un modelo ilustrativo de los procesos de replicación para el virus dengue y flavivirus en general ha sido diseñado con base a datos experimentales publicados en los últimos diez años. El modelo propone cuatro pasos bien definidos durante el proceso de replicación de ARN: 1 circularización del ARN viral mediada por un stem-loop del extremo 3' y la región 5' terminal TR, 2 linearización y síntesis de la forma intermedia replicativa mediada por el complejo NS3-NS5 y la glicoproteína NS1, 3 cambio de estado de NS5 a la forma rígida para la síntesis de formas replicativas y 4Desensamblaje del sistema de replicación NS3-NS5 mediado por fosforilación y la unión específica de NS5 con un receptor beta de la importina. Este modelo podría ser útil para el planteamiento de nuevos trabajos de investigación destinados al estudio de la replicación en flavivirus.

  17. STRUKTUR PROTEOMIK VIRUS DENGUE DAN MANFAATNYA SEBAGAI TARGET ANTIVIRUS

    Directory of Open Access Journals (Sweden)

    Novia Rachmayanti

    2014-09-01

    Full Text Available AbstrakVirus dengue (DENV telah menyebabkan sekitar 50 juta kasus infeksi demam berdarah setiap tahunnya, akan tetapi hingga saat ini belum terdapat vaksin maupun antivirus yang mampu mencegah atau mengobati penyakit tersebut. Selama pengembangan vaksin dan antivirus, diperoleh berbagai informasi tentang struktur protein DENV yang dapat dimanfaatkan sebagai target obat. Makalah membahas tentang struktur proteomik pada DENV, yaitu glikoprotein pada envelope, NS3 protease, NS3 helikase, NS5 metiltransferase, dan NS5 RNA-dependent RNA polimerase.AbstractDengue virus (DENV has caused over 50 millions infection every year. However, to date neither vaccine nor medicine could be used to prevent or cure the illness. During researches in finding the vaccine or antiviral for DENV, information on DENV protein structure has been obtained which is potentially used as drug target. This paper disscuss DENV proteomic structure that consist of envelope glicoprotein, NS3 protease, NS3 helicase, NS5 methyl-transferase, and NS5 RNA-dependent RNA polymerase.

  18. Post-treatment resistance analysis of hepatitis C virus from phase II and III clinical trials of ledipasvir/sofosbuvir.

    Science.gov (United States)

    Wyles, David; Dvory-Sobol, Hadas; Svarovskaia, Evguenia S; Doehle, Brian P; Martin, Ross; Afdhal, Nezam H; Kowdley, Kris V; Lawitz, Eric; Brainard, Diana M; Miller, Michael D; Mo, Hongmei; Gane, Edward J

    2017-04-01

    Ledipasvir/sofosbuvir combination treatment in phase III clinical trials resulted in sustained viral suppression in 94-99% of patients. This study characterized drug resistance in treatment failures, which may help to inform retreatment options. We performed NS5A and NS5B deep sequencing of hepatitis C virus (HCV) from patients infected with genotype (GT) 1 who participated in ledipasvir/sofosbuvir phase II and III clinical trials. Fifty-one of 2144 (2.4%) (42 GT1a and 9 GT1b) treated patients met the criteria for resistance analysis due to virologic failure following the end of treatment. The majority of patients with virologic failure (38 of 51; 74.5%) had detectable ledipasvir-specific resistance-associated substitutions (RASs) at the time of virologic failure (1% deep sequencing cut-off). The percent of patients with NS5A RASs at virologic failure were 37.5%, 66.7%, 94.7% and 100% in patients treated for 6, 8, 12 and 24weeks, respectively. The common substitutions detected at failure were Q30R/H, and/or Y93H/N in GT1a and Y93H in GT1b. At failure, 35.3% (18/51) of virologic failure patients' viruses had two or more NS5A RASs and the majority of patients harbored NS5A RASs conferring a 100-1000-fold (n=10) or >1000-fold (n=23) reduced susceptibility to ledipasvir. One patient in a phase II study with a known ledipasvir RAS at baseline (L31M) developed the S282T sofosbuvir (NS5B) RAS at failure. In GT1 HCV-infected patients treated with ledipasvir/sofosbuvir±ribavirin, virologic failure was rare. Ledipasvir resistance in NS5A was selected or enhanced in most patients with virologic failure, one of whom also developed resistance to sofosbuvir. Clinical studies have shown that combination treatment with ledipasvir/sofosbuvir efficiently cures most patients with genotype 1 hepatitis C infection. For the few patients failing treatment, we show that resistance to ledipasvir was observed in most patients, whereas resistance to sofosbuvir was less common. This has

  19. Efficacy and safety of daclatasvir and asunaprevir combination therapy in chronic hemodialysis patients with chronic hepatitis C.

    Science.gov (United States)

    Suda, Goki; Kudo, Mineo; Nagasaka, Atsushi; Furuya, Ken; Yamamoto, Yoshiya; Kobayashi, Tomoe; Shinada, Keisuke; Tateyama, Miki; Konno, Jun; Tsukuda, Yoko; Yamasaki, Kazushi; Kimura, Megumi; Umemura, Machiko; Izumi, Takaaki; Tsunematsu, Seiji; Sato, Fumiyuki; Terashita, Katsumi; Nakai, Masato; Horimoto, Hiromasa; Sho, Takuya; Natsuizaka, Mitsuteru; Morikawa, Kenichi; Ogawa, Koji; Sakamoto, Naoya

    2016-07-01

    HCV infection in chronic hemodialysis patients is high, has a poor prognosis and high risk of renal graft failure, and requires nosocomial infection control measures. However, options of anti-HCV therapy in such patients are limited and unsatisfactory. In this study, we report effectiveness and safety of HCV-NS5A-inhibitor daclatasvir (DCV) and protease-inhibitor asunaprevir (ASV) combination therapy for hemodialysis patients with HCV infection. This study was registered at the UMIN Clinical Trials Registry as UMIN000016355. Thirty-four dialysis patients were treated with DCV/ASV combination therapy between January 2015 and November 2015. Of those, 21 patients who were followed more than 12 weeks after treatment ended were included. We evaluated the 12-week sustained virologic response (SVR12) and adverse events during treatment. Of the 21 patients, four had compensated liver cirrhosis and three had resistance-associated variant of NS5A (NS5A RAVs)-Y93H at baseline. Overall, total of 95.5 % (20/21) of the patients achieved SVR12. Of note, all patients with cirrhosis or NS5A RAVs achieved SVR12. One relapser patient at 4 weeks post-treatment had NS3 D168E RAVs at baseline. A total of 20 patients (95.5 %) completed the 24-week therapy. One patient discontinued treatment at week 12 due to ALT elevations and achieved SVR12. DAV and ASV combination therapy for chronic hemodialysis patients with HCV infection was highly effective and well tolerated, even in elderly patients and patients with liver cirrhosis and NS5A-RAVs.

  20. Benefit-risk assessment for sofosbuvir/velpatasvir/voxilaprevir based on patient population and hepatitis C virus genotype: U. S. Food and Drug Administration's evaluation.

    Science.gov (United States)

    Struble, Kimberly; Chan-Tack, Kirk; Qi, Karen; Naeger, Lisa K; Birnkrant, Debra

    2017-10-23

    On July 18, 2017, the U.S. Food and Drug Administration (FDA) approved sofosbuvir/velpatasvir/voxilaprevir (SOF/VEL/VOX) (Vosevi) fixed-dose combination (FDC), an interferon-free, complete regimen for adult patients with chronic hepatitis C virus (HCV) infection without cirrhosis or with compensated cirrhosis (Child-Pugh A) who have: • genotype 1, 2, 3, 4, 5, or 6 infection and have previously been treated with an HCV regimen containing a nonstructural protein 5A (NS5A) inhibitor; and • genotype 1a or 3 infection and have previously been treated with an HCV regimen containing sofosbuvir without an NS5A inhibitor. Approval was based on an acceptable safety profile and high sustained virological response rates 12 weeks after the end of treatment (SVR12) in two phase 3 clinical trials in subjects previously treated with a direct-acting antiviral (DAA) regimen. In POLARIS-1, 96% of SOF/VEL/VOX-treated subjects achieved SVR12. In POLARIS-4, 98% of SOF/VEL/VOX-treated subjects achieved SVR12. A key and challenging question in evaluating the data was determining the contribution of VOX to SOF/VEL and how this differed depending on the genotype and patient population. In this article, we provide our perspective on the issues considered in making these determinations, especially regarding the POLARIS-4 data in subjects who have previously been treated with a chronic HCV regimen containing sofosbuvir without an NS5A inhibitor. We seek to provide context as to why a broad indication was given for NS5A inhibitor-experienced patients (HCV genotypes 1-6) while the indication for NS5A inhibitor- naïve patients was limited to HCV genotypes 1a and 3 only. (Hepatology 2017). Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  1. 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.

  2. 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.

  3. Evaluation of Adamantane Derivatives as Inhibitors of Dengue Virus mRNA Cap Methyltransferase by Docking and Molecular Dynamics Simulations.

    Science.gov (United States)

    Luzhkov, Victor; Decroly, Etienne; Canard, Bruno; Selisko, Barbara; Åqvist, Johan

    2013-02-01

    Binding of the Dengue virus S-adenosyl-L-methionine (AdoMet)-dependent mRNA cap methyltransferase (NS5MTaseDV ) with adamantane derivatives was explored using molecular modeling methods and (nucleoside-2'O)-methyltransferase bioassay. The studied compounds include urea derivatives of adamantane and the antiviral drugs amantadine and rimantadine. The urea derivatives of adamantanes had previously been identified as inhibitors of NS5MTaseDV . The docking simulations using GOLD, Glide, and Dock give consistent binding modes and binding affinities of adamantanes in the AdoMet-binding site of NS5MTaseDV and, in particular, yield similar positions for the previously found inhibitors. Combined, they perfectly correspond to the bioassay measurements of nucleoside-2'O-methyltransferase activity of NS5TaseDV , which confirmed inhibitory properties of the active urea adamantane but did not show inhibitory activity for amantadine and rimantadine. We also employed microscopic molecular dynamics (MD) simulations and a linear interaction energy (LIE) method to verify the docking results. The MD/LIE binding free energies of selected protein-inhibitor complexes agree overall with the binding affinities from docking and demonstrate that amantadine and rimantadine only weakly bind at the explored site. The MD simulations also demonstrated the flexible character of a protein loop that is located between the β2 and β3 strands and is part of the AdoMet-binding pocket of NS5MTaseDV . Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

    Science.gov (United States)

    Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François-Loïc

    2017-03-01

    Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral

  5. 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.

    Science.gov (United States)

    Margeridon-Thermet, Severine; Le Pogam, Sophie; Li, Lewyn; Liu, Tommy F; Shulman, Nancy; Shafer, Robert W; Najera, Isabel

    2014-01-01

    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.

  6. 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.

  7. Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies.

    Science.gov (United States)

    Di Maio, Velia C; Cento, Valeria; Lenci, Ilaria; Aragri, Marianna; Rossi, Piera; Barbaliscia, Silvia; Melis, Michela; Verucchi, Gabriella; Magni, Carlo F; Teti, Elisabetta; Bertoli, Ada; Antonucci, FrancescoPaolo; Bellocchi, Maria C; Micheli, Valeria; Masetti, Chiara; Landonio, Simona; Francioso, Simona; Santopaolo, Francesco; Pellicelli, Adriano M; Calvaruso, Vincenza; Gianserra, Laura; Siciliano, Massimo; Romagnoli, Dante; Cozzolongo, Raffaele; Grieco, Antonio; Vecchiet, Jacopo; Morisco, Filomena; Merli, Manuela; Brancaccio, Giuseppina; Di Biagio, Antonio; Loggi, Elisabetta; Mastroianni, Claudio M; Pace Palitti, Valeria; Tarquini, Pierluigi; Puoti, Massimo; Taliani, Gloria; Sarmati, Loredana; Picciotto, Antonino; Vullo, Vincenzo; Caporaso, Nicola; Paoloni, Maurizio; Pasquazzi, Caterina; Rizzardini, Giuliano; Parruti, Giustino; Craxì, Antonio; Babudieri, Sergio; Andreoni, Massimo; Angelico, Mario; Perno, Carlo F; Ceccherini-Silberstein, Francesca

    2017-04-01

    Despite the excellent efficacy of direct-acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance-associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real-life DAA failures. Of the 200 virological failures that were analyzed in 197 DAA-treated patients, 89 with pegylated-interferon+ribavirin (PegIFN+RBV) and 111 without (HCV-1a/1b/1g/2/3/4=58/83/1/6/24/25; 56.8% treatment experienced; 65.5% cirrhotic) were observed. Sanger sequencing of NS3/NS5A/NS5B was performed by home-made protocols, at failure (N=200) and whenever possible at baseline (N=70). The majority of the virological failures were relapsers (57.0%), 22.5% breakthroughs, 20.5% non-responders. RAS prevalence varied according to IFN/RBV use, DAA class, failure type and HCV genotype/subtype. It was 73.0% in IFN group vs 49.5% in IFN free, with the highest prevalence of NS5A-RASs (96.1%), compared to NS3-RASs (75.9% with IFN, 70.5% without) and NS5B-RASs (66.6% with IFN, 20.4% without, in sofosbuvir failures). In the IFN-free group, RASs were higher in breakthrough/non-responders than in relapsers (90.5% vs 40.0%, Presistance, including 11/11 NS3+NS5A failures. Furthermore, 20.0% of patients had baseline-RASs, which were always confirmed at failure. In our failure setting, RAS prevalence was remarkably high in all genes, with a partial exception for NS5B, whose limited resistance is still higher than previously reported. This multiclass resistance advocates for HCV resistance testing at failure, in all three genes for the best second-line therapeutic tailoring. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. The safety of daclatasvir for the treatment of hepatitis C.

    Science.gov (United States)

    Jafri, Syed-Mohammed; Gordon, Stuart C

    2015-01-01

    The direct acting antiviral daclatasvir is an NS5A replication inhibitor active against the entire range of hepatitis C virus genotypes. It is a key step in establishing the goal of an all-oral, ribavirin-free, pan-genotypic regimen against hepatitis C. We review current literature including published abstracts and manuscripts. Evidence was obtained through PubMed/Medline search using listed keywords and through review of published abstracts. Daclatasvir introduces a degree of pangenotypic potency currently lacking in other NS5A agents. Emerging literature suggests that daclatasvir in combination with other DAAs will represent a promising option in this difficult to treat populations including posttransplant, genotype 3 and HIV patients.

  9. Non-BPS D-brane Solutions in Six Dimensional Orbifolds

    CERN Document Server

    Lozano, Y

    2000-01-01

    Starting with the non-BPS D0-brane solution of IIB/$(-1)^{F_L}I_4$ constructed recently by Eyras and Panda we construct via T-duality the non-BPS D2-brane and D1-brane solutions of IIB/$(-1)^{F_L}I_4$ and IIA/$(-1)^{F_L}I_4$ predicted by Sen. The D2-brane couples magnetically to the vector field of the NS5B-brane living in the twisted sector of the Type IIB orbifold, whereas the D1-brane couples (electrically and magnetically) to the self-dual 2-form potential of the NS5A-brane that is present in the twisted sector of the Type IIA orbifold construction. Finally we discuss the eleven dimensional interpretation of these branes as originating from a non-BPS M1-brane solution of M-theory orientifolded by $\\Omega_\\rho I_5$.

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

    DEFF Research Database (Denmark)

    2013-01-01

    Genotype 7a has been identified recently, thus not much is known about the biology of this new, major HCV genotype. The present inventors developed hepatitis C virus 7a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced...... 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...... in HCV genotype 7, including vaccine studies and functional analyses...

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

    Energy Technology Data Exchange (ETDEWEB)

    Lozano, Yolanda [Department of Physics, University of Oviedo,Avda. Calvo Sotelo 18, Oviedo, 33007 (Spain); Macpherson, Niall T. [Dipartimento di Fisica, Università di Milano-Bicocca and INFN, sezione di Milano-Bicocca,Milano, I-20126 (Italy); Montero, Jesús [Department of Physics, University of Oviedo,Avda. Calvo Sotelo 18, Oviedo, 33007 (Spain); Núñez, Carlos [Department of Physics, Swansea University,Swansea, SA2 8PP United Kingdom (United Kingdom)

    2016-11-22

    In this paper we construct a new Type IIB background with an AdS{sub 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{sub 4}×S{sup 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{sub ρ}{sup ρ̂}(SU(N)) quiver field theory. This relates explicitly the AdS{sub 4} geometry to a D3-D5-NS5 brane intersection, illuminating what seems to be a more general phenomenon, relating AdS{sub p+1} backgrounds generated by non-Abelian T-duality to Dp-D(p+2)-NS5 branes intersections.

  13. Inhibition of viral RNA polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand RNA viruses beyond hepatitis C virus.

    Science.gov (United States)

    Deval, Jerome; Symons, Julian A; Beigelman, Leo

    2014-12-01

    A number of important human infections are caused by positive-strand RNA viruses, yet almost none can be treated with small molecule antiviral therapeutics. One exception is the chronic infection caused by hepatitis C virus (HCV), against which new generations of potent inhibitors are being developed. One of the main molecular targets for anti-HCV drugs is the viral RNA-dependent RNA polymerase, NS5B. This review summarizes the search for nucleoside and nucleotide analogs that inhibit HCV NS5B, which led to the FDA approval of sofosbuvir in 2013. Advances in anti-HCV therapeutics have also stimulated efforts to develop nucleoside analogs against other positive-strand RNA viruses. Although it remains to be validated in the clinic, the prospect of using nucleoside analogs to treat acute infections caused by RNA viruses represents an important paradigm shift and a new frontier for future antiviral therapies. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. A novel indirect ELISA for diagnosis of dengue fever.

    Science.gov (United States)

    Narayan, Rohan; Raja, Senthil; Kumar, Senthil; Sambasivam, Mohana; Jagadeesan, Raja; Arunagiri, Kavita; Krishnasamy, Kaveri; Palani, Gunasekaran

    2016-07-01

    Dengue fever (DF) is associated with significant morbidity and mortality in the tropical and sub-tropical regions of the world. Since there are no effective antiviral drugs for treatment, clinicians often rely on the accurate diagnosis of dengue fever to begin supportive therapy at early stages of the illness. The objective of this study was to develop an in-house dengue virus serotype 2 (DENV-2) non-structural protein- 5 (NS5) based indirect ELISA. DENV-2 was raised in Vero cells and the viral proteins were separated and subsequently the NS5 protein was eluted. Serum samples from primary and secondary dengue fever patients; and acute and convalescent samples from Japanese encephalitis (JE) and West Nile virus (WNV) cases were used to validate the ELISA. The assay was found to be 100 per cent specific in detecting DENV-2 specific antibodies from patient's serum. However, in terms of sensitivity, the assay could detect IgM antibodies only from 90 per cent of the primary dengue samples. The IgM/IgG ratio of the primary and secondary samples was 7.24 and 0.64, respectively. The results indicate that the DENV-2 NS5 ELISA is dengue group specific and can be used to differentiate dengue infection from other circulating Flavivirus infections. This NS5 ELISA can also be used to distinguish between primary and secondary dengue fever on the basis of IgM/IgG ratios. Further studies with larger sample sizes and different DENV serotypes are required to validate the ELISA.

  15. The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A.

    Science.gov (United States)

    Helbig, Karla J; Eyre, Nicholas S; Yip, Evelyn; Narayana, Sumudu; Li, Kui; Fiches, Guillaume; McCartney, Erin M; Jangra, Rohit K; Lemon, Stanley M; Beard, Michael R

    2011-11-01

    The interferon-stimulated gene, viperin, has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here, we demonstrate that viperin plays an integral part in the ability of interferon to limit the replication of cell-culture-derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis, we demonstrate that viperin localizes and interacts with HCV nonstructural protein 5A (NS5A) at the lipid-droplet (LD) interface. In addition, viperin also associates with NS5A and the proviral cellular factor, human vesicle-associated membrane protein-associated protein subtype A (VAP-A), at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus, as well as an N-terminal amphipathic helix. Removal of the amphipathic helix-redirected viperin from the cytosolic face of the endoplasmic reticulum and the LD to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the LD interface and replication complexes, but did not interact with NS5A proteins, as determined by FRET analysis. In conclusion, we propose that viperin interacts with NS5A and the host factor, VAP-A, to limit HCV replication at the replication complex. This highlights the complexity of the host control of viral replication by interferon-stimulated gene expression. Copyright © 2011 American Association for the Study of Liver Diseases.

  16. Characterization of virologic escape in hepatitis C virus genotype 1b patients treated with the direct-acting antivirals daclatasvir and asunaprevir.

    Science.gov (United States)

    Karino, Yoshiyasu; Toyota, Joji; Ikeda, Kenji; Suzuki, Fumitaka; Chayama, Kazuaki; Kawakami, Yoshiiku; Ishikawa, Hiroki; Watanabe, Hideaki; Hernandez, Dennis; Yu, Fei; McPhee, Fiona; Kumada, Hiromitsu

    2013-04-01

    Daclatasvir and asunaprevir are NS5A and NS3 protease-targeted antivirals currently under development for treatment of chronic hepatitis C virus infection. Clinical data on baseline and on-treatment correlates of drug resistance and response to these agents are currently limited. Hepatitis C virus genotype 1b Japanese patients (prior null responders to PegIFN-α/RBV [n=21] or PegIFN-α/RBV ineligible or intolerant [n=22]) were administered daclatasvir/asunaprevir for 24 weeks during a phase 2a open-label study. Genotypic and phenotypic analyses of NS3 and NS5A substitutions were performed at baseline, after virologic failure, and post-treatment through follow-up week 36. There were three viral breakthroughs and four relapsers. Baseline NS3 polymorphisms (T54S, Q80L, V170M) at amino acid positions previously associated with low-level resistance (daclatasvir resistance (daclatasvir-resistant substitutions persisted through 48weeks post-treatment, whereas asunaprevir-resistant substitutions were no longer detectable. Overall, 5/10 patients with baseline NS5A-Y93H experienced virologic failure, while 5/10 achieved a sustained virologic response. The potential association of a pre-existing NS5A-Y93H polymorphism with virologic failure on daclatasvir/asunaprevir combination treatment will be examined in larger studies. The persistence of treatment-emergent daclatasvir- and asunaprevir-resistant substitutions will require assessment in longer-term follow-up studies. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  17. Daclatasvir: potential role in hepatitis C [Corrigendum

    Directory of Open Access Journals (Sweden)

    Lee C

    2014-03-01

    Full Text Available Lee C. Drug Des Devel Ther. 2013;7:1223–1233. On page 1231 in the "Conclusion" section, line 3 contains incorrect information. The correct sentence is "This review summarizes key preclinical and clinical data of an HCV NS5A inhibitor, DCV, describing its discovery, mechanism of action, resistance profile, in vitro and in vivo efficacy and toxicity, and polymorphism."Read the original article

  18. Daclatasvir combined with peginterferon-? and ribavirin for the treatment of chronic hepatitis C: a meta-analysis

    OpenAIRE

    Peng, Qin; Li, Kang; Cao, Ming Rong; Bie, Cai Qun; Tang, Hui Jun; Tang, Shao Hui

    2016-01-01

    Daclatasvir, a HCV NS5A inhibitor, is a new direct-acting antiviral drug for chronic hepatitis C (CHC). This study aimed to evaluate the efficacy and safety of daclatasvir combined with peginterferon-? (pegIFN-?) and ribavirin (RBV) for the treatment of CHC. The databases of PUBMED, EMBASE, COCHRANE, WANFANG, and CNKI were retrieved to identify eligible studies. Pooled risk ratio (RR) and 95?% confidence interval (CI) were calculated using random or fixed models. A total of six RCTs including...

  19. Daclatasvir: potential role in hepatitis C [Corrigendum

    OpenAIRE

    Lee C

    2014-01-01

    Lee C. Drug Des Devel Ther. 2013;7:1223–1233. On page 1231 in the "Conclusion" section, line 3 contains incorrect information. The correct sentence is "This review summarizes key preclinical and clinical data of an HCV NS5A inhibitor, DCV, describing its discovery, mechanism of action, resistance profile, in vitro and in vivo efficacy and toxicity, and polymorphism."Read the original article

  20. A Designer’s Guide to Human Performance Modelling (La Modelisation des Performances Humaines: Manuel du Concepteur).

    Science.gov (United States)

    1998-12-01

    allow walk-throughs -it can be reassuring to use be achieved via the Internet , in the anticipation that the input data to produce data that match the...4 1 -20 4 -20 3 -20 3 3 -20 -20 4 1 geometry & layout 5 communicatio 2 2 1 2 -20 1 1 2 1 -20 1 -20 2 ns 5 environmental 2 3 2 3 2 2 -20 2 -20 1 1 1 2

  1. Sofosbuvir/Velpatasvir/Voxilaprevir: A Pan-Genotypic Direct-Acting Antiviral Combination for Hepatitis C.

    Science.gov (United States)

    Chahine, Elias B; Kelley, Denise; Childs-Kean, Lindsey M

    2017-11-01

    To review the efficacy and safety of sofosbuvir/velpatasvir/voxilaprevir in the treatment of hepatitis C virus (HCV) infection. A literature search through PubMed was conducted (August 2010 to August 2017) using the terms GS-9857, voxilaprevir, and NS3/4A protease inhibitor. Studies of sofosbuvir/velpatasvir/voxilaprevir were identified. Sofosbuvir/velpatasvir/voxilaprevir is indicated for adult patients with chronic HCV without cirrhosis or with compensated cirrhosis who have (1) genotype 1 through 6 and have previously been treated with an NS5A inhibitor or (2) genotype 1a or 3 and have previously been treated with sofosbuvir without an NS5A inhibitor. POLARIS-1 demonstrated that sofosbuvir/velpatasvir/voxilaprevir for 12 weeks was highly effective in patients with HCV genotype 1 through 6 who had prior exposure to an NS5A inhibitor. POLARIS-2 failed to demonstrate that sofosbuvir/velpatasvir/voxilaprevir for 8 weeks was noninferior to sofosbuvir/velpatasvir for 12 weeks in patients with HCV genotype 1 through 6 who had no prior exposure to direct-acting antivirals (DAAs). POLARIS-3 demonstrated that sofosbuvir/velpatasvir/voxilaprevir for 8 weeks was as effective as sofosbuvir/velpatasvir for 12 weeks in patients with HCV genotype 3 and compensated cirrhosis who had no prior exposure to DAAs. POLARIS-4 demonstrated that sofosbuvir/velpatasvir/voxilaprevir was as effective as sofosbuvir/velpatasvir for 12 weeks in patients with HCV genotype 1 through 3 who had prior exposure to DAAs but not an NS5A inhibitor. The most common adverse reactions were headache, fatigue, diarrhea, and nausea. Sofosbuvir/velpatasvir/voxilaprevir is safe and effective to treat HCV in patients who have previously been treated with DAAs.

  2. Screening of cellular proteins that interact with the classical swine ...

    Indian Academy of Sciences (India)

    2014-01-27

    Jan 27, 2014 ... 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, ...

  3. A novel indirect ELISA for diagnosis of dengue fever

    Directory of Open Access Journals (Sweden)

    Rohan Narayan

    2016-01-01

    Full Text Available Background & objectives: Dengue fever (DF is associated with significant morbidity and mortality in the tropical and sub-tropical regions of the world. Since there are no effective antiviral drugs for treatment, clinicians often rely on the accurate diagnosis of dengue fever to begin supportive therapy at early stages of the illness. The objective of this study was to develop an in-house dengue virus serotype 2 (DENV-2 non-structural protein- 5 (NS5 based indirect ELISA. Methods: DENV-2 was raised in Vero cells and the viral proteins were separated and subsequently the NS5 protein was eluted. Serum samples from primary and secondary dengue fever patients; and acute and convalescent samples from Japanese encephalitis (JE and West Nile virus (WNV cases were used to validate the ELISA. Results: The assay was found to be 100 per cent specific in detecting DENV-2 specific antibodies from patient′s serum. However, in terms of sensitivity, the assay could detect IgM antibodies only from 90 per cent of the primary dengue samples. The IgM/IgG ratio of the primary and secondary samples was 7.24 and 0.64, respectively. Interpretation & conclusions: The results indicate that the DENV-2 NS5 ELISA is dengue group specific and can be used to differentiate dengue infection from other circulating Flavivirus infections. This NS5 ELISA can also be used to distinguish between primary and secondary dengue fever on the basis of IgM/IgG ratios. Further studies with larger sample sizes and different DENV serotypes are required to validate the ELISA.

  4. A trigger system using cathode read-out chambers and fast computing of kinematical quantities

    CERN Document Server

    Boucrot, J; Caillet, J; Callot, O; Delcros, P; Dube, R; Hrisoho, A; Lefrançois, J

    1980-01-01

    The authors describe a system of cathode read-out multiwire proportional chambers which performs the trigger of a spectrometer installed at the CERN SPS to study high mass dimuon pairs. These chambers, associated with fast ECL circuitry, allow to select particles with high transverse momentum in less than 110 ns. A further development of the electronics will permit to trigger on high dimuon masses with a response time of the order of 170 ns. (5 refs).

  5. 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.

  6. Successful Retreatment of Chronic HCV Genotype-1 Infection With Ledipasvir and Sofosbuvir After Initial Short Course Therapy With Direct-Acting Antiviral Regimens.

    Science.gov (United States)

    Wilson, Eleanor M; Kattakuzhy, Sarah; Sidharthan, Sreetha; Sims, Zayani; Tang, Lydia; McLaughlin, Mary; Price, Angie; Nelson, Amy; Silk, Rachel; Gross, Chloe; Akoth, Elizabeth; Mo, Hongmei; Subramanian, G Mani; Pang, Phillip S; McHutchison, John G; Osinusi, Anu; Masur, Henry; Kohli, Anita; Kottilil, Shyam

    2016-02-01

    The optimal retreatment strategy for chronic hepatitis C virus (HCV) patients who fail directly-acting antiviral agent (DAA)-based treatment is unknown. In this study, we assessed the efficacy and safety of ledipasvir (LDV) and sofosbuvir (SOF) for 12 weeks in HCV genotype-1 (GT-1) patients who failed LDV/SOF-containing therapy. In this single-center, open-label, phase 2a trial, 34 participants with HCV (GT-1) and early-stage liver fibrosis who previously failed 4-6 weeks of LDV/SOF with GS-9669 and/or GS-9451 received LDV/SOF for 12 weeks. The primary endpoint was HCV viral load below the lower limit of quantification 12 weeks after completion of therapy (sustained virological response [SVR]12). Deep sequencing of the NS3, NS5A, and NS5B regions were performed at baseline, at initial relapse, prior to retreatment, and at second relapse with Illumina next-generation sequencing technology. Thirty-two of 34 enrolled participants completed therapy. Two patients withdrew after day 0. Participants were predominantly male and black, with median baseline HCV viral load of 1.3 × 10(6) IU/mL and Metavir fibrosis stage 1 and genotype-1a. Median time from relapse to retreatment was 22 weeks. Prior to retreatment, 29 patients (85%) had NS5A-resistant variants. The SVR12 rate was 91% (31/34; intention to treat, ITT) after retreatment. One patient relapsed. In patients who previously failed short-course combination DAA therapy, we demonstrate a high SVR rate in response to 12 weeks of LDV/SOF, even for patients with NS5A resistance-associated variants. NCT01805882. Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  7. Ledipasvir and sofosbuvir for untreated HCV genotype 1 infection.

    OpenAIRE

    Afdhal, Nezam; Zeuzem, Stefan; Kwo, Paul Y.; Chojkier, Mario; Gitlin, Norman; Puoti, Massimo; Romero Gomez, Manuel; Zarski, Jean Pierre; Agarwal, Kosh; Buggisch, Peter; Foster, Graham R.; Bräu, Norbert; Buti, Maria; Jacobson, Ira M.; Subramanian, G.Mani

    2014-01-01

    BACKGROUND: In phase 2 studies, treatment with the all-oral combination of the nucleotide polymerase inhibitor sofosbuvir and the NS5A inhibitor ledipasvir resulted in high rates of sustained virologic response among previously untreated patients with hepatitis C virus (HCV) genotype 1 infection. METHODS: We conducted a phase 3, open-label study involving previously untreated patients with chronic HCV genotype 1 infection. Patients were randomly assigned in a 1:1:1:1 ratio to receive ledipasv...

  8. 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.

  9. Simple protocol for population (Sanger sequencing for Zika virus genomic regions

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    Gabriela Bastos Cabral

    2017-11-01

    Full Text Available BACKGROUND A number of Zika virus (ZIKV sequences were obtained using Next-generation sequencing (NGS, a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTIVE The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5. In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. METHODS Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. FINDINGS Seven complete ZIKV envelope protein (1,571 kb and six partial NS5 (0,798 Kb were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. MAIN CONCLUSIONS The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.

  10. Simple protocol for population (Sanger) sequencing for Zika virus genomic regions.

    Science.gov (United States)

    Cabral, Gabriela Bastos; Ferreira, João Leandro de Paula; Souza, Renato Pereira de; Cunha, Mariana Sequetin; Luchs, Adriana; Figueiredo, Cristina Adelaide; Brígido, Luís Fernando de Macedo

    2018-01-01

    A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.

  11. 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

  12. AGOR 28: SIO Shipyard Representative Bi-Weekly Progress Report

    Science.gov (United States)

    2015-07-02

    for Sally Ride. ii. Working on NS5 Hierarchy 4. Operator Concerns: • Acoustic Tiles – No additional tiles have been removed this reporting...period. The yard has covered the tiles in the HVAC and MSD spaces with insulation and Quad=zero. The yard has covered the tiles on the upper level of...are the on only areas to have exposed tile that is to be covered by insulation and Quad-zero. Port Side Main Machinery Room, Upper Level

  13. Naturally Occurring Resistance-Associated Variants to Hepatitis C Virus Direct-Acting Antiviral Agents in Treatment-Naive HCV Genotype 6a-Infected Patients

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    Zhanyi Li

    2017-01-01

    Full Text Available Background and Objective. The direct-acting antiviral agents (DAAs antiviral therapy has drastically improved the prognosis of hepatitis C virus (HCV patients. However, the viral drug resistance-associated variants (RAVs can limit the efficacy of DAAs. For the HCV-6a is not the predominant prevalent genotype; the data on the prevalence of naturally occurring RAVs in it is scarce. Our study aims to assess the prevalence of RAVs in treatment-naive HCV-6a patients. Methods. Nested PCR assays were performed on 95 HCV-6a patients to amplify HCV viral regions of NS3, NS5A, and NS5B. Results. In NS3/4A region, we detected Q80K in 95.5% isolates (84/88 and D168E in 2.3% isolates (2/88. In NS5A region, we detected Q30R in 93.2% isolates (82/88, L31M in 4.6% isolates (4/88, and H58P in 6.8% isolates (6/88. In NS5B region, we detected A15G in 2.3% isolates (2/88, S96T in 1.1% isolates (1/88, and S282T in 20.7% isolates (17/88 and we detected I482L in 100% isolates (4/4, V494A in 50% isolates (2/4, and V499A in 100% isolates (4/4. Conclusions. RAVs to DAAs preexist in treatment-naive HCV-6a patients. Further studies should address the issue of the impact of RAVs in response to DAA therapies for HCV-6a patients.

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

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    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.

  15. The practical management of chronic hepatitis C infection in Japan - dual therapy of daclatasvir + asunaprevir.

    Science.gov (United States)

    Hayes, C Nelson; Imamura, Michio; Chayama, Kazuaki

    2017-02-01

    Without treatment, many of the 200 million people worldwide with chronic hepatitis C virus (HCV) infection will develop cirrhosis or liver cancer. Japan was the first nation to approve an interferon-free therapy for HCV, and sustained viral response (SVR) rates >90% have been achieved with asunaprevir, a protease inhibitor, plus daclatasvir, an inhibitor of the non-structural 5A (NS5A) protein. Areas covered: This review provides an overview of the results from both clinical trials and real world experience with asunaprevir and daclatasvir therapy focused primarily on Japan. A literature search using the keywords 'asunaprevir,' 'daclatasvir,' 'interferon-free therapy,' and 'direct-acting antiviral drugs' was initially used to select relevant literature for inclusion in the review. Expert commentary: While not approved in the United States, dual therapy with asunaprevir plus daclatasvir has already been successfully used in Japan and throughout East Asia to treat many thousands of patients. Pre-existing or treatment-emergent NS5A-Y93 or -L31 resistance-associated variants (RAVs) may lead to viral breakthrough, and alternative therapies should be considered for these patients, but patients who harbor NS5A RAVs only at low frequency are likely to achieve SVR. The therapy has also been shown to be safe and effective with renal dysfunction or liver cirrhosis.

  16. Efficacy of daclatasvir in hepatitis C virus.

    Science.gov (United States)

    Izumi, Namiki

    2014-09-01

    Daclatasvir is a novel NS5A inhibitor of hepatitis C virus (HCV). Daclatasvir combined with peginterferon α-2a and ribavirin in Japanese patients infected with genotype 1b HCV achieved sustained virological response (SVR) in 100% of treatment-naïve patients, due to high rates of favorable IL28B allele and genotype 1b. SVR 24 was achieved by asunaprevir and daclatasvir in 87.4% of intolerant and 80.5% of nonresponder patients. Baseline NS5A-resistant variants were detected and they failed to achieve SVR. Most patients with genotype 1a experienced virological breakthrough by dual oral treatment, and should be treated QUAD or replaced by all oral regimens that are more potent and have fewer side effects. IFN-free regimens including daclatasvir and asunaprevir for genotype 1 null responders should be tailored to subtype, and preexisting NS5A-resistant variants should be evaluated carefully before choosing the drugs. This regimen alone is unlikely to move forward without additional agents.

  17. A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity.

    Directory of Open Access Journals (Sweden)

    Hangfei Qi

    2014-04-01

    Full Text Available Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052 as a potent nonstructural protein 5A (NS5A inhibitor for Hepatitis C virus (HCV infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients.

  18. Daclatasvir plus Asunaprevir Treatment for Real-World HCV Genotype 1-Infected Patients in Japan.

    Science.gov (United States)

    Kanda, Tatsuo; Yasui, Shin; Nakamura, Masato; Suzuki, Eiichiro; Arai, Makoto; Haga, Yuki; Sasaki, Reina; Wu, Shuang; Nakamoto, Shingo; Imazeki, Fumio; Yokosuka, Osamu

    2016-01-01

    Background. All-oral combination of direct-acting antivirals could lead to higher sustained virologic response (SVR) in hepatitis C virus (HCV)-infected patients. In the present study, we examined the efficacy and safety of the dual oral treatment with HCV nonstructural protein (NS) 5A inhibitor daclatasvir (DCV) plus HCV NS3/4A inhibitor asunaprevir (ASV) for 24 weeks in real-world HCV genotype 1-infected Japanese individuals. Methods. After screening for HCV NS5A resistance-associated variants (RAVs) by PCR invader assay, a total of 54 Japanese patients infected with HCV genotype 1 treated with DCV plus ASV were retrospectively analyzed. SVR12 was used for evaluation of the virologic response. Results. Of the total 54 patients, 46 patients (85.2%) were treated with DCV plus ASV for 24 weeks and achieved SVR12. The other 8 patients (14.8%) discontinued this treatment before 24 weeks due to adverse events. Of these 8 patients, 5 and 3 patients did and did not achieve SVR12, respectively. Finally, 51 of 54 (94.4%) patients achieved SVR12. Conclusion. Treatment with DCV and ASV after screening for HCV NS5A RAVs by PCR invader assay is effective and safe in the treatment of real-world HCV genotype 1-infected patients in Japan.

  19. 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.

  20. 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...

  1. Post-translational modifications of hepatitis C viral proteins and their biological significance.

    Science.gov (United States)

    Hundt, Jana; Li, Zhubing; Liu, Qiang

    2013-12-21

    Replication of hepatitis C virus (HCV) depends on the interaction of viral proteins with various host cellular proteins and signalling pathways. Similar to cellular proteins, post-translational modifications (PTMs) of HCV proteins are essential for proper protein function and regulation, thus, directly affecting viral life cycle and the generation of infectious virus particles. Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positive-stranded RNA genome. The key modifications include the regulated intramembranous proteolytic cleavage of core protein, disulfide bond formation of core, glycosylation of HCV envelope proteins E1 and E2, methylation of nonstructural protein 3 (NS3), biotinylation of NS4A, ubiquitination of NS5B and phosphorylation of core and NS5B. Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well. For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3, we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear. In this review, we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.

  2. 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.

  3. 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 timeZIKV infection and for monitoring immune responses in vaccine trials. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. A Multiplex Microsphere Immunoassay for Zika Virus Diagnosis

    Directory of Open Access Journals (Sweden)

    Susan J. Wong

    2017-02-01

    Full Text Available 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 < 4 h and requires small specimen volume (10 μl 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.

  5. Phylogenesys and homology modeling in Zika virus epidemic: food for thought.

    Science.gov (United States)

    Angeletti, Silvia; Lo Presti, Alessandra; Giovanetti, Marta; Grifoni, Alba; Amicosante, Massimo; Ciotti, Marco; Alcantara, Luiz-Carlos J; Cella, Eleonora; Ciccozzi, Massimo

    Zika virus (ZIKV) is an emerging Flavivirus that have recently caused an outbreak in Brazil and rapid spread in several countries. In this study, the consequences of ZIKV evolution on protein recognition by the host immune system have been analyzed. Evolutionary analysis was combined with homology modeling and T-B cells epitope predictions. Two separate clades, the African one with the Uganda sequence, as the most probable ancestor, and the second one containing all the most recent sequences from the equatorial belt were identified. Brazilian strains clustered all together and closely related to the French Polynesia isolates. A strong presence of a negatively selected site in the envelope gene (Env) protein was evidenced, suggesting a probable purging of deleterious polymorphisms in functionally important genes. Our results show relative conservancy of ZIKV sequences when envelope and other non-structural proteins (NS3 and NS5) are analyzed by homology modeling. However, some regions within the consensus sequence of NS5 protein and to a lesser extent in the envelope protein, show localized high mutation frequency corresponding to a considerable alteration in protein stability. In terms of viral immune escape, envelope protein is under a higher selective pressure than NS5 and NS3 proteins for HLA class I and II molecules. Moreover, envelope mutations that are not strictly related to T-cell immune responses are mostly located on the surface of the protein in putative B-cell epitopes, suggesting an important contribution of B cells in the immune response as well.

  6. Characterization of a novel flavivirus isolated from Culex (Melanoconion) ocossa mosquitoes from Iquitos, Peru.

    Science.gov (United States)

    Evangelista, Julio; Cruz, Cristhopher; Guevara, Carolina; Astete, Helvio; Carey, Cristiam; Kochel, Tadeusz J; Morrison, Amy C; Williams, Maya; Halsey, Eric S; Forshey, Brett M

    2013-06-01

    We describe the isolation and characterization of a novel flavivirus, isolated from a pool of Culex (Melanoconion) ocossa Dyar and Knab mosquitoes collected in 2009 in an urban area of the Amazon basin city of Iquitos, Peru. Flavivirus infection was detected by indirect immunofluorescent assay of inoculated C6/36 cells using polyclonal flavivirus antibodies (St. Louis encephalitis virus, yellow fever virus and dengue virus type 1) and confirmed by RT-PCR. Based on partial sequencing of the E and NS5 gene regions, the virus isolate was most closely related to the mosquito-borne flaviviruses but divergent from known species, with less than 45 and 71 % pairwise amino acid identity in the E and NS5 gene products, respectively. Phylogenetic analysis of E and NS5 amino acid sequences demonstrated that this flavivirus grouped with mosquito-borne flaviviruses, forming a clade with Nounané virus (NOUV). Like NOUV, no replication was detected in a variety of mammalian cells (Vero-76, Vero-E6, BHK, LLCMK, MDCK, A549 and RD) or in intracerebrally inoculated newborn mice. We tentatively designate this genetically distinct flavivirus as representing a novel species, Nanay virus, after the river near where it was first detected.

  7. 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

    (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......, 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...

  8. Comments On String Propagation Near Defects

    CERN Document Server

    Sahakyan, D

    2004-01-01

    This thesis investigates various aspects of string propagation near defects. In particular we study the effective theory describing NS5 branes in certain decoupling limit gs → 0, which is called Little String Theory (LST). The LST has a holographical description in terms of closed strings propagating in the near horizon geometry of NS5 branes. Using this description, we show that the high energy thermodynamics of LST is unstable; we exhibit a mode localized near the horizon of the black hole, which has mass that vanishes at high energy. We argue that the high temperature phase of the theory involves the condensation of this mode. Another aspect of the LST, which is addressed in the thesis, is the topological version of this theory. We argue that the topological LST is described in terms of N = 2 string in the background of NS5 branes. We show that this topological string can be used to efficiently compute the half-BPS F 4 terms in the low-energy effective action of the LST. Using the strong-weak c...

  9. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Directory of Open Access Journals (Sweden)

    Kengo Morohashi

    Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  10. Abbeylands Nursing Home and Alzheimers Unit, Carhoo, Kildorrery, Cork

    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.

  11. Resistance analysis of hepatitis C virus genotype 1 prior treatment null responders receiving daclatasvir and asunaprevir.

    Science.gov (United States)

    McPhee, Fiona; Hernandez, Dennis; Yu, Fei; Ueland, Joseph; Monikowski, Aaron; Carifa, Arlene; Falk, Paul; Wang, Chunfu; Fridell, Robert; Eley, Timothy; Zhou, Nannan; Gardiner, David

    2013-09-01

    In a sentinel cohort, hepatitis C virus (HCV) patients (primarily genotype [GT] 1a) were treated with daclatasvir (NS5A inhibitor) and asunaprevir (NS3 protease inhibitor). Preexistence, emergence, and persistence of resistance variants in patients who failed this treatment are described. HCV-infected null responders received daclatasvir (60 mg once daily) and asunaprevir (600 mg twice daily) alone (Group A, 11 patients) or with peginterferon alfa-2a and ribavirin (Group B, 10 patients) for 24 weeks. Resistance testing was performed on baseline samples and samples with HCV RNA ≥1,000 IU/mL at Week 1 through posttreatment Week 48. Resistance substitution susceptibility to inhibition by asunaprevir and daclatasvir was assessed using HCV replicon assays. In Group A, six GT1a patients experiencing viral breakthrough and one GT1a patient who relapsed had detectable NS5A (Q30E/R, L31V/M, Y93C/N) and NS3 (R155K, D168A/E/V/Y) resistance-associated variants at failure. Two of six viral breakthrough patients achieved SVR48 after treatment intensification with peginterferon alfa-2a and ribavirin. For 2/4 viral breakthrough patients not responding to treatment intensification, NS3 resistance variants changed (D168Y to D168T; R155K to V36M-R155K). At posttreatment Week 48, daclatasvir-resistant variants persisted while asunaprevir-resistant variants were generally replaced by wild-type sequences. The NS3 sequence remained unchanged in the one patient with NS3-R155K at baseline, relapse, and posttreatment Week 48. In Group B, no viral breakthrough was observed. The treatment failure of daclatasvir and asunaprevir in HCV GT1a patients was associated with both NS5A and NS3 resistance variants in prior null responders. NS5A resistance variants persisted while NS3 resistance variants generally decayed, suggesting a higher relative fitness of NS5A variants. Copyright © 2013 by the American Association for the Study of Liver Diseases.

  12. 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

  13. Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing.

    Science.gov (United States)

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-05-01

    With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test. Copyright © 2017 American Society for Microbiology.

  14. Ultradeep Sequencing Study of Chronic Hepatitis C Virus Genotype 1 Infection in Patients Treated with Daclatasvir, Peginterferon, and Ribavirin

    Science.gov (United States)

    Murakami, Eisuke; Imamura, Michio; Hayes, C. Nelson; Abe, Hiromi; Hiraga, Nobuhiko; Honda, Yoji; Ono, Atsushi; Kosaka, Keiichi; Kawaoka, Tomokazu; Tsuge, Masataka; Aikata, Hiroshi; Takahashi, Shoichi; Miki, Daiki; Ochi, Hidenori; Matsui, Hirotaka; Kanai, Akinori; Inaba, Toshiya; McPhee, Fiona

    2014-01-01

    Direct-acting antivirals (DAAs) are either part of the current standard of care or are in advanced clinical development for the treatment of patients chronically infected with hepatitis C virus (HCV) genotype 1, but concern exists with respect to the patients who fail these regimens with emergent drug-resistant variants. In the present study, ultradeep sequencing was performed to analyze resistance to daclatasvir (DCV), which is a highly selective nonstructural protein 5A (NS5A) inhibitor. Eight patients with HCV genotype 1b, who were either treatment naive or prior nonresponders to pegylated interferon plus ribavirin (Rebetol; Schering-Plough) (PEG-IFN/RBV) therapy, were treated with DCV combined with PEG-IFN alpha-2b (Pegintron; Schering-Plough, Kenilworth, NJ) and RBV. To identify the cause of viral breakthrough, the preexistence and emergence of DCV-resistant variants at NS5A amino acids were analyzed by ultradeep sequencing. Sustained virological response (SVR) was achieved in 6 of 8 patients (75%), with viral breakthrough occurring in the other 2 patients (25%). DCV-resistant variant Y93H preexisted as a minor population at higher frequencies (0.1% to 0.5%) in patients who achieved SVR. In patients with viral breakthrough, DCV-resistant variant mixtures emerged at NS5A-31 over time that persisted posttreatment with Y93H. Although enrichment of DCV-resistant variants was detected, the preexistence of a minor population of the variant did not appear to be associated with virologic response in patients treated with DCV/PEG-IFN/RBV. Ultradeep sequencing results shed light on the complexity of DCV-resistant quasispecies emerging over time, suggesting that multiple resistance pathways are possible within a patient who does not rapidly respond to a DCV-containing regimen. (This study has been registered at ClinicalTrials.gov under registration no. NCT01016912.) PMID:24468783

  15. Efficacy and safety of ledipasvir/sofosbuvir with ribavirin in chronic hepatitis C patients who failed daclatasvir/asunaprevir therapy: pilot study.

    Science.gov (United States)

    Kawakami, Yoshiiku; Ochi, Hidenori; Hayes, Clair Nelson; Imamura, Michio; Tsuge, Masataka; Nakahara, Takashi; Katamura, Yoshio; Kohno, Hiroshi; Kohno, Hirotaka; Tsuji, Keiji; Takaki, Shintaro; Mori, Nami; Honda, Yohji; Arataki, Keiko; Takahashi, Shoichi; Kira, Shinsuke; Tamura, Toru; Masuda, Kazunari; Nakamura, Toshio; Kikkawa, Masaya; Chayama, Kazuaki

    2017-08-16

    In Japan, daclatasvir (DCV) and asunaprevir (ASV) therapy was the first IFN-free treatment to be approved, and thousands of patients have since been successfully treated, with an SVR rate of around 90%. The converse, however, is that around 10% of patients fail to achieve viral eradication and must be retreated using a different approach. This study is to evaluate treatment efficacy of ledipasvir/sofosbuvir and ribavirin in patients who failed to respond to DCV and ASV therapy. Thirty patients were treated with 12 weeks of ledipasvir/sofosbuvir and ribavirin. We evaluated the rate of sustained virological response 12 weeks after the end of treatment (SVR 12 ) and examined the incidence of adverse events during ledipasvir/sofosbuvir and ribavirin treatment. NS5A and NS5B resistance-associated variants (RAVs) in treatment failure cases were examined. The overall SVR 12 rate was 86.7% (26/30). Large decreases in mean log 10 HCV RNA levels were observed in patients without cirrhosis, and the SVR 12 rate for these patients was 100% (12/12). In cases of cirrhosis, SVR 12 rate was 72.2% (13/18). The common factors in treatment failure cases were the presence of liver cirrhosis and both NS5A L31M/I and Y93H RAVs. The frequency of RAVs did not change before and after treatment among patients who relapsed. Ledipasvir/sofosbuvir with ribavirin is an effective retreatment option for patients with chronic hepatitis C who failed to respond to prior daclatasvir and asunaprevir therapy.

  16. Natural Polymorphisms Conferring Resistance to HCV Protease and Polymerase Inhibitors in Treatment-Naïve 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.

  17. 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.

  18. 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

  19. Surface Conductivity of Biological Macromolecules Measured by Nanopipette Dielectrophoresis

    Science.gov (United States)

    Clarke, Richard W.; Piper, Joe D.; Ying, Liming; Klenerman, David

    2007-05-01

    We report the measurement of the surface conductivity of biological macromolecules by dielectrophoretic trapping at the tip of a glass nanopipet. We find that the threshold voltage for trapping is a function of salt concentration and can be directly linked to the effective conductivity of the biomolecule and its solvation shell. The surface conductivities obtained for 20-mer single-stranded DNA, 40-mer double-stranded DNA, and yellow fluorescent protein are 7.9±1.9nS, 5.3±0.7nS, and 21.5±1.6nS, respectively.

  20. Twistor approach to string compactifications: A review

    Energy Technology Data Exchange (ETDEWEB)

    Alexandrov, Sergei, E-mail: salexand@univ-montp2.fr

    2013-01-01

    We review a progress in obtaining the complete non-perturbative effective action of type II string theory compactified on a Calabi–Yau manifold. This problem is equivalent to understanding quantum corrections to the metric on the hypermultiplet moduli space. We show how all these corrections, which include D-brane and NS5-brane instantons, are incorporated in the framework of the twistor approach, which provides a powerful mathematical description of hyperkähler and quaternion-Kähler manifolds. We also present new insights on S-duality, quantum mirror symmetry, connections to integrable models and topological strings.

  1. Modularity, quaternion-Kähler spaces, and mirror symmetry

    Energy Technology Data Exchange (ETDEWEB)

    Alexandrov, Sergei; Banerjee, Sibasish [Université Montpellier 2, Laboratoire Charles Coulomb UMR 5221, F-34095 Montpellier (France)

    2013-10-15

    We provide an explicit twistorial construction of quaternion-Kähler manifolds obtained by deformation of c-map spaces and carrying an isometric action of the modular group SL(2,Z). The deformation is not assumed to preserve any continuous isometry and therefore this construction presents a general framework for describing NS5-brane instanton effects in string compactifications with N= 2 supersymmetry. In this context the modular invariant parametrization of twistor lines found in this work yields the complete non-perturbative mirror map between type IIA and type IIB physical fields.

  2. Surface conductivity of biological macromolecules measured by nanopipette dielectrophoresis.

    Science.gov (United States)

    Clarke, Richard W; Piper, Joe D; Ying, Liming; Klenerman, David

    2007-05-11

    We report the measurement of the surface conductivity of biological macromolecules by dielectrophoretic trapping at the tip of a glass nanopipet. We find that the threshold voltage for trapping is a function of salt concentration and can be directly linked to the effective conductivity of the biomolecule and its solvation shell. The surface conductivities obtained for 20-mer single-stranded DNA, 40-mer double-stranded DNA, and yellow fluorescent protein are 7.9+/-1.9 nS, 5.3+/-0.7 nS, and 21.5+/-1.6 nS, respectively.

  3. c-Map as c=1 string

    Energy Technology Data Exchange (ETDEWEB)

    Alexandrov, Sergei, E-mail: Sergey.Alexandrov@univ-montp2.fr [Universite Montpellier 2 and CNRS, Laboratoire Charles Coulomb UMR 5221, F-34095, Montpellier (France)

    2012-10-01

    We show the existence of a duality between the c-map space describing the universal hypermultiplet at tree level and the matrix model description of two-dimensional string theory compactified at a self-dual radius and perturbed by a sine-Liouville potential. It appears as a particular case of a general relation between the twistor description of four-dimensional quaternionic geometries and the Lax formalism for Toda hierarchy. Furthermore, we give an evidence that the instanton corrections to the c-map metric coming from NS5-branes can be encoded into the Baker-Akhiezer function of the integrable hierarchy.

  4. D-Branes in the Background of NS Fivebranes

    CERN Document Server

    Elitzur, Shmuel; Rabinovici, Eliezer; Sarkisian, G; Kutasov, D; Elitzur, Shmuel; Giveon, Amit; Kutasov, David; Rabinovici, Eliezer; Sarkissian, Gor

    2000-01-01

    We study the dynamics of $D$-branes in the near-horizon geometry of $NS$ fivebranes. This leads to a holographically dual description of the physics of $D$-branes ending on and/or intersecting $NS5$-branes. We use it to verify some properties of such $D$-branes which were deduced indirectly in the past, and discuss some instabilities of non-supersymmetric brane configurations. Our construction also describes vacua of Little String Theory which are dual to open plus closed string theory in asymptotically linear dilaton spacetimes.

  5. Characterization of a Tembusu virus isolated from naturally infected house sparrows (Passer domesticus) in Northern China.

    Science.gov (United States)

    Tang, Y; Diao, Y; Yu, C; Gao, X; Ju, X; Xue, C; Liu, X; Ge, P; Qu, J; Zhang, D

    2013-04-01

    The house sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Tembusu virus (TMUV) strain, TMUV-SDHS, was isolated from house sparrows living around the poultry farms in Shandong Province, Northern China. Genetic analysis of E and NS5 genes showed that it had a close relationship with that of the YY5 strain, which can cause severe egg drop in ducks. Pathogenicity studies showed that the virus is highly virulent when experimentally inoculated into the ducks. These findings show that house sparrows carrying the Tembusu virus may play an important role in transmitting the virus among other species. © 2012 Blackwell Verlag GmbH.

  6. 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...15500 41 10000 14000 42 11500 Is500 43 16500 22000 1 15000 20000 52 16000 22000 53 21000 28500 16 At1. 19500 2650 16 Gulf 16500 25500 17 Atl. 20500

  7. Sierra Army Depot, Phase 1, Remedial Investigation/Feasibility Study, Lassen County, California. Appendices G-Q

    Science.gov (United States)

    1991-10-01

    b) lam..-a.o.101*1. ISISI OF tPICI ION$ (roeld .1l~ .5* il ... J. Col. A) ~ i1ai w.* le d m1 41,7 "" I I I:, 5 *NS5..I..j~.# CUP , l~b (ZgnufL~Job...and Kirschner, 1985) Investigations in humans following prenatal exposure to mercury suggests increased spontaneous abortion and menstrual ...severe symptoms such as methemoglobinemia, jaundice, aplastic anemia, cataract formation, menstrual disorders, neurological dysfunction and nephrotoxicity

  8. Identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction.

    Science.gov (United States)

    Pierre, V; Drouet, M T; Deubel, V

    1994-01-01

    A reverse transcription/polymerase chain reaction (RT/PCR) protocol for the rapid detection and identification of flaviviruses was developed using a set of universal oligonucleotide primers. These primers correspond to sequences in the 3' non-coding region and in the NS5 gene which are highly conserved among the mosquito-borne flaviviruses. The sequences of the resulting amplified products were analysed for dengue 1, dengue 2, dengue 3, dengue 4, Japanese encephalitis, West Nile, yellow fever and Zika viruses, and compared with the published sequences of other flaviviruses. The 291-297 nucleotides corresponding to the C-terminus of NS5 gene showed 56 to 76% similarity, whereas the 3' non-coding region (190 to 421 nucleotides) showed only 20 to 36% similarity. Genetic classification of the Zika virus supported its traditional serological grouping. Recombinant plasmids containing the flavivirus sequences were used in a nucleic acid hybridization test to identify the RT/PCR products derived from viral RNA extracted from experimentally infected mosquitoes. The plasmids were dotted on a strip of nitrocellulose membrane and incubated with the RT/PCR product labelled with digoxigenin during the PCR step. This is a valuable method for the rapid and specific identification of mosquito-borne flaviviruses in biological specimens and for subsequent sequence analysis.

  9. Successful treatment of three patients with human immunodeficiency virus and hepatitis C virus genotype 1b co-infection by daclatasvir plus asunaprevir.

    Science.gov (United States)

    Hirashima, Noboru; Iwase, Hiroaki; Shimada, Masaaki; Ryuge, Nobumitsu; Imamura, Junji; Ikeda, Hiroki; Tanaka, Yasuhito; Matsumoto, Nobuyuki; Okuse, Chiaki; Itoh, Fumio; Yokomaku, Yoshiyuki; Watanabe, Tsunamasa

    2017-02-01

    Co-infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) often accelerates the course of HCV-associated liver disease. Daclatasvir (DCV) plus asunaprevir (ASV) have been shown to be highly effective for HCV-infected patients with genotype 1b. Three patients co-infected with HIV/HCV genotype 1b were enrolled in this study. Prior to initiation of HCV treatment, the variants associated with L31 and Y93 in the non-structural protein 5A (NS5A) region of the HCV genome were confirmed to be absent using a direct sequencing method. Taking into consideration the lower risk of drug-drug interaction and the need for immediate treatment, the patients received 60 mg DCV once daily plus 100 mg ASV twice daily for 24 weeks. In one patient, the alanine aminotransferase level was elevated to 228 IU/L at 24 weeks after the start of treatment, but he completed the 24-week treatment course. All three patients achieved sustained viral response, without severe complications (including HIV virological rebound). Thus, in cases where NS5A variants are confirmed to be absent and patients are antiretroviral therapy-naïve, with CD4+ over 500/μL or HIV well controlled by RAL-based cART, DCV plus ASV may represent a good treatment option for HIV and HCV genotype 1b co-infected patients.

  10. Safety and efficacy of dual therapy with daclatasvir and asunaprevir for older patients with chronic hepatitis C.

    Science.gov (United States)

    Morio, Reona; Imamura, Michio; Kawakami, Yoshiiku; Morio, Kei; Kobayashi, Tomoki; Yokoyama, Satoe; Kimura, Yuki; Nagaoki, Yuko; Kawaoka, Tomokazu; Tsuge, Masataka; Hiramatsu, Akira; Nelson Hayes, C; Aikata, Hiroshi; Takahashi, Shoichi; Miki, Daiki; Ochi, Hidenori; Mori, Nami; Takaki, Shintaro; Tsuji, Keiji; Chayama, Kazuaki

    2017-04-01

    Daclatasvir and asunaprevir combination therapy has shown a high virological response for chronic genotype 1 hepatitis C virus (HCV)-infected patients. However, the safety and efficacy of the therapy for older patients are unknown. One hundred seventy patients younger than 75 years and 139 patients aged 75 years or older with genotype 1 HCV infection were treated for 24 weeks with daclatasvir plus asunaprevir. Pretreatment drug-resistance-associated variants at NS5A-L31 and NS5A-Y93 were determined by the Invader assay. Virological response and adverse events according to age were analyzed. The sustained virological response (SVR) rate for older patients was similar to that for younger patients (97.1 and 92.4 % respectively). In multivariate regression analysis, prior simeprevir treatment (odds ratio 56.6 for absence; P daclatasvir plus asunaprevir therapy similar to those of younger patients. Even though RAVs were detected, virological response similar to that for patients with no detectable RAVs may still be expected for patients with RAVs as long as the population frequency is low.

  11. 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

  12. DWSB in heterotic flux compactifications

    CERN Document Server

    Held, Johannes; Marchesano, Fernando; Martucci, Luca

    2010-01-01

    We address the construction of non-supersymmetric vacua in heterotic compactifications with intrinsic torsion and background fluxes. In particular, we implement the approach of domain-wall supersymmetry breaking (DWSB) previously developed in the context of type II flux compactifications. This approach is based on considering backgrounds where probe NS5-branes wrapping internal three-cycles and showing up as four-dimensional domain-walls do not develop a BPS bound, while all the other BPS bounds characterizing the N=1 supersymmetric compactifications are preserved at tree-level. Via a scalar potential analysis we provide the conditions for these backgrounds to solve the ten-dimensional equations of motion including order \\alpha' corrections. We also consider backgrounds where some of the NS5-domain-walls develop a BPS bound, show their relation to no-scale SUSY-breaking vacua and construct explicit examples via elliptic fibrations. Finally, we consider backgrounds with a non-trivial gaugino condensate and dis...

  13. A comparative analysis of the substrate permissiveness of HCV and GBV-B NS3/4A proteases reveals genetic evidence for an interaction with NS4B protein during genome replication.

    Science.gov (United States)

    Benureau, Yann; Warter, Lucile; Malcolm, Bruce A; Martin, Annette

    2010-10-25

    The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Hepatitis C virus genotypes in Tirana, Albania.

    Science.gov (United States)

    Haldeda, Migena; Baume, Julien; Tamalet, Catherine; Bizhga, Melpomeni; Colson, Philippe

    2014-01-01

    Hepatitis C virus (HCV) infection is a worldwide concern. Knowledge of the HCV genotype is clinically important because it predicts the rate of response to therapy and guides the treatment duration. Moreover, it allows molecular epidemiology to be performed. To our knowledge, the prevalence of HCV genotypes has been assessed only once in Albania, using a line probe genotyping assay. We determined HCV genotypes by population sequencing of HCV-infected patients in Tirana, Albania. HCV genotype and sequence analyses were performed for serum samples collected from January 2011 through May 2012 from 61 HCV-seropositive patients using population sequencing of the NS3 protease gene and alternatively the NS5b gene and the 5' untranslated region (UTR). HCV RNA was retrieved from the blood samples of 50 patients. The HCV NS3 protease gene was sequenced for 28 patients and NS5b and/or 5'UTR fragments were sequenced for an additional 22 patients. The predominant genotype was 1b in 25 patients (50%), followed by genotypes 2c, 4a, 3a, and 1a in 18%, 14%, 8%, and 6% of cases, respectively. Best matches for these HCV RNAs in GenBank were obtained in different countries worldwide. One NS3 protease naturally harbored an amino acid conferring minor drug resistance to newly available HCV protease inhibitors. In conclusion, HCV-1b was predominant in the present Albanian population, as in southeastern Europe. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. 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.

  16. 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.

  17. Oncogenic Potential of Hepatitis C Virus Proteins

    Directory of Open Access Journals (Sweden)

    Ranjit Ray

    2010-09-01

    Full Text Available Chronic hepatitis C virus (HCV infection is a major risk factor for liver disease progression, and may lead to cirrhosis and hepatocellular carcinoma (HCC. The HCV genome contains a single-stranded positive sense RNA with a cytoplasmic lifecycle. HCV proteins interact with many host-cell factors and are involved in a wide range of activities, including cell cycle regulation, transcriptional regulation, cell proliferation, apoptosis, lipid metabolism, and cell growth promotion. Increasing experimental evidences suggest that HCV contributes to HCC by modulating pathways that may promote malignant transformation of hepatocytes. At least four of the 10 HCV gene products, namely core, NS3, NS5A and NS5B play roles in several potentially oncogenic pathways. Induction of both endoplasmic reticulum (ER stress and oxidative stress by HCV proteins may also contribute to hepatocyte growth promotion. The current review identifies important functions of the viral proteins connecting HCV infections and potential for development of HCC. However, most of the putative transforming potentials of the HCV proteins have been defined in artificial cellular systems, and need to be established relevant to infection and disease models. The new insight into the mechanisms for HCV mediated disease progression may offer novel therapeutic targets for one of the most devastating human malignancies in the world today.

  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. Synthesis and Characterization of Celecoxib Derivatives as Possible Anti-Inflammatory, Analgesic, Antioxidant, Anticancer and Anti-HCV Agents

    Directory of Open Access Journals (Sweden)

    Amartya Basu

    2013-03-01

    Full Text Available A series of novel N-(3-substituted aryl/alkyl-4-oxo-1,3-thiazolidin-2-ylidene-4-[5-(4-methylphenyl-3-(trifluoromethyl-1H-pyrazol-1-yl]benzenesulfonamides 2a–e were synthesized by the addition of ethyl a-bromoacetate and anhydrous sodium acetate in dry ethanol to N-(substituted aryl/alkylcarbamothioyl-4-[5-(4-methylphenyl-3-(trifluoro-methyl-1H-pyrazol-1-yl]benzene sulfonamides 1a–e, which were synthesized by the reaction of alkyl/aryl isothiocyanates with celecoxib. The structures of the isolated products were determined by spectral methods and their anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV NS5B RNA-dependent RNA polymerase (RdRp activities evaluated. The compounds were also tested for gastric toxicity and selected compound 1a was screened for its anticancer activity against 60 human tumor cell lines. These investigations revealed that compound 1a exhibited anti-inflammatory and analgesic activities and further did not cause tissue damage in liver, kidney, colon and brain compared to untreated controls or celecoxib. Compounds 1c and 1d displayed modest inhibition of HCV NS5B RdRp activity. In conclusion, N-(ethylcarbamothioyl-4-[5-(4-methylphenyl-3-(trifluoromethyl-1H-pyrazol-1-yl]benzenesulfonamide (1a may have the potential to be developed into a therapeutic agent.

  20. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

    DEFF Research Database (Denmark)

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

    2012-01-01

    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......) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5' untranslated region (5'UTR)-NS5A and JFH1 NS5B-3'UTR; recovered...

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

    Science.gov (United States)

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

    2013-01-01

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

  2. 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.

  3. Ledipasvir plus sofosbuvir for 12 weeks in patients with hepatitis C genotype 4 infection.

    Science.gov (United States)

    Abergel, Armand; Metivier, Sophie; Samuel, Didier; Jiang, Deyuan; Kersey, Kathryn; Pang, Phillip S; Svarovskaia, Evguenia; Knox, Steven J; Loustaud-Ratti, Veronique; Asselah, Tarik

    2016-10-01

    Genotype 4 hepatitis C virus (HCV) was considered difficult to treat in the era of pegylated interferon-alpha (Peg-IFN-α) and ribavirin regimens. We evaluated the efficacy and safety of therapy with the nonstructural (NS) 5A inhibitor, ledipasvir, combined with the NS5B polymerase inhibitor, sofosbuvir, in patients with HCV genotype 4. In this phase 2, open-label study, 44 patients (22 treatment naïve and 22 treatment experienced) received a fixed-dose combination tablet of 90 mg of ledipasvir and 400 mg of sofosbuvir orally once-daily for 12 weeks. The primary endpoint was the percentage of patients with HCV RNA ledipasvir and sofosbuvir is an effective and safe treatment for a wide range of HCV 4 subtypes in both treatment-naïve and -experienced patients, including those with compensated cirrhosis. (EudraCT number: 2013-003978-27; Clinicaltrials.gov NCT02081079) (Hepatology 2016;64:1049-1056). © 2016 by the American Association for the Study of Liver Diseases.

  4. 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. Copyright 2016 Prous Science, S.A.U. or its licensors. All rights reserved.

  5. Combination ledipasvir-sofosbuvir for the treatment of chronic hepatitis C virus infection: a review and clinical perspective.

    Science.gov (United States)

    Nkuize, Marcel; Sersté, Thomas; Buset, Michel; Mulkay, Jean-Pierre

    2016-01-01

    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.

  6. Effect of nitrendipine, nimodipine and nisoldipine on water and electrolytes excretion in rats.

    Science.gov (United States)

    Singh, J; Kaur, A H; Bansal, S; Lal, H

    1996-01-01

    In water loaded (5 ml/100 g) unanesthetized rats. nitrendipine (NT), nimodipine (NM) and nisoldipine (NS) (5 mg/ kg, i.p.) caused significant (P < 0.01) increase in water and Na+ excretion. However, there was no significant increase in K+ excretion after NT, NM and NS administration. NS was more potent in increasing excretion of water load as compared to NT and NM. The glomerular filtration rate as assessed by creatinine clearance, was significantly (P < 0.01) increased in NT, NM and NS (5 mg/kg, i.p.) treated groups as compared to control. The mean creatinine clearance values after NT, NM and NS were 26.95 +/- 0.35, 22.11 +/- 0.72 and 28.13 +/- 0.95 respectively as compared to 22.19 +/- 0.51, 18.77 +/- 0.42 and 22.97 +/- 0.60 in corresponding control groups. The results of the study suggest that in addition to other effects, NT, NM and NS have a selective inhibitory effect on Na+ handling mechanisms in the nephron.

  7. Direct-acting antivirals: the endgame for hepatitis C?

    Science.gov (United States)

    D'Ambrosio, Roberta; Degasperi, Elisabetta; Colombo, Massimo; Aghemo, Alessio

    2017-06-01

    Directly-acting antivirals (DAA) have finally allowed all patients to be potentially cured from chronic hepatitis C (HCV) infection. All-oral, Interferon (IFN)-free regimens are based upon the combination of molecules targeting different sites of the HCV replication process. Three classes of DAA exist: protease inhibitors (anti-NS3/4A), RNA-dependent polymerase inhibitors (anti-NS5B) and anti-NS5A inhibitors, which are characterized by different antiviral potency and barrier to resistance and therefore are usually combined in different treatment schedules. Treatment regimens are still largely dependent on HCV genotype and stage of liver disease, with duration ranging between 12 weeks and 24 weeks, while overall treatment efficacy has climbed to nearly 95% in most patient groups, including historically difficult-to-treat categories (HCV genotype 1, advanced liver disease). The elimination of IFN has allowed safe and efficacious treatment of patients formerly contraindicated to antiviral therapy, such as decompensated cirrhosis and solid organ transplant recipients. Availability of potent and safe antiviral drugs combined with improvement of worldwide access to treatment could finally lead to HCV elimination in the next decades. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Approved Antiviral Drugs over the Past 50 Years

    Science.gov (United States)

    2016-01-01

    SUMMARY Since the first antiviral drug, idoxuridine, was approved in 1963, 90 antiviral drugs categorized into 13 functional groups have been formally approved for the treatment of the following 9 human infectious diseases: (i) HIV infections (protease inhibitors, integrase inhibitors, entry inhibitors, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and acyclic nucleoside phosphonate analogues), (ii) hepatitis B virus (HBV) infections (lamivudine, interferons, nucleoside analogues, and acyclic nucleoside phosphonate analogues), (iii) hepatitis C virus (HCV) infections (ribavirin, interferons, NS3/4A protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors), (iv) herpesvirus infections (5-substituted 2′-deoxyuridine analogues, entry inhibitors, nucleoside analogues, pyrophosphate analogues, and acyclic guanosine analogues), (v) influenza virus infections (ribavirin, matrix 2 protein inhibitors, RNA polymerase inhibitors, and neuraminidase inhibitors), (vi) human cytomegalovirus infections (acyclic guanosine analogues, acyclic nucleoside phosphonate analogues, pyrophosphate analogues, and oligonucleotides), (vii) varicella-zoster virus infections (acyclic guanosine analogues, nucleoside analogues, 5-substituted 2′-deoxyuridine analogues, and antibodies), (viii) respiratory syncytial virus infections (ribavirin and antibodies), and (ix) external anogenital warts caused by human papillomavirus infections (imiquimod, sinecatechins, and podofilox). Here, we present for the first time a comprehensive overview of antiviral drugs approved over the past 50 years, shedding light on the development of effective antiviral treatments against current and emerging infectious diseases worldwide. PMID:27281742

  9. 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.

  10. Structural models for the design of novel antiviral agents against Greek Goat Encephalitis

    Directory of Open Access Journals (Sweden)

    Louis Papageorgiou

    2014-11-01

    Full Text Available The Greek Goat Encephalitis virus (GGE belongs to the Flaviviridae family of the genus Flavivirus. The GGE virus constitutes an important pathogen of livestock that infects the goat’s central nervous system. The viral enzymes of GGE, helicase and RNA-dependent RNA polymerase (RdRP, are ideal targets for inhibitor design, since those enzymes are crucial for the virus’ survival, proliferation and transmission. In an effort to understand the molecular structure underlying the functions of those viral enzymes, the three dimensional structures of GGE NS3 helicase and NS5 RdRP have been modelled. The models were constructed in silico using conventional homology modelling techniques and the known 3D crystal structures of solved proteins from closely related species as templates. The established structural models of the GGE NS3 helicase and NS5 RdRP have been evaluated for their viability using a repertoire of in silico tools. The goal of this study is to present the 3D conformations of the GGE viral enzymes as reliable structural models that could provide the platform for the design of novel anti-GGE agents.

  11. Recurrent hepatitis C after liver transplant

    Science.gov (United States)

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

    2014-01-01

    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. PMID:25152571

  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. Antigenicity of envelop and non-structural proteins of dengue serotypes and their potentiality to elicit specifi antibody

    Directory of Open Access Journals (Sweden)

    Ramesh Venkatachalam

    2015-06-01

    Full Text Available Objective: To find out the antigenic nature of envelop (E and non-structural (NS proteins and their ability to induce specific antibodies, and to investigate specific antibody produced by specific dengue virus (DENV serotypes. Methods: Amino acid sequences of E and NS proteins of dengue serotypes were analysed by using VaxiJen antigen predicition server. The transmembrane of topology analyses were conducted by using transmembrane prediction using hidden markov models. The Hex dock server was used for docking. Results: The antigenicity score and exomembrane potentiality of E and NS proteins were calculated. All those proteins were antigenic; these antigens were made to interact with antibodies such as immunoglobulin A, immunoglobulin G and immunoglobulin M. Higher energy values of immunoglobulin M were found in DENV-1 and DENV-2, and more energy values were found in immunoglobulin G of DENV-3, DENV-4, NS-1, NS-3 and NS-5. Conclusions: In the present study, DENV-1 and DENV-2 are positive to immunoglobulin M and involved in the primary infection. DENV 3, DENV 4 and all the NS proteins (NS-1, NS-3, NS-5 which elicit immunoglobulin G are involved in the secondary infection.

  14. Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging.

    Science.gov (United States)

    Wang, Hongliang; Tai, Andrew W

    2017-01-01

    Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication. In chronically infected cells, it is not known whether these viral replication organelles are being continually resupplied by newly synthesized viral proteins in situ, or whether they are generated de novo. Here we aimed to study temporal events in replication organelles formation and maturation. Here we use pulse-chase labeling in combination with confocal microscopy, correlative light electron microscopy and biochemical methods to identify temporally distinct populations of replication organelles in living cells and study the formation, morphogenesis as well as compositional and functional changes of replication organelles over time. We found that HCV replication organelles are continuously generated de novo at spatially distinct sites from preformed ones. This process is accompanied by accumulated intracellular membrane alteration, increased cholesterol delivery, NS5A phosphorylation, and positive-strand RNA content, and by eventual association with HCV core protein around lipid droplets. Generation of spatially segregated foci requires viral NS5A and the host factors phosphatidylinositol 4-kinase and oxysterol-binding protein, while association of foci with lipid droplets requires cholesterol. Our results reveal that HCV replication organelles are not static structures, but instead are continuously generated and dynamically change in composition and possibly also in function. Hepatitis C virus replication membrane structures are continuously generated at spatially distinct sites. New replication organelles are different in composition, and possibly also in function, compared to old replication organelles. Published by Elsevier B.V.

  15. 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.

  16. Aeginetia indica Decoction Inhibits Hepatitis C Virus Life Cycle.

    Science.gov (United States)

    Lin, Cheng-Wei; Lo, Chieh-Wen; Tsai, Chia-Ni; Pan, Ting-Chun; Chen, Pin-Yin; Yu, Ming-Jiun

    2018-01-09

    Chronic hepatitis C virus (HCV) infection is still a global epidemic despite the introduction of several highly effective direct-acting antivirals that are tagged with sky-high prices. The present study aimed to identify an herbal decoction that ameliorates HCV infection. Among six herbal decoctions tested, the Aeginetia indica decoction had the most profound effect on the HCV reporter activity in infected Huh7.5.1 liver cells in a dose- and time-dependent manner. The Aeginetia indica decoction exerted multiple inhibitory effects on the HCV life cycle. Pretreatment of the cells with the Aeginetia indica decoction prior to HCV infection reduced the HCV RNA and non-structural protein 3 (NS3) protein levels in the infected cells. The Aeginetia indica decoction reduced HCV internal ribosome entry site-mediated protein translation activity. It also reduced the HCV RNA level in the infected cells in association with reduced NS5A phosphorylation at serine 235, a predominant phosphorylation event indispensable to HCV replication. Thus, the Aeginetia indica decoction inhibits HCV infection, translation, and replication. Mechanistically, the Aeginetia indica decoction probably reduced HCV replication via reducing NS5A phosphorylation at serine 235.

  17. Non-geometric five-branes in heterotic supergravity

    Science.gov (United States)

    Sasaki, Shin; Yata, Masaya

    2016-11-01

    We study T-duality chains of five-branes in heterotic supergravity where the first order α'-corrections are present. By performing the α'-corrected T-duality transformations of the heterotic NS5-brane solutions, we obtain the KK5-brane and the exotic 5 2 2 -brane solutions associated with the symmetric, the neutral and the gauge NS5-branes. We find that the Yang-Mills gauge field in these solutions satisfies the self-duality condition in the three- and two-dimensional transverse spaces to the brane world-volumes. The O(2 , 2) monodromy structures of the 5 2 2 -brane solutions are investigated by the α'-corrected generalized metric. Our analysis shows that the symmetric 5 2 2 -brane solution, which satisfies the standard embedding condition, is a T-fold and it exhibits the non-geometric nature. We also find that the neutral 5 2 2 -brane solution is a T-fold at least at O({α}^') . On the other hand, the gauge 5 2 2 -brane solution is not a T-fold but show unusual structures of space-time.

  18. Non-geometric five-branes in heterotic supergravity

    Energy Technology Data Exchange (ETDEWEB)

    Sasaki, Shin; Yata, Masaya [Department of Physics, Kitasato University,Sagamihara 252-0373 (Japan); Department of Physics, National University of Singapore,2, Science Drive 3, Singapore 117542 (Singapore)

    2016-11-10

    We study T-duality chains of five-branes in heterotic supergravity where the first order α{sup ′}-corrections are present. By performing the α{sup ′}-corrected T-duality transformations of the heterotic NS5-brane solutions, we obtain the KK5-brane and the exotic 5{sub 2}{sup 2}-brane solutions associated with the symmetric, the neutral and the gauge NS5-branes. We find that the Yang-Mills gauge field in these solutions satisfies the self-duality condition in the three- and two-dimensional transverse spaces to the brane world-volumes. The O(2,2) monodromy structures of the 5{sub 2}{sup 2}-brane solutions are investigated by the α{sup ′}-corrected generalized metric. Our analysis shows that the symmetric 5{sub 2}{sup 2}-brane solution, which satisfies the standard embedding condition, is a T-fold and it exhibits the non-geometric nature. We also find that the neutral 5{sub 2}{sup 2}-brane solution is a T-fold at least at O(α{sup ′}). On the other hand, the gauge 5{sub 2}{sup 2}-brane solution is not a T-fold but show unusual structures of space-time.

  19. 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.

  20. Collisional deactivation of Ba 5d7p (3)D1 by noble gases.

    Science.gov (United States)

    Smedley, John E; Coulter, Sarah K; Felton, Edward J; Zomlefer, Kayla S

    2008-10-02

    Collisional deactivation of the 5d7p (3)D1 state of Ba by noble gases is studied by time- and wavelength-resolved fluorescence techniques. A pulsed, frequency-doubled dye laser at 273.9 nm excites the 5d7p (3)D1 level from the ground state, and fluorescence at 364.1 and 366.6 nm from the 5d7p (3)D1 --> 6s5d (3)D1 and 5d7p (3)D1 --> 6s5d (3)D2 transitions, respectively, is monitored in real time to obtain the deactivation rate constants. At 835 K these rate constants are as follows: He, (1.69 +/- 0.08) x 10(-9) cm(3) s(-1); Ne, (3.93 +/- 0.14) x 10(-10) cm(3) s(-1); Ar, (4.53 +/- 0.15) x 10(-10) cm(3) s(-1); Kr, (4.64 +/- 0.13) x 10(-10) cm(3) s(-1); Xe, (5.59 +/- 0.22) x 10(-10) cm(3) s(-1). From time-resolved 5d7p (3)D1 emission in the absence of noble gas and from the intercepts of the quenching plots, the lifetime of this state is determined to be 100 +/- 1 ns. Using time- and wavelength-resolved Ba emission with a low background pressure of noble gas, radiative lifetimes of several near-resonant states are determined from the exponential rise of the fluorescence signals. These results are as follows: 5d6d (3)D3, 28 +/- 3 ns; 5d7p (3)P1, 46 +/- 2 ns; 5d6d (3)G3, 21.5 +/- 0.8 ns; 5d7p (3)F3, 48 +/- 1 ns. Integrated fluorescence signals are used to infer the relative rate constants for population transfer from the 5d7p (3)D1 state to eleven near-resonant fine structure states.

  1. Sofosbuvir, Velpatasvir, and Voxilaprevir for Previously Treated HCV Infection.

    Science.gov (United States)

    Bourlière, Marc; Gordon, Stuart C; Flamm, Steven L; Cooper, Curtis L; Ramji, Alnoor; Tong, Myron; Ravendhran, Natarajan; Vierling, John M; Tran, Tram T; Pianko, Stephen; Bansal, Meena B; de Lédinghen, Victor; Hyland, Robert H; Stamm, Luisa M; Dvory-Sobol, Hadas; Svarovskaia, Evguenia; Zhang, Jie; Huang, K C; Subramanian, G Mani; Brainard, Diana M; McHutchison, John G; Verna, Elizabeth C; Buggisch, Peter; Landis, Charles S; Younes, Ziad H; Curry, Michael P; Strasser, Simone I; Schiff, Eugene R; Reddy, K Rajender; Manns, Michael P; Kowdley, Kris V; Zeuzem, Stefan

    2017-06-01

    Patients who are chronically infected with hepatitis C virus (HCV) and who do not have a sustained virologic response after treatment with regimens containing direct-acting antiviral agents (DAAs) have limited retreatment options. We conducted two phase 3 trials involving patients who had been previously treated with a DAA-containing regimen. In POLARIS-1, patients with HCV genotype 1 infection who had previously received a regimen containing an NS5A inhibitor were randomly assigned in a 1:1 ratio to receive either the nucleotide polymerase inhibitor sofosbuvir, the NS5A inhibitor velpatasvir, and the protease inhibitor voxilaprevir (150 patients) or matching placebo (150 patients) once daily for 12 weeks. Patients who were infected with HCV of other genotypes (114 patients) were enrolled in the sofosbuvir-velpatasvir-voxilaprevir group. In POLARIS-4, patients with HCV genotype 1, 2, or 3 infection who had previously received a DAA regimen but not an NS5A inhibitor were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir-voxilaprevir (163 patients) or sofosbuvir-velpatasvir (151 patients) for 12 weeks. An additional 19 patients with HCV genotype 4 infection were enrolled in the sofosbuvir-velpatasvir-voxilaprevir group. In the three active-treatment groups, 46% of the patients had compensated cirrhosis. In POLARIS-1, the rate of sustained virologic response was 96% with sofosbuvir-velpatasvir-voxilaprevir, as compared with 0% with placebo. In POLARIS-4, the rate of response was 98% with sofosbuvir-velpatasvir-voxilaprevir and 90% with sofosbuvir-velpatasvir. The most common adverse events were headache, fatigue, diarrhea, and nausea. In the active-treatment groups in both trials, the percentage of patients who discontinued treatment owing to adverse events was 1% or lower. Sofosbuvir-velpatasvir-voxilaprevir taken for 12 weeks provided high rates of sustained virologic response among patients across HCV genotypes in whom treatment with a DAA regimen

  2. All-oral daclatasvir plus asunaprevir for chronic hepatitis C virus (HCV) genotype 1b infection: a sub-analysis in Asian patients from the HALLMARK DUAL study.

    Science.gov (United States)

    Kao, Jia-Horng; Lee, Youn-Jae; Heo, Jeong; Ahn, Sang-Hoon; Lim, Young-Suk; Peng, Cheng-Yuan; Chang, Ting-Tsung; Torbeyns, Anne; Hughes, Eric; Bhore, Rafia; Noviello, Stephanie

    2016-10-01

    Daclatasvir plus asunaprevir (DCV + ASV) has demonstrated potent antiviral activity in patients with hepatitis C virus (HCV) genotype 1b (GT-1b) infection in the HALLMARK DUAL trial. This post hoc analysis was conducted to determine the efficacy and safety of this treatment in Asian patients. Treatment-naive patients were randomly assigned (2:1; double-blinded) to receive DCV (60 mg once daily) plus ASV (100 mg twice daily) or placebo for 12 weeks. Subsequently, placebo patients entered another study, and the remaining patients continued treatment for an additional 12 weeks. Non-responders to peginterferon/ribavirin and ineligible/intolerant patients received dual therapy for 24 weeks. Sustained virological response at post-treatment Week 12 [sustained virological response (SVR)12] and safety outcomes were evaluated. This post hoc analysis included 186 Asian patients (Korean, 78; Taiwanese, 85; others, 23), of whom 32.3% were cirrhotic. SVR12 was observed in 92.3, 78.6 and 80.0% of treatment-naive, ineligible/intolerant and non-responder patients, respectively, and was comparable with non-Asian patients. SVR12 by baseline factors including age, viral load, interleukin-28B genotype and cirrhosis status was similar between the Asian sub-cohorts. Among 18 Asian patients with NS5A-Y93H or NS5A-L31M/V resistance-associated variants (RAVs), seven patients achieved SVR12. Multivariate regression analysis showed a significant influence of NS5A RAVs in both Asian and non-Asian cohorts. The incidence of serious adverse events in Asian patients was low (7.2%). Two Taiwanese patients had elevated alanine aminotransferase (≥5.1 × ULN); both achieved SVR12. All-oral dual therapy with DCV + ASV resulted in high SVR rates and was well tolerated in Asian patients with HCV GT-1b infection. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. A phase 3, open-label study of daclatasvir plus asunaprevir in Asian patients with chronic hepatitis C virus genotype 1b infection who are ineligible for or intolerant to interferon alfa therapies with or without ribavirin.

    Science.gov (United States)

    Wei, Lai; Zhang, Mingxiang; Xu, Min; Chuang, Wan-Long; Lu, Wei; Xie, Wen; Jia, Zhansheng; Gong, Guozhong; Li, Yueqi; Bae, Si Hyun; Yang, Yong-Feng; Xie, Qing; Lin, Shumei; Chen, Xinyue; Niu, Junqi; Jia, Jidong; Garimella, Tushar; Torbeyns, Anne; McPhee, Fiona; Treitel, Michelle; Yin, Philip D; Mo, Ling

    2016-11-01

    Daclatasvir plus asunaprevir has demonstrated efficacy and safety in patients with chronic hepatitis C virus genotype 1b infection. This study focused on evaluating daclatasvir plus asunaprevir in interferon (±ribavirin)-ineligible or -intolerant Asian patients with genotype 1b infection from mainland China, Korea, and Taiwan. Interferon (±ribavirin)-ineligible and -intolerant patients with genotype 1b infection received daclatasvir 60 mg tablets once daily plus asunaprevir 100 mg soft capsules twice daily for 24 weeks. The primary endpoint was sustained virologic response at post-treatment week 24 (SVR24). Of the 159 patients treated, 89.3% were Chinese, 65.4% were female, and 73.6% were interferon-intolerant. Cirrhosis was present in 32.7% of patients, and 40.3% had IL28B non-CC genotypes. SVR24 was achieved by 145/159 (91.2%) patients (100% concordance with SVR12) and was similarly high in cirrhotic patients (47/52, 90.4%). SVR24 was higher in patients without baseline NS5A (L31M or Y93H) resistance-associated variants (RAVs) (137/139, 98.6%), including those with cirrhosis (43/44, 97.7%). Prevalence of baseline NS5A RAVs was low (19/159, 11.9%), particularly in mainland China (10/127, 7.9%). One death (0.6%), five serious adverse events (3.1%), and three grade 4 laboratory abnormalities (1.9%) occurred on treatment; none were considered related to study drugs. Two patients (1.3%) discontinued because of adverse events. Treatment was generally well tolerated regardless of cirrhosis status. Daclatasvir plus asunaprevir achieved a SVR24 rate of 91.2%, rising to 98.6% in patients without baseline NS5A RAVs, and was generally well tolerated in interferon (±ribavirin)-ineligible or -intolerant patients with genotype 1b infection from mainland China, Korea, and Taiwan. © 2016 The Authors Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  4. Ledipasvir-sofosbuvir in patients with hepatitis C virus genotype 5 infection: an open-label, multicentre, single-arm, phase 2 study.

    Science.gov (United States)

    Abergel, Armand; Asselah, Tarik; Metivier, Sophie; Kersey, Kathryn; Jiang, Deyuan; Mo, Hongmei; Pang, Phillip S; Samuel, Didier; Loustaud-Ratti, Véronique

    2016-04-01

    Data about the response of hepatitis C virus (HCV) genotype 5 to approved and experimental treatment regimens are scarce. We assessed the efficacy and safety of combination therapy with the NS5A inhibitor ledipasvir and the NS5B polymerase inhibitor sofosbuvir in patients with HCV genotype 5. We did this open-label, multicentre, single-arm, phase 2 trial at five hospitals in France. Eligible patients were at least 18 years old and had chronic infection with HCV genotype 5, with plasma HCV RNA of at least 10,000 IU/mL. We used BLAST analyses of NS5B partial sequences to establish the genotype and subtype at screening. Patients were given a fixed-dose combination tablet of 90 mg ledipasvir and 400 mg sofosbuvir orally once per day for 12 weeks. The primary endpoint was the proportion of patients with a sustained viral response, defined as HCV RNA concentration less than 15 IU/mL at 12 weeks after the end of treatment (SVR12). We analysed efficacy and safety in all patients who received at least one dose of ledipasvir-sofosbuvir. This trial is registered with EudraCT, number 2013-003978-27, and with ClinicalTrials.gov, number NCT02081079. From March 7 to June 10, 2014, we recruited 41 patients, including 21 who were treatment naive and 20 who were treatment experienced. All patients were of white ethnic origins. All 41 patients who started treatment completed the full 12 weeks of treatment and had undetectable HCV RNA at their final treatment visit. In the overall study population, 39 (95%, 95% CI 83-99) of 41 patients achieved SVR12. SVR12 was achieved by 20 (95%, 76-100) of the 21 patients who were treatment naive and 19 (95%, 75-100) of the 20 patients who were treatment experienced. Eight (89%) of nine patients with cirrhosis achieved SVR12, whereas 31 (97%) of the 32 patients without cirrhosis achieved SVR12. The two patients who did not reach SVR12 both had IL28B TT genotype and had viral relapse within 4 weeks of the end of treatment. The most common adverse

  5. The global spread of hepatitis C virus 1a and 1b: a phylodynamic and phylogeographic analysis.

    Directory of Open Access Journals (Sweden)

    Gkikas Magiorkinis

    2009-12-01

    Full Text Available BACKGROUND: Hepatitis C virus (HCV is estimated to affect 130-180 million people worldwide. Although its origin is unknown, patterns of viral diversity suggest that HCV genotype 1 probably originated from West Africa. Previous attempts to estimate the spatiotemporal parameters of the virus, both globally and regionally, have suggested that epidemic HCV transmission began in 1900 and grew steadily until the late 1980s. However, epidemiological data suggest that the expansion of HCV may have occurred after the Second World War. The aim of our study was to elucidate the timescale and route of the global spread of HCV. METHODS AND FINDINGS: We show that the rarely sequenced HCV region (E2P7NS2 is more informative for molecular epidemiology studies than the more commonly used NS5B region. We applied phylodynamic methods to a substantial set of new E2P7NS2 and NS5B sequences, together with all available global HCV sequences with information in both of these genomic regions, in order to estimate the timescale and nature of the global expansion of the most prevalent HCV subtypes, 1a and 1b. We showed that transmission of subtypes 1a and 1b "exploded" between 1940 and 1980, with the spread of 1b preceding that of 1a by at least 16 y (95% confidence interval 15-17. Phylogeographic analysis of all available NS5B sequences suggests that HCV subtypes 1a and 1b disseminated from the developed world to the developing countries. CONCLUSIONS: The evolutionary rate of HCV appears faster than previously suggested. The global spread of HCV coincided with the widespread use of transfused blood and blood products and with the expansion of intravenous drug use but slowed prior to the wide implementation of anti-HCV screening. Differences in the transmission routes associated with subtypes 1a and 1b provide an explanation of the relatively earlier expansion of 1b. Our data show that the most plausible route of the HCV dispersal was from developed countries to the

  6. Grazoprevir-Elbasvir Combination Therapy for Treatment-Naive Cirrhotic and Noncirrhotic Patients With Chronic Hepatitis C Virus Genotype 1, 4, or 6 Infection: A Randomized Trial.

    Science.gov (United States)

    Zeuzem, Stefan; Ghalib, Reem; Reddy, K Rajender; Pockros, Paul J; Ben Ari, Ziv; Zhao, Yue; Brown, Deborah D; Wan, Shuyan; DiNubile, Mark J; Nguyen, Bach-Yen; Robertson, Michael N; Wahl, Janice; Barr, Eliav; Butterton, Joan R

    2015-07-07

    Novel interferon- and ribavirin-free regimens are needed to treat hepatitis C virus (HCV) infection. To evaluate the safety and efficacy of grazoprevir (NS3/4A protease inhibitor) and elbasvir (NS5A inhibitor) in treatment-naive patients. Randomized, blinded, placebo-controlled trial. (ClinicalTrials.gov: NCT02105467). 60 centers in the United States, Europe, Australia, Scandinavia, and Asia. Cirrhotic and noncirrhotic treatment-naive adults with genotype 1, 4, or 6 infection. Oral, once-daily, fixed-dose grazoprevir 100 mg/elbasvir 50 mg for 12 weeks, stratified by fibrosis and genotype. Patients were randomly assigned 3:1 to immediate or deferred therapy. Proportion of patients in the immediate-treatment group achieving unquantifiable HCV RNA 12 weeks after treatment (SVR12); adverse events in both groups. Among 421 participants, 194 (46%) were women, 157 (37%) were nonwhite, 382 (91%) had genotype 1 infection, and 92 (22%) had cirrhosis. Of 316 patients receiving immediate treatment, 299 of 316 (95% [95% CI, 92% to 97%]) achieved SVR12, including 144 of 157 (92% [CI, 86% to 96%]) with genotype 1a, 129 of 131 (99% [CI, 95% to 100%]) with genotype 1b, 18 of 18 (100% [CI, 82% to 100%]) with genotype 4, 8 of 10 (80% [CI, 44% to 98%]) with genotype 6, 68 of 70 (97% [CI, 90% to 100%]) with cirrhosis, and 231 of 246 (94% [CI, 90% to 97%]) without cirrhosis. Virologic failure occurred in 13 patients (4%), including 1 case of breakthrough infection and 12 relapses, and was associated with baseline NS5A polymorphisms and emergent NS3 or NS5A variants or both. Serious adverse events occurred in 9 (2.8%) and 3 (2.9%) patients in the active and placebo groups, respectively (difference 1 percentage points]); none were considered drug related. The most common adverse events in the active group were headache (17%), fatigue (16%), and nausea (9%). The study lacked an active-comparator control group and included relatively few genotype 4 and 6 infections. Grazoprevir

  7. Non-geometric branes are DFT monopoles

    Energy Technology Data Exchange (ETDEWEB)

    Bakhmatov, Ilya [Kazan Federal University, Institute of Physics, General Relativity Department,Kremlevskaya 16a, 420111, Kazan (Russian Federation); Kleinschmidt, Axel [Max-Planck-Institut für Gravitationsphysik (Albert-Einstein-Institut),Am Mühlenberg 1, DE-14476 Potsdam (Germany); International Solvay Institutes,Campus Plaine C.P. 231, Boulevard du Triomphe, 1050 Bruxelles (Belgium); Musaev, Edvard T. [Max-Planck-Institut für Gravitationsphysik (Albert-Einstein-Institut),Am Mühlenberg 1, DE-14476 Potsdam (Germany); Kazan Federal University, Institute of Physics, General Relativity Department,Kremlevskaya 16a, 420111, Kazan (Russian Federation)

    2016-10-14

    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{sub 2}{sup 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.

  8. Daclatasvir for the treatment of hepatitis C virus infection.

    Science.gov (United States)

    Adler, Hugh; Lambert, John S

    2014-09-01

    Daclatasvir was pivotal to the trial that established proof-of-concept that an interferon-free regimen could induce a sustained virologic response in patients with chronic HCV infection. This NS5A inhibitor is not currently licensed for the treatment of HCV, but has shown promising efficacy and minimal side-effects in clinical trials to date, where it has been tested in combination with a variety of different HCV therapies. An all-oral, interferon-free curative combination therapy for HCV is now tantalizingly close to becoming part of routine clinical practice, with multiple highly-efficacious direct-acting antiviral agents emerging virtually simultaneously. In this article we will discuss daclatasvir's background and review the clinical trials published to date, concluding with our predictions regarding its future place in the treatment armamentarium against HCV.

  9. Daclatasvir for the treatment of chronic hepatitis C virus infection.

    Science.gov (United States)

    Temesgen, Z; Rizza, S A

    2015-05-01

    Daclatasvir is a nonstructural protein 5A (NS5A) replication complex inhibitor that has shown potent in vitro activity against multiple hepatitis C virus (HCV) genotypes (GT). It is currently in advanced clinical development as a component of combination treatment regimens in a variety of HCV-infected patient populations. In studies conducted thus far, it has been generally well tolerated. It has been approved for the treatment of HCV GTs 1-4 in the European Union. The combination of daclatasvir and asunaprevir (an HCV NS3/4A protease inhibitor) has been approved in Japan for the treatment of patients with GT1 HCV infection. Here we review the available literature on daclatasvir, including its information on its discovery, mechanism of action, pharmacology, preclinical and clinical activity, resistance and safety. Copyright 2015 Prous Science, S.A.U. or its licensors. All rights reserved.

  10. Daclatasvir: potential role in hepatitis C

    Science.gov (United States)

    Lee, Choongho

    2013-01-01

    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. PMID:24204123

  11. Drug-induced lung injury associated with combination therapy of daclatasvir and asunaprevir: The first case report.

    Science.gov (United States)

    Kamada, Takahiro; Furuta, Kenjiro; Tomioka, Hiromi

    2016-05-01

    Combination therapy with direct acting antiviral agents (DAAs) without interferon (IFN) has emerged as a treatment for chronic hepatitis C because of its high overall sustained virologic response rates and favorable side effect profile as compared to that with interferon. We report the first case of drug-induced lung injury (DLI) associated with IFN-free therapy with the DAAs, daclatasvir (NS5A inhibitor) and asunaprevir (NS3/4A protease inhibitor). Although this combination therapy of DAAs has been considered to have fewer side effects than IFN, more attention should be paid to DLI as an important side effect. Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

  12. Synthesis of isotopically labeled daclatasvir for use in human clinical studies.

    Science.gov (United States)

    Easter, John A; Burrell, Richard C; Bonacorsi, Samuel J

    2016-04-01

    Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol-Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon-14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 μCi/mg. To support a human absolute bioavailability study, 5.56 g of [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Hemoglobin Decrease with Iron Deficiency Induced by Daclatasvir plus Asunaprevir Combination Therapy for Chronic Hepatitis C Virus Genotype 1b.

    Directory of Open Access Journals (Sweden)

    Nobuyuki Matsumoto

    Full Text Available Decreased hemoglobin (Hb level has been supposed to be a relatively rare side effect of a combination therapy against hepatitis C virus that consists of the NS5A inhibitor daclatasvir (DCV and the NS3/4A protease inhibitor asunaprevir (ASV.The study was conducted in 75 patients with genotype 1b chronic hepatitis C virus infection who had started combination therapy with DCV and ASV at St. Marianna University School of Medicine Hospital between September 2014 and December 2014.Among the patients examined, decreased Hb level by ≥1.5 g/dL from the values at treatment initiation was observed in 11 individuals. This was accompanied by decreased mean corpuscular volume, and iron and ferritin levels.These findings suggest that the mechanism of the phenomenon is caused by iron deficiency. The underlying mechanism and clinical impacts will need to be further examined.

  14. 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).

  15. iHADAMAC: A complementary tool for sequential resonance assignment of globular and highly disordered proteins

    Science.gov (United States)

    Feuerstein, Sophie; Plevin, Michael J.; Willbold, Dieter; Brutscher, Bernhard

    2012-01-01

    An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the 1H- 15N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate resonance assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.

  16. Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

    Energy Technology Data Exchange (ETDEWEB)

    Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

    2017-08-15

    Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

  17. 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.

  18. Little strings on D n orbifolds

    Science.gov (United States)

    Kim, Joonho; Lee, Kimyeong

    2017-10-01

    We explore two classes of 6d N=(1,0) little string theories obtained from type IIA/IIB NS5-branes probing D n singularities. Their tensor branches are described by effective gauge theories whose instanton solitons are macroscopic little strings. We specifically study two families of 2d N=(0,4) gauge theories which describe at low energy the worldsheet dynamics of the type IIA/IIB little strings. These gauge theories are useful to calculate the supersymmetric partition functions of the little string theories on R 4 × T 2. We establish the T-duality of the little string theories by utilizing their BPS spectra as a probe.

  19. 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.

  20. Favorable efficacy of daclatasvir plus asunaprevir in treatment of elderly Japanese patients infected with HCV genotype 1b aged 70 and older.

    Science.gov (United States)

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

    2017-01-01

    The combination of daclatasvir and asunaprevir is efficacious in the treatment of hepatitis C virus (HCV) infection, but its efficacy and predictors of efficacy in the elderly (≥70 years) remain unclear. In this study, 844 patients with chronic HCV genotype 1b infection, were treated with daclatasvir (60 mg once daily) plus asunaprevir (100 mg twice daily) for 24 weeks. Using the intention-to-treat analysis, the sustained virological response (SVR) rates were 87% and 88% for all 844 patients and 411 elderly (>70 years of age), respectively. In both groups, multivariate analysis identified NS5A-Y93H mutation (elderly patients. Viral factors negatively influenced the response to treatment. Treatment improved AFP level and liver stiffness (surrogate markers of hepatocellular carcinoma), regardless of treatment efficacy. J. Med. Virol. 89:91-98, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Safety and efficacy of an 8-week regimen of grazoprevir plus ruzasvir plus uprifosbuvir compared with grazoprevir plus elbasvir plus uprifosbuvir in participants without cirrhosis infected with hepatitis C virus genotypes 1, 2, or 3 (C-CREST-1 and C-CREST-2, part A)

    DEFF Research Database (Denmark)

    Gane, Edward J; Pianko, Stephen; Roberts, Stuart K

    2017-01-01

    BACKGROUND: New hepatitis C virus (HCV) therapies with pan-genotypic efficacy are needed. The goals of part A of C-CREST-1 and C-CREST-2 were to compare the efficacies of two doses (300 mg or 450 mg once daily) of uprifosbuvir (MK-3682; NS5B inhibitor) in an 8-week regimen combined with grazoprevir...... mg/day); grazoprevir (100 mg/day) plus elbasvir (50 mg/day) plus uprifosbuvir (300 mg/day); or grazoprevir (100 mg/day) plus elbasvir (50 mg/day) plus uprifosbuvir (450 mg/day), according to a computer-generated allocation schedule. Randomisation was centrally implemented using an interactive voice...

  2. Novel inhibitors of dengue virus methyltransferase: discovery by in vitro-driven virtual screening on a desktop computer grid.

    Science.gov (United States)

    Podvinec, Michael; Lim, Siew Pheng; Schmidt, Tobias; Scarsi, Marco; Wen, Daying; Sonntag, Louis-Sebastian; Sanschagrin, Paul; Shenkin, Peter S; Schwede, Torsten

    2010-02-25

    Dengue fever is a viral disease that affects 50-100 million people annually and is one of the most important emerging infectious diseases in many areas of the world. Currently, neither specific drugs nor vaccines are available. Here, we report on the discovery of new inhibitors of the viral NS5 RNA methyltransferase, a promising flavivirus drug target. We have used a multistage molecular docking approach to screen a library of more than 5 million commercially available compounds against the two binding sites of this enzyme. In 263 compounds chosen for experimental verification, we found 10 inhibitors with IC(50) values of <100 microM, of which four exhibited IC(50) values of <10 microM in in vitro assays. The initial hit list also contained 25 nonspecific aggregators. We discuss why this likely occurred for this particular target. We also describe our attempts to use aggregation prediction to further guide the study, following this finding.

  3. Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

    Science.gov (United States)

    Tang, Jing; Vernekar, Sanjeev Kumar V; Chen, Yue-Lei; Miller, Lena; Huber, Andrew D; Myshakina, Nataliya; Sarafianos, Stefan G; Parniak, Michael A; Wang, Zhengqiang

    2017-06-16

    Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8-9 as exemplified with compounds 8c and 9c. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. High-throughput sequencing analysis reveals the genetic diversity of different regions of the murine norovirus genome during in vitro replication.

    Science.gov (United States)

    Mauroy, Axel; Taminiau, Bernard; Nezer, Carine; Ghurburrun, Elsa; Baurain, Denis; Daube, Georges; Thiry, Etienne

    2017-04-01

    In this study, we report the genetic diversity and nucleotide mutation rates of five representative regions of the murine norovirus genome during in vitro passages. The mutation rates were similar in genomic regions encompassing partial coding sequences for non-structural (NS) 1-2, NS5, NS6, NS7 proteins within open reading frame (ORF) 1. In a region encoding a portion of the major capsid protein (VP1) within ORF2 (also including the ORF4 region) and a portion of the minor structural protein (VP2), the mutation rates were estimated to be at least one order of magnitude higher. The VP2 coding region was found to have the highest mutation rate.

  5. 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

    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... rerandomised at Week 12 to continue daclatasvir/peginterferon-alfa-2a/ribavirin for 24 weeks total duration or to placebo/peginterferon-alfa-2a/ribavirin for another 12 weeks. Patients without PDR and placebo patients continued peginterferon-alfa/ribavirin through Week 48. Primary efficacy endpoints were...

  6. Defect (p,q five-branes

    Directory of Open Access Journals (Sweden)

    Tetsuji Kimura

    2015-04-01

    Full Text Available We study a local description of composite five-branes of codimension two. The formulation is constructed by virtue of SL(2,Z×SL(2,Z monodromy associated with two-torus. Applying conjugate monodromy transformations to the complex structures of the two-torus, we obtain a field configuration of a defect (p,q five-brane. This is a composite state of p defect NS5-branes and q exotic 522-branes. We also obtain a new example of hyper-Kähler 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 a single defect KK5-brane.

  7. SL(2, Z) invariant rotating (m, n) strings in AdS{sub 3} x S{sup 3} with mixed flux

    Energy Technology Data Exchange (ETDEWEB)

    Barik, M.S.P.; Panigrahi, Kamal L. [Indian Institute of Technology Kharagpur, Department of Physics, Kharagpur (India); Khouchen, Malak; Kluson, Josef [Masaryk University, Department of Theoretical Physics and Astrophysics, Faculty of Science, Brno (Czech Republic)

    2017-05-15

    We study rigidly rotating and pulsating (m, n) strings in AdS{sub 3} x S{sup 3} with mixed three form flux. The AdS{sub 3} x S{sup 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 the dyonic giant magnon and single spike solutions corresponding to the equations of motion of a probe string in this background and find various relationships among the conserved charges. We further study a class of pulsating (m, n) string in AdS{sub 3} with mixed three form flux. (orig.)

  8. Holography, brane intersections and six-dimensional SCFTs

    Energy Technology Data Exchange (ETDEWEB)

    Bobev, Nikolay [Instituut voor Theoretische Fysica, K.University Leuven,Celestijnenlaan 200D, BE-3001 Leuven (Belgium); Dibitetto, Giuseppe [Department of Physics and Astronomy, Uppsala University,Box 516, SE-75120 Uppsala (Sweden); Gautason, Friðrik Freyr [Instituut voor Theoretische Fysica, K.University Leuven,Celestijnenlaan 200D, BE-3001 Leuven (Belgium); Institut de Physique Théorique, Université Paris Saclay, CEA, CNRS,Orme des Merisiers, F-91191 Gif-sur-Yvette (France); Truijen, Brecht [Instituut voor Theoretische Fysica, K.University Leuven,Celestijnenlaan 200D, BE-3001 Leuven (Belgium)

    2017-02-23

    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.

  9. 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.

  10. Branes for Higgs phases and exact conformal field theories

    CERN Document Server

    Sfetsos, K

    1999-01-01

    We consider multicenter supergravity solutions corresponding to Higgs phases of supersymmetric Yang-Mills theories with Z_N symmetric vacuua. In certain energy regimes, we find a description in terms of a generalized wormhole solution that corresponds to the SL(2,R)/U(1) \\times SU(2)/U(1) exact conformal field theory. We show that U-dualities map these backgrounds to purely gravitational ones and comment on the relation to the black holes arising from intersecting D1 and D5 branes. We also discuss supersymmetric properties of the various solutions and the relation to 2-dim solitons, on flat space, of the reduced axion-dilaton-gravity equations. Finally, we address the problem of understanding other supergravity solutions from the multicenter ones. As prototype examples we use rotating D3 branes and NS5 and D5 branes associated to non-Abelian duals of 4-dim hyper-Kahler metrics with SO(3) isometry.

  11. [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.

  12. Methods for Solving the Viscoelasticity Equations for Cylinder and Sphere Problems

    Science.gov (United States)

    1976-03-22

    EXPRILSiOD C.ANB GIVEN VALID IN ANY N COMPONENTK. 6 fo QI6VLL.ACfMf/A1T RFAArioNS 5) 5 T RE IS -m DI SP L;ýLC ?-’t, 4V T~TOC As AU VtINI ) ADN !~ ’ýUf bu *r~*r...oNrtt o q *3v "s1IANS or W~LAYPX% tNOI’(Ac4 ArN Lcal% CoNSTAUts 9 K 1 iLl qQjRLAT1o TO Tv(3 ELAS’toC Akfl V-iLC~us_ U’-iAidtAL Xrw¶Mto4’ lE COq’¾jA.a5

  13. Functional analysis of replication determinantsin classical swine fever virus

    DEFF Research Database (Denmark)

    Hadsbjerg, Johanne

    boar with great socio-economic importance for countries, like Denmark, that have a significant trade in agricultural production. CSFV has, like other RNA viruses, a high mutation rate allowing the virus to adapt to changes in selection pressure. Hence obtaining a more thorough knowledge of sequence...... in the coding regions for NS2 and NS5B that restored the coding sequence to that of the parental field strain. When rescued viruses, containing mutant IRES elements, were introduced into pigs no induction of clinical disease could be observed and only limited levels of viral RNA were detected. In contrast......, inoculation with the parental virus caused similar clinical symptoms as that observed with the parental field strain. Analysing the adaptation of the viruses throughout the entire viralgenome during virus replication allowed a more comprehensive understanding of the virusproperties beyond what can...

  14. 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. Published by Elsevier B.V.

  15. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells

    DEFF Research Database (Denmark)

    Saeed, Mohsan; Scheel, Troels K H; Gottwein, Judith M

    2012-01-01

    to express a chimeric fusion protein of firefly luciferase and neomycin phosphotransferase to yield stable replicon-expressing cells. Using these constructs, the inhibitory effects of beta interferon (IFN-β), an NS3 protease inhibitor, and an NS5B nucleoside polymerase inhibitor were readily detected...... culture adaptive mutations originally reported for genotype 1b replicons. RNA replication was confirmed by quantitative reverse transcription-PCR and detection of viral protein. Sequencing of multiple independent replicon clones revealed the presence of additional nonsynonymous mutations. Interestingly......, all potentially adaptive mutations mapped to the NS3 protein. These mutations, when introduced back into original constructs, substantially increased colony formation efficiency. To make these replicons useful for high-throughput screening and evaluation of antiviral compounds, they were modified...

  16. 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.

  17. Dielectric 5-Branes and Giant Gravitons in ABJM

    CERN Document Server

    Herrero, Mario; Picos, Marco

    2011-01-01

    We construct a supersymmetric NS5-brane wrapped on a twisted 5-sphere expanding in the $CP^3$ in $AdS_4\\times CP^3$, with D0-brane charge. This configuration provides a realization of the stringy exclusion principle in terms of giant D0-branes. In the maximal case the twisted 5-sphere reduces to a $CP^2$ and its energy can be accounted for both by a bound state of $k$ D4-branes wrapping the $CP^2$ and a bound state of $N$ D0-branes, a realization on the gravity side of the symmetry of Young diagrams with $N$ rows and $k$ columns. We discuss some generalizations of this configuration in M-theory carrying angular momentum, some of them with an interpretation as giant gravitons. We provide the microscopical description that allows to explore the region of finite 't Hooft coupling.

  18. Novel Flaviviruses Detected in Different Species of Mosquitoes in Spain

    Science.gov (United States)

    Sánchez-Seco, María-Paz; Palacios, Gustavo; Molero, Francisca; Reyes, Noelia; Ruiz, Santiago; Aranda, Carles; Marqués, Eduard; Escosa, Raul; Moreno, Juana; Figuerola, Jordi; Tenorio, Antonio

    2012-01-01

    Abstract We report the characterization of three novel flaviviruses isolated in Spain. Marisma Mosquito virus, a novel mosquito borne virus, was isolated from Ochlerotatus caspius mosquitoes; Spanish Ochlerotatus flavivirus and Spanish Culex flavivirus, two novel insect flaviviruses, were isolated from Oc. caspius and Culex pipiens, respectively. During this investigation, we designed a sensitive RT-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus NS5 gene and could be directly used in clinical or environmental samples for flavivirus characterization and surveillance. Analysis of the sequence generated from that amplicon contains enough phylogenetic information for proper taxonomic studies. Moreover, the use of this tool allowed the detection of additional flavivirus DNA forms in Culex, Culiseta, and Ochlerotatus mosquitoes. PMID:22022811

  19. Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus.

    Science.gov (United States)

    Ansari, M Azim; Pedergnana, Vincent; L C Ip, Camilla; Magri, Andrea; Von Delft, Annette; Bonsall, David; Chaturvedi, Nimisha; Bartha, Istvan; Smith, David; Nicholson, George; McVean, Gilean; Trebes, Amy; Piazza, Paolo; Fellay, Jacques; Cooke, Graham; Foster, Graham R; Hudson, Emma; McLauchlan, John; Simmonds, Peter; Bowden, Rory; Klenerman, Paul; Barnes, Eleanor; Spencer, Chris C A

    2017-05-01

    Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control.

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

    Directory of Open Access Journals (Sweden)

    Eliane Silva

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

  1. Three-charge black holes and quarter BPS states in Little String Theory

    Energy Technology Data Exchange (ETDEWEB)

    Giveon, Amit [Racah Institute of Physics, The Hebrew University,Jerusalem, 91904 (Israel); Harvey, Jeffrey; Kutasov, David; Lee, Sungjay [Enrico Fermi Institute and Department of Physics, The University of Chicago,5620 S. Ellis Av., Chicago, Illinois 60637 (United States)

    2015-12-22

    We show that the system of k NS5-branes wrapping T{sup 4}×S{sup 1} has non-trivial vacuum structure. Different vacua have different spectra of 1/4 BPS states that carry momentum and winding around the S{sup 1}. In one vacuum, such states are described by black holes; in another, they can be thought of as perturbative BPS states in Double Scaled Little String Theory. In general, both kinds of states are present. We compute the degeneracy of perturbative BPS states exactly, and show that it differs from that of the corresponding black holes. We comment on the implication of our results to the black hole microstate program, UV/IR mixing in Little String Theory, string thermodynamics, the string/black hole transition, and other issues.

  2. Human cases of Wesselsbron disease, South Africa 2010-2011.

    Science.gov (United States)

    Weyer, Jacqueline; Thomas, Juno; Leman, Patricia A; Grobbelaar, Antoinette A; Kemp, Alan; Paweska, Janusz T

    2013-05-01

    Wesselsbron disease is a neglected, mosquito-borne zoonotic infection reported from Africa. The disease primarily affects sheep and other ruminants with incidental spillover to humans. As for other arboviral diseases in Africa, little or no active surveillance is conducted, and the public and veterinary health burden of this disease remains unclear. We report on the clinical histories of 2 human cases of Wesselsbron disease that were laboratory confirmed during the 2010-2011 Rift Valley fever outbreak investigation in South Africa. This report describes the first confirmed human cases of Wesselsbron disease since 1996. Molecular sequencing and analysis of the partial NS5 gene of the Wesselsbron genome was used to identify 2 circulating clades of the virus in southern Africa. Clade I included isolates collected from South Africa and Zimbabwe, whereas clade II only included isolates from the KwaZulu Natal Province of South Africa.

  3. A new 6d fixed point from holography

    Energy Technology Data Exchange (ETDEWEB)

    Apruzzi, Fabio [Department of Physics, University of North Carolina,Chapel Hill, NC 27599 (United States); CUNY Graduate Center, Initiative for the Theoretical Sciences,New York, NY 10016 (United States); Department of Physics, Columbia University,New York, NY 10027 (United States); Dibitetto, Giuseppe; Tizzano, Luigi [Department of Physics and Astronomy, Uppsala university,Box 516, SE-75120 Uppsala (Sweden)

    2016-11-22

    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 AdS{sub 7}×M{sub 3}, where M{sub 3} is topologically an S{sup 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 (D6)-bar 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.

  4. Exact half-BPS type IIB interface solutions II: flux solutions and multi-Janus

    Energy Technology Data Exchange (ETDEWEB)

    D' Hoker, Eric; Estes, John; Gutperle, Michael [Department of Physics and Astronomy, University of California, Los Angeles, CA 90095 (United States)

    2007-06-15

    Regularity and topology conditions are imposed on the exact Type IIB solutions on AdS{sub 4} x S{sup 2} x S{sup 2} x {sigma} with 16 supersymmetries, which were derived in a companion paper [1]. We construct an infinite class of regular solutions with varying dilaton, and non-zero 3-form fluxes. Our solutions may be viewed as the fully back-reacted geometries of AdS{sub 5} x S{sup 5} (or more generally, Janus) doped with D5 and/or NS5 branes. The solutions are parametrized by the choice of an arbitrary genus g hyper-elliptic Riemann surface {sigma} with boundary, all of whose branch points are restricted to lie on a line. For genus 0, the Janus solution with 16 supersymmetries and 6 real parameters is recovered; its topology coincides with that of AdS{sub 5} x S{sup 5}. The genus g {>=} 1 solutions are parametrized by a total of 4g + 6 real numbers, 2g-1 of which are the real moduli of {sigma}. The solutions have 2g + 2 asymptotic AdS{sub 5} x S{sup 5} regions, g three-spheres with RR 3-form charge, and another g with NSNS 3-form charge. Collapse of consecutive branch points of {sigma} yields singularities which correspond to D5 and NS5 branes in the probe limit. It is argued that the AdS/CFT dual gauge theory to each of our solutions consists of a 2+1-dimensional planar interface on which terminate 2g + 2 half-Minkowski 3+1-dimensional space-time N = 4 super-Yang-Mills theories. Generally, the N = 4 theory in each Minkowski half-space-time may have an independent value of the gauge coupling, and the interface may support various operators, whose interface couplings are further free parameters of the dual gauge theory.

  5. Transmission of GBV-C/HGV from drug-addicted mothers to their babies.

    Science.gov (United States)

    Viazov, S; Riffelmann, M; Sarr, S; Ballauff, A; Meisel, H; Roggendorf, M

    1997-07-01

    In 1995, a new flavivirus, GBV-C/HGV was identified. Little information is available on the clinical manifestations and epidemiology of GBV-C/HGV infection. We investigated the risk of mother-to-infant transmission in a group of GBV-C/HGV RNA positive women and followed up the GBV-C/HGV infected babies. Twenty-eight anti-HCV positive women, of whom 25 have been intravenous drug users, and their children were included in the study. RNA was extracted from serum, reverse transcribed and amplified with primers from the NS5 region of GBV-C/HGV and 5'-UTR of HCV in a nested polymerase chain reaction. Amplified DNA fragments were gel purified and sequenced; the sequences obtained were subjected to a phylogenetic analysis. Transmission of GBV-C/HGV occurred in 10 (56%) of 18 infants born to GBV-C/HGV positive mothers; all these women were drug abusers. Only one (5%) of 19 babies whose mothers were HCV RNA positive by polymerase chain reaction, was infected with HCV during the follow up. High sequence homology in the NS5 region of GBV-C/HGV isolates in 10 mother-child pairs suggested mother-to-infant transmission. All 10 babies remained GBV-C/HGV RNA positive during follow up (2-12 months). None of the GBV-C/HGV infected infants became icteric or demonstrated any clinical or biochemical signs of liver disease. Mother-to-infant transmission of GBV-C/HGV may be high, at least in HCV-infected, drug-addicted women. In GBV-C/HGV RNA positive infants the rate of GBV-C/HGV persistent infection is high, but the infection is not accompanied by any symptoms of liver disease.

  6. Modification of S-Adenosyl-l-Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation

    Science.gov (United States)

    Tambunan, Usman Sumo Friend; Nasution, Mochammad Arfin Fardiansyah; Azhima, Fauziah; Parikesit, Arli Aditya; Toepak, Erwin Prasetya; Idrus, Syarifuddin; Kerami, Djati

    2017-01-01

    Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world’s population in tropical and subtropical countries. Nonstructural protein 5 (NS5) methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S-adenosyl-l-methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2′OH, resulting in S-adenosyl-l-homocysteine (SAH). The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) test. The 2 simulations were performed using Molecular Operating Environment (MOE) 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356) based on ΔGbinding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever. PMID:28469408

  7. Daclatasvir–sofosbuvir combination therapy with or without ribavirin for hepatitis C virus infection: from the clinical trials to real life

    Directory of Open Access Journals (Sweden)

    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

  8. Impact of resistance-associated variant dominancy on treatment in patients with HCV genotype 1b receiving daclatasvir/asunaprevir.

    Science.gov (United States)

    Ikeda, Hiroki; Watanabe, Tsunamasa; Okuse, Chiaki; Matsumoto, Nobuyuki; Ishii, Toshiya; Yamada, Norie; Shigefuku, Ryuta; Hattori, Nobuhiro; Matsunaga, Kotaro; Nakano, Hiroyasu; Hiraishi, Tetsuya; Kobayashi, Minoru; Yasuda, Kiyomi; Yamamoto, Hiroyuki; Yasuda, Hiroshi; Kurosaki, Masayuki; Izumi, Namiki; Yotsuyanagi, Hiroshi; Suzuki, Michihiro; Itoh, Fumio

    2017-01-01

    Sustained virological responses (SVR) by daclatasvir (DCV) and asunaprevir (ASV) therapy for genotype 1b hepatitis C virus (HCV) infected patients has been significantly affected by pre-existence of Y93 H resistance-associated variants (RAVs) in the non-structural protein 5A (NS5A) region. The aim of this study was to elucidate the dominancy of naturally occurring RAVs in viral quasispecies on treatment outcomes in patients with HCV. In total, 138 patients were prospectively selected from 152 patients treated with DCV and ASV, where evaluation of treatment outcomes at 12 weeks post-treatment was possible. Pre-treatment RAVs in the non-structural protein 3 and NS5A regions were detected by polymerase chain reaction (PCR)-Invader assays, and the ratio of Y93H RAVs in viral quasispecies was measured by quantitative PCR-Invader assay. Among 25 patients detected the Y93H RAV, the Y93H ratio was 1-25% in 5 patients, 26-75% in 7 patients, and ≥76% in 13 patients. Overall, SVR at 12 weeks after the completion of treatment (SVR12) was 91% (125/138), and those with Y93H ratios of <1%, 1-25%, 26-75%, and ≥76% were 99%, 100%, 71%, and 23%, respectively. Thus, the SVR12 decreased as the HCV Y93H ratio increased (P < 0.0001). The dominancy of pre-treatment RAVs of DCV and ASV affected its treatment outcomes, suggesting that evaluating the dominancy of HCV RAVs could be required for every other direct-acting antiviral agent treatments. J. Med. Virol. 89:99-105, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. On-treatment HCV RNA as a predictor of sustained virological response in HCV genotype 3-infected patients treated with daclatasvir and sofosbuvir.

    Science.gov (United States)

    Kowdley, Kris V; Nelson, David R; Lalezari, Jacob P; Box, Terry; Gitlin, Norman; Poleynard, Gary; Rabinovitz, Mordechai; Ravendhran, Natarajan; Sheikh, Aasim M; Siddique, Asma; Bhore, Rafia; Noviello, Stephanie; Rana, Khurram

    2016-11-01

    Many currently available direct-acting antiviral (DAA) regimens are less effective against HCV genotype 3 than against other HCV genotypes. The all-oral, pangenotypic DAA combination of daclatasvir (NS5A inhibitor) + sofosbuvir (nucleotide NS5B inhibitor) was studied in genotype 3-infected treatment-naive and -experienced patients (ALLY-3) who achieved rates of sustained virological response at post-treatment Week 12 (SVR12) of 90 and 86% respectively. In this analysis, we assessed whether on-treatment responses to daclatasvir + sofosbuvir in genotype 3-infected patients could predict treatment outcome. In ALLY-3, treatment-naive and -experienced patients, with or without cirrhosis, were treated with daclatasvir + sofosbuvir for 12 weeks. HCV RNA kinetics and categorical virological responses on treatment were assessed. The proportions of responders and nonresponders by study week, and time to first undetectable HCV RNA, were analysed for utility in predicting treatment outcome. Overall, HCV RNA levels declined rapidly during Week 1 of treatment in both treatment-naive and -experienced cohorts. Although patients with cirrhosis had a slower initial virological response as measured by the proportion of patients with HCV RNA below the lower limit of quantification at Week 1, responses converged thereafter. Positive and negative predictive values calculated for on-treatment responses were generally comparable with the overall SVR12 rate and were therefore limited indicators of outcome. SVR12 rates were not impacted by time to first undetectable HCV RNA. On-treatment responses are not useful predictors of ultimate virological response to the daclatasvir + sofosbuvir regimen. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Culex flavivirus and West Nile virus in Culex quinquefasciatus populations in the southeastern United States.

    Science.gov (United States)

    Crockett, Rebekah Kent; Burkhalter, Kristen; Mead, Daniel; Kelly, Rosmarie; Brown, Jeffrey; Varnado, Wendy; Roy, Alma; Horiuchi, Kalanthe; Biggerstaff, Brad J; Miller, Barry; Nasci, Roger

    2012-01-01

    Little is known of the interactions between insect-only flaviviruses and other arboviruses in their mosquito hosts, or the potential public health significance of these associations. The specific aims of this study were to describe the geographic distribution, prevalence, and seasonal infection rates of Culex flavivirus (CxFV) and West Nile virus (WNV) in Culex quinquefasciatus Say in the Southeastern United States, investigate the potential association between CxFV and WNV prevalence in Cx. quinquefasciatus and describe the phylogenetic relationship among CxFV and WNV isolates from the Southeastern United States and around the world. Using ArboNET records, 11 locations were selected across Georgia, Mississippi, and Louisiana that represented a range of WNV human case incidence levels. Cx. quinquefasciatus were trapped weekly throughout the summer of 2009 and pools were screened for flavivirus RNA by reverse transcriptase polymerase chain reaction. Cx. quinquefasciatus from Georgia had significantly higher CxFV infection rates than either Mississippi or Louisiana. CxFV was not detected in Mississippi after July, and no CxFV was detected in Cx. quinquefasciatus in Louisiana. In Georgia, CxFV infection rates were variable between and within counties and over time. WNV infection rates were not significantly different across states or months, and WNV sequences from all three states were identical to each other in the envelope and NS5 gene regions. Phylogenetically, NS5 and E gene sequences from Georgia CxFV isolates clustered with CxFV from Japan, Iowa, and Texas. Multiple CxFV genetic variants were found circulating simultaneously in Georgia. No evidence was found supporting an association between WNV and CxFV infection prevalence in Cx. quinquefasciatus.

  11. [Serological and pathological follow-up studies of the patients with single GBV-C/HGV RNA infection].

    Science.gov (United States)

    Zhao, J; Wang, S; Xin, S

    2001-03-01

    To elucidate the pathogenicity of GBV-C/HGV in causing viral hepatitis. The sera and bioptic liver tissues, which involved 8 cases of the second liver puncture with an interval of 2 years, from 24 cases of single GBV-C/HGV RNA positive patients were observed for more than 2 years. GBV-C/HGV RNA in the sera and liver tissues was detected by using RT-PCR and in situ hybridization, respectively. Under light and electron microscope, the pathology of the liver tissues was observed. At the beginning of the observation, the mean serum alanine transaminase(ALT) level was 60.17 IU/ml(range, 42.00-87.00 IU/ml), and the positive rates of anti-E2 antibody and GBV-C/HGV RNA in the sera and NS3 and/or NS5 in the liver tissues were 4.17%, 75.00% and 54.17%, respectively. In all the examined specimens, 79.17% of them showed mild acute or chronic hepatitis, mainly presenting with degeneration of hepatocytes, mild portal inflammation. GBV-C/HGV RNA and its NS3 and/or NS5 antigens were mainly detected in hepatocytes and a few mononuclear cells. In contrast, after more than 2 years, the natural change rate (negative rate of GBV-C/HGV) of 24 cases was 66.67% (P GBV-C/HGV could induce mild and self-limited hepatitis, which showed that GBV-C/HGV may be a light and self-limited pathogenic agent to human liver. E2 antibody appearance may indicate a recovery and provide a protection from GBV-C/HGV infection. Whether there is any other recovery marker in serum requires further study.

  12. Empirical fitness models for hepatitis C virus immunogen design.

    Science.gov (United States)

    Hart, Gregory R; Ferguson, Andrew L

    2015-11-24

    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. HCV-hepatitis C virus, HLA-human leukocyte antigen, CTL-cytotoxic T lymphocyte, NS5B-nonstructural protein 5B, MSA-multiple sequence alignment, PEG-IFN-pegylated interferon.

  13. Genotypic distribution of hepatitis C virus in Thailand and Southeast Asia.

    Science.gov (United States)

    Wasitthankasem, Rujipat; Vongpunsawad, Sompong; Siripon, Nipaporn; Suya, Chutima; Chulothok, Phrutsada; Chaiear, Kasemporn; Rujirojindakul, Pairaya; Kanjana, Sawan; Theamboonlers, Apiradee; Tangkijvanich, Pisit; Poovorawan, Yong

    2015-01-01

    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.

  14. Investigation of a Case of Genotype 5a Hepatitis C Virus Transmission in a French Hemodialysis Unit Using Epidemiologic Data and Deep Sequencing.

    Science.gov (United States)

    Aho-Glélé, L S; Giraudon, H; Astruc, K; Soltani, Z; Lefebvre, A; Pothier, P; Bour, J B; Manoha, C

    2016-02-01

    BACKGROUND Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. A patient was recently found to be HCV seropositive during hemodialysis follow-up. OBJECTIVE To determine whether nosocomial transmission had occurred and which viral populations were transmitted. DESIGN HCV transmission case. SETTING A dialysis unit in a French hospital. METHODS Molecular and epidemiologic investigations were conducted to determine whether 2 cases were related. Risk analysis and auditing procedures were performed to determine the transmission pathway(s). RESULTS Sequence analyses of the NS5b region revealed a 5a genotype in the newly infected patient. Epidemiologic investigations suggested that a highly viremic genotype 5a HCV-infected patient who underwent dialysis in the same unit was the source of the infection. Phylogenetic analysis of NS5b and hypervariable region-1 sequences revealed a genetically related virus (>99.9% nucleotide identity). Deep sequencing of hypervariable region-1 indicated that HCV quasispecies were found in the source whereas a single hypervariable region-1 HCV variant was found in the newly infected patient, and that this was identical to the major variant identified in the source patient. Risk analysis and auditing procedures were performed to determine the transmission pathway(s). Nosocomial patient-to-patient transmission via healthcare workers' hands was the most likely explanation. In our dialysis unit, this unique incident led to the adjustment of infection control policy. CONCLUSIONS The data support transmission of a unique variant from a source with a high viral load and genetic diversity. This investigation also underlines the need to periodically evaluate prevention and control practices.

  15. 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.

  16. 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.

  17. Modification of S-Adenosyl-l-Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation.

    Science.gov (United States)

    Tambunan, Usman Sumo Friend; Nasution, Mochammad Arfin Fardiansyah; Azhima, Fauziah; Parikesit, Arli Aditya; Toepak, Erwin Prasetya; Idrus, Syarifuddin; Kerami, Djati

    2017-01-01

    Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world's population in tropical and subtropical countries. Nonstructural protein 5 (NS5) methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S-adenosyl-l-methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2'OH, resulting in S-adenosyl-l-homocysteine (SAH). The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) test. The 2 simulations were performed using Molecular Operating Environment (MOE) 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356) based on ΔGbinding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever.

  18. Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains.

    Science.gov (United States)

    Setoh, Yin Xiang; Prow, Natalie A; Rawle, Daniel J; Tan, Cindy Si En; Edmonds, Judith H; Hall, Roy A; Khromykh, Alexander A

    2015-06-01

    A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven individual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed a predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent. © 2015 The Authors.

  19. High prevalence of hepatitis C virus genotype 1b infection in a small town of Argentina. Phylogenetic and Bayesian coalescent analysis.

    Directory of Open Access Journals (Sweden)

    Marcelo D Golemba

    Full Text Available Previous studies in Argentina have documented a general prevalence of Hepatitis C Virus (HCV infection close to 2%. In addition, a high prevalence of HCV has been recently reported in different Argentinean small rural communities. In this work, we performed a study aimed at analyzing the origins and diversification patterns of an HCV outbreak in Wheelwright, a small rural town located in Santa Fe province (Argentina.A total of 89 out of 1814 blood samples collected from people living in Wheelwright, were positive for HCV infection. The highest prevalence (4.9% was observed in people older than 50 years, with the highest level for the group aged between 70-79 years (22%. The RFLP analyses showed that 91% of the positive samples belonged to the HCV-1b genotype. The E1/E2 and NS5B genes were sequenced, and their phylogenetic analysis showed that the HCV-1b sequences from Wheelwright were monophyletic. Bayesian coalescent-based methods were used to estimate substitution rates and time of the most recent common ancestor (tMRCA. The mean estimated substitution rates and the tMRCA for E1/E2 with and without HVR1 and NS5B were 7.41E-03 s/s/y and 61 years, 5.05E-03 s/s/y and 58 years and 3.24E-03 s/s/y and 53 years, respectively. In summary, the tMRCA values, the demographic model with constant population size, and the fact that the highest prevalence of infection was observed in elder people support the hypothesis that the HCV-1b introduction in Wheelwright initially occurred at least five decades ago and that the early epidemic was characterized by a fast rate of virus transmission. The epidemic seems to have been controlled later on down to the standard transmission rates observed elsewhere.

  20. High Prevalence of Hepatitis C Virus Genotype 1b Infection in a Small Town of Argentina. Phylogenetic and Bayesian Coalescent Analysis

    Science.gov (United States)

    Golemba, Marcelo D.; Di Lello, Federico A.; Bessone, Fernando; Fay, Fabian; Benetti, Silvina; Jones, Leandro R.; Campos, Rodolfo H.

    2010-01-01

    Previous studies in Argentina have documented a general prevalence of Hepatitis C Virus (HCV) infection close to 2%. In addition, a high prevalence of HCV has been recently reported in different Argentinean small rural communities. In this work, we performed a study aimed at analyzing the origins and diversification patterns of an HCV outbreak in Wheelwright, a small rural town located in Santa Fe province (Argentina). A total of 89 out of 1814 blood samples collected from people living in Wheelwright, were positive for HCV infection. The highest prevalence (4.9%) was observed in people older than 50 years, with the highest level for the group aged between 70–79 years (22%). The RFLP analyses showed that 91% of the positive samples belonged to the HCV-1b genotype. The E1/E2 and NS5B genes were sequenced, and their phylogenetic analysis showed that the HCV-1b sequences from Wheelwright were monophyletic. Bayesian coalescent-based methods were used to estimate substitution rates and time of the most recent common ancestor (tMRCA). The mean estimated substitution rates and the tMRCA for E1/E2 with and without HVR1 and NS5B were 7.41E-03 s/s/y and 61 years, 5.05E-03 s/s/y and 58 years and 3.24E-03 s/s/y and 53 years, respectively. In summary, the tMRCA values, the demographic model with constant population size, and the fact that the highest prevalence of infection was observed in elder people support the hypothesis that the HCV-1b introduction in Wheelwright initially occurred at least five decades ago and that the early epidemic was characterized by a fast rate of virus transmission. The epidemic seems to have been controlled later on down to the standard transmission rates observed elsewhere. PMID:20090919

  1. 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.

  2. Combination ledipasvir-sofosbuvir for the treatment of chronic hepatitis C virus infection: a review and clinical perspective

    Directory of Open Access Journals (Sweden)

    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

  3. Clinical Pharmacokinetics and Pharmacodynamics of Ledipasvir/Sofosbuvir, a Fixed-Dose Combination Tablet for the Treatment of Hepatitis C.

    Science.gov (United States)

    German, Polina; Mathias, Anita; Brainard, Diana; Kearney, Brian P

    2016-11-01

    Ledipasvir/sofosbuvir (Harvoni ® ), a fixed-dose combination tablet of an NS5A inhibitor ledipasvir and an NS5B polymerase inhibitor sofosbuvir, is approved in the US, European Union, Canada, and other regions for the treatment of chronic hepatitis C virus infection in adults. Following absorption, ledipasvir reaches maximum plasma concentrations (T max ) 4-4.5 h post-dose and is eliminated with a terminal half-life (t 1/2 ) of 47 h. Sofosbuvir undergoes intracellular activation to an active triphosphate GS-461203 (not detected in plasma) and ultimately to GS-331007, a predominant circulating metabolite, which is the primary analyte of interest in clinical pharmacology studies. Sofosbuvir is rapidly absorbed and eliminated from plasma (T max : 0.8-1 h; t 1/2 : 0.5 h). The peak plasma concentrations for GS-331007 are achieved between 3.5 and 4 h post-dose; the elimination t 1/2 for GS-331007 is 27 h. Ledipasvir/sofosbuvir exhibits a favorable clinical pharmacology profile; it can be administered once daily without regard to food and does not require dose modification in hepatitis C virus-infected patients with any degree of hepatic impairment or mild to moderate renal impairment. The pharmacokinetic profiles of ledipasvir, sofosbuvir, and GS-331007 (predominant circulating metabolite of sofosbuvir) are not significantly affected by demographic variables; pharmacokinetic/pharmacodynamic analyses reveal no exposure-response relationships for efficacy or safety. The review summarizes the clinical pharmacokinetics, pharmacodynamics, and pharmacokinetic/pharmacodynamic analyses for ledipasvir/sofosbuvir.

  4. Ledipasvir/Sofosbuvir: a review of its use in chronic hepatitis C.

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    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 ledipasvir/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.

  5. Geno2pheno[HCV] - A Web-based Interpretation System to Support Hepatitis C Treatment Decisions in the Era of Direct-Acting Antiviral Agents.

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    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

  6. Retreatment of HCV Genotype-1 with Sofosbuvir and Ledipasvir after Relapse with Sofosbuvir and Ribavirin: A Pilot Study

    Science.gov (United States)

    Osinusi, Anu; Kohli, Anita; Marti, Miriam M; Nelson, Amy; Zhang, Xiaozhen; Meissner, Eric G; Silk, Rachel; Townsend, Kerry; Pang, Phillip S; Subramanian, Mani; McHutchison, John G.; Fauci, Anthony S; Masur, Henry; Kottilil, Shyam

    2015-01-01

    Background The interferon (IFN)-free regimen of sofosbuvir and ribavirin for 24 weeks was recently approved to treat chronic hepatitis C virus (HCV), genotype-1 (GT-1) infection for interferon-ineligible patients . However, sofosbuvir/ribavirin therapy is associated with treatment relapse in 15-30% of HCV GT-1 study subjects. Neither the mechanism of relapse nor the optimal retreatment strategy for these subjects is defined. Objective To assess the safety and efficacy of sofosbuvir and ledipasvir in chronic HCV GT-1 infected subjects who relapsed following sofosbuvir/ribavirin therapy. Design Phase 2a, open-label study (ClinicalTrials.gov, number NCT01805882). Setting Single U.S site. Subjects Fourteen HCV, GT-1 subjects who relapsed following treatment with 24 weeks of sofosbuvir/ribavirin were treated with 12 weeks of sofosbuvir/ledipasvir. Measurments HCV RNA concentration and population sequencing to detect NS5B S282T mutations. Results All 14 subjects treated with sofosbuvir/ledipasvir for 12 weeks achieved a sustained virologic response, including seven with advanced liver disease (HAI-fibrosis 3-4) and one with a detectable NS5B S282T mutation post sofosbuvir/ribavirin therapy. Sofosbuvir/ledipasvir was well tolerated with few adverse events. Four grade 3 events (elevated serum creatinine in a subject with baseline renal insufficiency, hypercholesterolemia and hypophosphatemia (n=2)) occurred. There were no grade 4 events or treatment discontinuations. Limitations Small sample size. Conclusions The fixed-dose combination of sofosbuvir/ledipasvir was efficacious in a small cohort of subjects with HCV GT-1 infections who relapse following sofosbuvir/ribavirin therapy, even in the setting of advanced liver disease. Larger studies are needed to confirm these preliminary efficacy results. Primary Funding Source National Institute of Allergy and Infectious Diseases (NIAID), National Cancer Institute and Clinical Center Intramural Program and a Collaborative

  7. Genetic History of Hepatitis C Virus in Venezuela: High Diversity and Long Time of Evolution of HCV Genotype 2

    Science.gov (United States)

    Sulbarán, Maria Z.; Di Lello, Federico A.; Sulbarán, Yoneira; Cosson, Clarisa; Loureiro, Carmen L.; Rangel, Héctor R.; Cantaloube, Jean F.; Campos, Rodolfo H.; Moratorio, Gonzalo; Cristina, Juan; Pujol, Flor H.

    2010-01-01

    Background The subtype diversity of the hepatitis C virus (HCV) genotypes is unknown in Venezuela. Methodology/Principal Findings Partial sequencing of the NS5B region was performed in 310 isolates circulating in patients from 1995 to 2007. In the samples collected between 2005 and 2007, HCV genotype 1 (G1) was the most common genotype (63%), composed as expected of mainly G1a and G1b. G2 was the second most common genotype (33%), being G2a almost absent and G2j the most frequent subtype. Sequence analysis of the core region confirmed the subtype assignment performed within the NS5b region in 63 isolates. The complete genome sequence of G2j was obtained. G2j has been described in France, Canada and Burkina Fasso, but it was not found in Martinique, where several subtypes of G2 circulate in the general population. Bayesian coalescence analysis indicated a most recent common ancestor (MRCA) of G2j around 1785, before the introduction of G1b (1869) and G1a (1922). While HCV G1a and G1b experienced a growth reduction since 1990, coincident with the time when blood testing was implemented in Venezuela, HCV G2j did not seem to reach growth equilibrium during this period. Conclusions/Significance Assuming the introduction of G2j from Africa during the slave trade, the high frequency of G2j found in Venezuela could suggest: 1- the introduction of African ethnic groups different from the ones introduced to Martinique or 2- the occurrence of a founder effect. This study represents an in-depth analysis of the subtype diversity of HCV in Venezuela, which is still unexplored in the Americas and deserves further studies. PMID:21179440

  8. Molecular cloning and characterization of the genes encoding the proteins of Zika virus.

    Science.gov (United States)

    Hou, Wangheng; Cruz-Cosme, Ruth; Armstrong, Najealicka; Obwolo, Lilian Akello; Wen, Fayuan; Hu, Wenhui; Luo, Min-Hua; Tang, Qiyi

    2017-09-10

    Zika virus (ZIKV) encodes a precursor protein (also called polyprotein) of about 3424 amino acids that is processed by proteases to generate 10 mature proteins and a small peptide. In the present study, we characterized the chemical features, suborganelle distribution and potential function of each protein using Flag-tagged protein expression system. Western blot analysis revealed the molecular weight of the proteins and the polymerization of E, NS1, and NS3 proteins. In addition, we performed multi-labeled fluorescent immunocytochemistry and subcellular fractionation to determine the subcellular localization of these proteins in host cells. We found that 1) the capsid protein colocalizes with 3 different cellular organelles: nucleoli, Golgi apparatus, and lipid droplet; NS2b and NS4a are associated with the Golgi apparatus; 2) the capsid and NS1proteins distribute in both cytoplasm and nucleus, NS5 is a nuclear protein; 3) NS3 protein colocalizes with tubulin and affects Lamin A; 4) Envelope, PrM, and NS2a proteins co-localize with the endoplasmic reticulum; 5) NS1 is associated with autophagosomes and NS4b is related to early endosome; 6) NS5 forms punctate structures in the nucleus that associate with splicing compartments shown by SC35, leading to reduction of SC35 protein level and trafficking of SC35 from the nucleus to the cytoplasm. These data suggest that ZIKV generates 10 functional viral proteins that exhibit distinctive subcellular distribution in host cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Differential in vitro effects of intravenous versus oral formulations of silibinin on the HCV life cycle and inflammation.

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    Jessica Wagoner

    Full Text Available Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp activity better than silibinin, with IC50 values of 40-85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43-73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases.

  10. Low prevalence of HCV infection with predominance of genotype 4 among HIV patients living in Libreville, Gabon.

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    Angélique Ndjoyi-Mbiguino

    Full Text Available Gabon is an endemic area for human immunodeficiency virus (HIV and hepatitis C virus (HCV and the risk of co-infection is high.Between November 2015 and April 2016, we conducted retrospective study on HCV infection among people living with HIV/AIDS (PLHA. A total of 491 PLHA were included in this study and tested for the presence of HCV infection. HIV viral loads were obtained using the Generic HIV viral Load® assay and the CD4+ T cells count was performed using BD FACSCount™ CD4 reagents. HCV screening was performed using the MP Diagnostics HCV ELISA 4.0 kit. HCV genotypes were determined by sequence analysis of NS5B and Core regions. The Mann-Whitney test was used to compare the groups. Chi-2 test and Fisher's Exact Test were used to compare prevalence.HCV seroprevalence was 2.9% (14/491, (95% confidence interval (CI:1.4-4.3%. The percentage of HCV viremic patients, defined by the detection of HCV RNA in plasma, was 57% (8/14, representing 1.6% of the total population. HCV seroprevalence and replicative infection were not statistically differ with gender. The percentage of co-infection increased with age. No correlation with CD4+ T cells count and HIV viral load level was registered in this study. Identified HCV strains were predominantly of genotype 4 (87.5% including 4k, 4e, 4g, 4p, 4f and 4c subtypes. Only one strain belonged to genotype 2 (subtype 2q. Analysis of the NS5B region did not reveal the presence of resistance-associated substitutions for sofosbuvir.A systematic screening of hepatitis C is therefore strongly recommended as well as genotyping of HCV strains in order to adapt treatments for the specific case of people living with HIV/AIDS in Central Africa.

  11. Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals.

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    Scott Seronello

    Full Text Available 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.

  12. Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release.

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    Foster, Toshana L; Thompson, Gary S; Kalverda, Arnout P; Kankanala, Jayakanth; Bentham, Matthew; Wetherill, Laura F; Thompson, Joseph; Barker, Amy M; Clarke, Dean; Noerenberg, Marko; Pearson, Arwen R; Rowlands, David J; Homans, Steven W; Harris, Mark; Foster, Richard; Griffin, Stephen

    2014-02-01

    Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)-based methods. This represents atomic resolution information for a full-length virus-coded ion channel, or "viroporin," whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug-protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000-fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. This proof-of-principle that structure-guided design can lead to drug-like molecules affirms p7 as a much-needed new target in the burgeoning era of HCV DAA. Copyright © 2013 The Authors. HEPATOLOGY published by Wiley on behalf of the American Association for the Study of Liver Diseases.

  13. Modification of -Adenosyl--Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation

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    Usman Sumo Friend Tambunan

    2017-04-01

    Full Text Available Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world’s population in tropical and subtropical countries. Nonstructural protein 5 (NS5 methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S -adenosyl- l -methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2′OH, resulting in S -adenosyl- l -homocysteine (SAH. The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity test. The 2 simulations were performed using Molecular Operating Environment (MOE 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356 based on ΔG binding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever.

  14. 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.

  15. Transmission dynamics of hepatitis C virus among intra venous drug users in the border state of Manipur, India.

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

  16. Meta-Analysis of Grazoprevir plus Elbasvir for Treatment of Hepatitis C Virus Genotype 1 Infection.

    Science.gov (United States)

    Ahmed, Hussien; Abushouk, Abdelrahman Ibrahim; Menshawy, Amr; Attia, Attia; Mohamed, Arwa; Negida, Ahmed; Abdel-Daim, Mohamed M

    2018-01-01

    Grazoprevir is an NS3/4A protease inhibitor (PI), while elbasvir is an NS5A inhibitor. We performed this meta-analysis to directly compare grazoprevir plus elbasvir and ribavirin regimen vs. grazoprevir and elbasvir without ribavirin in the treatment of hepatitis C virus genotype 1 infection and to precisely evaluate the efficacy of the latter regimen in cirrhotic, IL28 CC genotype patients and those coinfected with human immunodeficiency virus. A computer literature search of PubMed, Scopus, EBSCO, Embase, and Cochrane central was conducted. Studies were screened for eligibility. Sustained virologic response (SVR) rates were pooled using OpenMeta[Analyst] software for windows. A subgroup analysis was performed to stratify the treatment efficacy according to the different baseline characteristics of HCV patients. Eight randomized controlled trials (n = 1,297 patients) were pooled in the final analysis. The overall SVR rate was 96.6% with 95% CI [95.5% to 98%]. For cirrhotic patients, the SVR rate was 95.7% with 95% CI [93.9% to 97.5%] and for non-cirrhotic patients, the SVR rate was 97% with 95% CI [95.9% to 98.4%]. Furthermore, the addition of ribavirin (RBV) to the treatment regimen did not significantly improve the SVR (RR 1.003, 95% CI [0.944 to 1.065]). The dual regimen was effective in patient populations with NS3 resistance-associated (RAS). However, this regimen achieved lower SVR rates (< 90%) in patients with NS5A RAS. We conclude that the 12-week treatment regimen of the fixed dose combination of grazoprevir plus elbasvir achieved high SVR rates in patients with HCV genotype 1 infection. The addition of ribavirin to this regimen did not add a significant benefit.

  17. Multiple ascending dose study of BMS-790052, a nonstructural protein 5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1.

    Science.gov (United States)

    Nettles, Richard E; Gao, Min; Bifano, Marc; Chung, Ellen; Persson, Anna; Marbury, Thomas C; Goldwater, Ronald; DeMicco, Michael P; Rodriguez-Torres, Maribel; Vutikullird, Apinya; Fuentes, Ernesto; Lawitz, Eric; Lopez-Talavera, Juan Carlos; Grasela, Dennis M

    2011-12-01

    The antiviral activity, resistance profile, pharmacokinetics (PK), safety, and tolerability of BMS-790052, a nonstructural protein 5A (NS5A) replication complex inhibitor, were evaluated in a double-blind, placebo-controlled, sequential panel, multiple ascending dose study. Thirty patients with chronic hepatitis C virus (HCV) genotype 1 infection were randomized to receive a 14-day course of BMS-790052 (1, 10, 30, 60, or 100 mg once daily or 30 mg twice daily) or placebo in a ratio of 4:1. The mean maximum decline from baseline in HCV RNA ranged from 2.8 to 4.1 log(10) IU/mL; the placebo group showed no evidence of antiviral activity. Most patients experienced viral rebound on or before day 7 of treatment with BMS-790052 monotherapy; viral rebound was associated with viral variants that had been previously implicated in resistance development in the in vitro replicon system. The PK profile was supportive of once-daily dosing with median peak plasma concentrations at 1-2 hours postdose and mean terminal half-life of 12-15 hours. Steady state was achieved following 3-4 days of daily dosing. BMS-790052 was well tolerated in all dose groups, with adverse events occurring with a similar frequency in BMS-790052- and placebo-treated groups. There were no clinically relevant changes in vital signs, laboratory, or electrocardiogram parameters. BMS-7590052 is the first NS5A replication complex inhibitor with multiple dose proof-of-concept in clinic. At doses of 1-100 mg daily, BMS-790052 was well tolerated, had a PK profile supportive of once-daily dosing, and produced a rapid and substantial decrease in HCV-RNA levels in patients chronically infected with HCV genotype 1. Copyright © 2011 American Association for the Study of Liver Diseases.

  18. Identification of class I HLA T cell control epitopes for West Nile virus.

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    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.

  19. Genetically modified VSV(NJ) vector is capable of accommodating a large foreign gene insert and allows high level gene expression.

    Science.gov (United States)

    An, Hwa-Yong; Kim, Gyoung Nyoun; Wu, Kunyu; Kang, C Yong

    2013-01-01

    It is desirable to develop a RNA virus vector capable of accommodating large foreign genes for high level gene expression. Vesicular stomatitis virus (VSV) has been used as a gene expression vector, especially Indiana serotype (VSV(Ind)), but less with New Jersey serotype (VSV(NJ)). Here, we report constructions of genetically modified rVSV(NJ) vector carrying various lengths of human hepatitis C virus (HCV) non-structural (NS) protein genes, level of inserted gene expression and characterization of rVSV(NJ). We modified the M gene of VSV(NJ) by changing methionine to arginine at positions 48 and 51 (rVSV(NJ)-M) (Kim and Kang, 2007) for construction of rVSV(NJ) with various lengths of HCV non-structural genes. The NS polyprotein genes of HCV were inserted between the G and L genes of the rVSV(NJ)-M vector, and recombinant VSV(NJ)-M viruses with HCV gene inserts were recovered by the reverse genetics. The recombinant VSV(NJ)-M vector with the HCV NS genes express high levels of all different forms of the NS proteins. The electron microscopic examination showed that lengths of recombinant VSV(NJ)-M without gene of interests, VSV(NJ)-M with a gene of HCV NS3 and NS4A (VSV(NJ)-M-NS3/4A), VSV(NJ)-M with a gene of HCV NS4AB plus NS5AB (VSV(NJ)-M-NS4AB/5AB), and VSV(NJ)-M carrying a gene of HCV NS3, NS4AB, and NS5AB (VSV(NJ)-M-NS3/4AB/5AB) were 172±10.5 nm, 201±12.5 nm, 226±12.9 nm, and 247±18.2 nm, respectively. The lengths of recombinant VSVs increased approximately 10nm by insertion of 1kb of foreign genes. The diameter of these recombinant viruses also increased slightly by longer HCV gene inserts. Our results showed that the recombinant VSV(NJ)-M vector can accommodate as much as 6000 bases of the foreign gene. We compared the magnitude of the IFN induction in mouse fibroblast L(Y) cells infected with rVSV(NJ) wild type and rVSV(NJ) M mutant viruses and show that the rVSV(NJ) M mutant virus infection induced a higher level of the IFN-β compare to the wild type

  20. HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes

    Science.gov (United States)

    Zhu, Zhaowen; Tran, Huy; Mathahs, M. Meleah; Moninger, Thomas O.; Schmidt, Warren N.

    2017-01-01

    Introduction Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. Results Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. Conclusions HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT

  1. All-Oral 12-Week Treatment With Daclatasvir Plus Sofosbuvir in Patients With Hepatitis C Virus Genotype 3 Infection: ALLY-3 Phase III Study

    Science.gov (United States)

    Nelson, David R; Cooper, James N; Lalezari, Jacob P; Lawitz, Eric; Pockros, Paul J; Gitlin, Norman; Freilich, Bradley F; Younes, Ziad H; Harlan, William; Ghalib, Reem; Oguchi, Godson; Thuluvath, Paul J; Ortiz-Lasanta, Grisell; Rabinovitz, Mordechai; Bernstein, David; Bennett, Michael; Hawkins, Trevor; Ravendhran, Natarajan; Sheikh, Aasim M; Varunok, Peter; Kowdley, Kris V; Hennicken, Delphine; McPhee, Fiona; Rana, Khurram; Hughes, Eric A

    2015-01-01

    Treatment options for patients with hepatitis C virus (HCV) genotype 3 infection are limited, with the currently approved all-oral regimens requiring 24-week treatment and the addition of ribavirin (RBV). This phase III study (ALLY-3; http://ClinicalTrials.gov: NCT02032901) evaluated the 12-week regimen of daclatasvir (DCV; pangenotypic nonstructural protein [NS]5A inhibitor) plus sofosbuvir (SOF; pangenotypic NS5B inhibitor) in patients infected with genotype 3. Patients were either treatment naïve (n = 101) or treatment experienced (n = 51) and received DCV 60 mg plus SOF 400 mg once-daily for 12 weeks. Coprimary endpoints were the proportions of treatment-naïve and treatment-experienced patients achieving a sustained virological response (SVR) at post-treatment week 12 (SVR12). SVR12 rates were 90% (91 of 101) and 86% (44 of 51) in treatment-naïve and treatment-experienced patients, respectively; no virological breakthrough was observed, and ≥99% of patients had a virological response (VR) at the end of treatment. SVR12 rates were higher in patients without cirrhosis (96%; 105 of 109) than in those with cirrhosis (63%; 20 of 32). Five of seven patients who previously failed treatment with an SOF-containing regimen and 2 of 2 who previously failed treatment with an alisporivir-containing regimen achieved SVR12. Baseline characteristics, including gender, age, HCV-RNA levels, and interleukin-28B genotype, did not impact virological outcome. DCV plus SOF was well tolerated; there were no adverse events (AEs) leading to discontinuation and only 1 serious AE on-treatment, which was unrelated to study medications. The few treatment-emergent grade 3/4 laboratory abnormalities that were observed were transient. Conclusion: A 12-week regimen of DCV plus SOF achieved SVR12 in 96% of patients with genotype 3 infection without cirrhosis and was well tolerated. Additional evaluation to optimize efficacy in genotype 3–infected patients with cirrhosis is underway

  2. Daclatasvir plus Sofosbuvir for HCV in Patients Coinfected with HIV-1.

    Science.gov (United States)

    Wyles, David L; Ruane, Peter J; Sulkowski, Mark S; Dieterich, Douglas; Luetkemeyer, Anne; Morgan, Timothy R; Sherman, Kenneth E; Dretler, Robin; Fishbein, Dawn; Gathe, Joseph C; Henn, Sarah; Hinestrosa, Federico; Huynh, Charles; McDonald, Cheryl; Mills, Anthony; Overton, Edgar Turner; Ramgopal, Moti; Rashbaum, Bruce; Ray, Graham; Scarsella, Anthony; Yozviak, Joseph; McPhee, Fiona; Liu, Zhaohui; Hughes, Eric; Yin, Philip D; Noviello, Stephanie; Ackerman, Peter

    2015-08-20

    The combination of daclatasvir, a hepatitis C virus (HCV) NS5A inhibitor, and the NS5B inhibitor sofosbuvir has shown efficacy in patients with HCV monoinfection. Data are lacking on the efficacy and safety of this combination in patients coinfected with human immunodeficiency virus type 1 (HIV-1). This was an open-label study involving 151 patients who had not received HCV treatment and 52 previously treated patients, all of whom were coinfected with HIV-1. Previously untreated patients were randomly assigned in a 2:1 ratio to receive either 12 weeks or 8 weeks of daclatasvir at a standard dose of 60 mg daily (with dose adjustment for concomitant antiretroviral medications) plus 400 mg of sofosbuvir daily. Previously treated patients were assigned to undergo 12 weeks of therapy at the same doses. The primary end point was a sustained virologic response at week 12 after the end of therapy among previously untreated patients with HCV genotype 1 who were treated for 12 weeks. Patients had HCV genotypes 1 through 4 (83% with genotype 1), and 14% had compensated cirrhosis; 98% were receiving antiretroviral therapy. Among patients with genotype 1, a sustained virologic response was reported in 96.4% (95% confidence interval [CI], 89.8 to 99.2) who were treated for 12 weeks and in 75.6% (95% CI, 59.7 to 87.6) who were treated for 8 weeks among previously untreated patients and in 97.7% (95% CI, 88.0 to 99.9) who were treated for 12 weeks among previously treated patients. Rates of sustained virologic response across all genotypes were 97.0% (95% CI, 91.6 to 99.4), 76.0% (95% CI, 61.8 to 86.9), and 98.1% (95% CI, 89.7 to 100), respectively. The most common adverse events were fatigue, nausea, and headache. There were no study-drug discontinuations because of adverse events. HIV-1 suppression was not compromised. Among previously untreated HIV-HCV coinfected patients receiving daclatasvir plus sofosbuvir for HCV infection, the rate of sustained virologic response across

  3. Daclatasvir for previously untreated chronic hepatitis C genotype-1 infection: a randomised, parallel-group, double-blind, placebo-controlled, dose-finding, phase 2a trial.

    Science.gov (United States)

    Pol, Stanislas; Ghalib, Reem H; Rustgi, Vinod K; Martorell, Claudia; Everson, Greg T; Tatum, Harvey A; Hézode, Christophe; Lim, Joseph K; Bronowicki, Jean-Pierre; Abrams, Gary A; Bräu, Norbert; Morris, David W; Thuluvath, Paul J; Reindollar, Robert W; Yin, Philip D; Diva, Ulysses; Hindes, Robert; McPhee, Fiona; Hernandez, Dennis; Wind-Rotolo, Megan; Hughes, Eric A; Schnittman, Steven

    2012-09-01

    Several direct-acting antivirals for chronic hepatitis C virus (HCV) infection are available, but they are limited by tolerability and dosing schedules. Once-daily daclatasvir, a potent NS5A replication complex inhibitor, was generally well tolerated in phase 1 studies. We assessed daclatasvir in combination with pegylated interferon (peginterferon) and ribavirin for chronic HCV. In this double-blind, parallel-group, dose-finding, phase 2a study, treatment-naive patients with HCV genotype-1 infection (without cirrhosis) from 14 centres in the USA and France were randomly assigned (1:1:1:1) to receive peginterferon alfa-2a (180 μg per week) and ribavirin (1000-1200 mg daily) plus placebo or 3 mg, 10 mg, or 60 mg of daclatasvir taken once daily, for 48 weeks. The primary efficacy endpoint was undetectable HCV RNA at 4 weeks and 12 weeks after start of treatment (extended rapid virological response, eRVR). Analysis was of all participants who received one dose of study drug. We used descriptive analyses to compare results. This study is registered with ClinicalTrials.gov, number NCT00874770. 48 patients were randomly assigned (12 per group); all received at least one dose of study drug. 15 patients discontinued treatment before week 48. Five of 12 patients (42%, 80% CI 22-64%) who received 3 mg daclatasvir achieved eRVR, compared with ten of 12 (83%, 61-96%) who received 10 mg daclatasvir, nine of 12 (75%, 53-90%) who received 60 mg daclatasvir, and one of 12 (8%, 1-29%) who received placebo. Adverse events and discontinuations as a result of adverse events occurred with similar frequency across groups. Daclatasvir seems to be a potent NS5A replication complex inhibitor that increases the antiviral potency of peginterferon and ribavirin. Our findings support the further development of regimens containing 60 mg daclatasvir for the treatment of chronic genotype-1 HCV infection. Bristol-Myers Squibb. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. 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

  5. A high frequency of GBV-C/HGV coinfection in hepatitis C patients in Germany.

    Science.gov (United States)

    Yan, Jie; Dennin, Reinhard H

    2000-12-01

    AIM:To detect infection rate of GBV-C/HGV in hepatitis C patients, to determine the methods of higher sensitivity and the primers of higher efficiency for GBV-C/HGV RNA detection and to study the dominant subtype and mutation of GBV-C/HGV.METHODS:Quantitative RT-PCR for detection pf HCV RNA concentration in serum samples, RT-nested PCR with two sets of primers for detection of GBV-C RNA, RT-PCR ELISA with two sets of primers for detection of HGV RNA, nucleotide sequence and putative amino acid sequence analysis.RESULTS:The positive rates of GBV-C RNA at the 5'-NCR and NS3 region in 211 serums amples from the patients with HCV infection were 31.8% and 22.8% respectively. The positive rates of HGV RNA at the 5'-NCR and NS5 region in the same samples were 47.9% and 31.8% respectively. The total positive rate of GBV-C/HGV RNA was as high as 55.5%. HCV copy numbers in the patients without GBV-C/HGV coinfection were statistically higher than that in the patients with GBV-C/HGV coinfection (PGBV-C NS3 region nucleotide sequences. All nucleotide sequences from amplification products showed higher homology to HGV genome than to GBV-C genome even though part of the sequences were amplified with GBV-C primers.CONCLUSION:A high frequency of GBV-C/HGV coinfection existed in the hepatitis C patients. RT-PCR ELISA was more sensitive than RT-nested PCR for detection of GBV-C/HGV RNA. The primers derived from the 5'-NCR was more efficient than those derived from the NS3 and NS5 regions. A reverse relationship was found to exist between HCV RNA concentration and GBV-C/HGV infection frequency. HGV was the dominant subtype of the virus in the local area. The major mutations of GBV-C/HGV genomes were random mutation of nucleotide residue.

  6. 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

  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 Caracterização molecular de duas cepas do flavivírus Rocio, isoladas durante a epidemia de encefalite no Estado de São Paulo, Brasil e desenvolvimento do teste one-step RT-PCR para diagnóstico

    Directory of Open Access Journals (Sweden)

    Terezinha Lisieux Moraes Coimbra

    2008-04-01

    Full Text Available 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.O vírus Rocio (ROCV foi responsável por uma explosiva epidemia de encefalite que ocorreu nos anos 70 afetando cerca de 1.000 habitantes de 20 municípios litorâneos do Estado de São Paulo, Brasil. ROCV foi isolado em 1975 de cerebelo de caso humano fatal de encefalite. As manifestações clínicas da doença são semelhantes àquelas descritas para encefalite St. Louis. ROCV apresenta intensa reatividade cruzada com os vírus do Complexo da Encefalite Japonesa (JEV, particularmente com o vírus Ilhéus (ILHV e com os vírus das encefalites St. Louis, Murray Valley e West Nile. Neste estudo, relatamos o desenvolvimento de um teste de RT-PCR específico para diagnóstico de ROCV e a caracterização molecular das cepas SPAn37630 e SPH37623. Foi realizada a análise filogenética das seqüências parciais dos genes NS5 e E, de ambas as cepas. Os resultados indicaram que essas cepas são intimamente relacionadas ao complexo JEV, mas formando um subgrupo com o

  8. 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

  9. Use of a Recombinant Gamma-2 Herpesvirus Vaccine Vector against Dengue Virus in Rhesus Monkeys.

    Science.gov (United States)

    Bischof, Georg F; Magnani, Diogo M; Ricciardi, Michael; Shin, Young C; Domingues, Aline; Bailey, Varian K; Gonzalez-Nieto, Lucas; Rakasz, Eva G; Watkins, David I; Desrosiers, Ronald C

    2017-08-15

    Research on vaccine approaches that can provide long-term protection against dengue virus infection is needed. Here we describe the construction, immunogenicity, and preliminary information on the protective capacity of recombinant, replication-competent rhesus monkey rhadinovirus (RRV), a persisting herpesvirus. One RRV construct expressed nonstructural protein 5 (NS5), while a second recombinant expressed a soluble variant of the E protein (E85) of dengue virus 2 (DENV2). Four rhesus macaques received a single vaccination with a mixture of both recombinant RRVs and were subsequently challenged 19 weeks later with 1 × 10 5 PFU of DENV2. During the vaccine phase, plasma of all vaccinated monkeys showed neutralizing activity against DENV2. Cellular immune responses against NS5 were also elicited, as evidenced by major histocompatibility complex class I (MHC-I) tetramer staining in the one vaccinated monkey that was Mamu-A*01 positive. Unlike two of two unvaccinated controls, two of the four vaccinated monkeys showed no detectable viral RNA sequences in plasma after challenge. One of these two monkeys also showed no anamnestic increases in antibody levels following challenge and thus appeared to be protected against the acquisition of DENV2 following high-dose challenge. Continued study will be needed to evaluate the performance of herpesviral and other persisting vectors for achieving long-term protection against dengue virus infection. IMPORTANCE Continuing studies of vaccine approaches against dengue virus (DENV) infection are warranted, particularly ones that may provide long-term immunity against all four serotypes. Here we investigated whether recombinant rhesus monkey rhadinovirus (RRV) could be used as a vaccine against DENV2 infection in rhesus monkeys. Upon vaccination, all animals generated antibodies capable of neutralizing DENV2. Two of four vaccinated monkeys showed no detectable viral RNA after subsequent high-dose DENV2 challenge at 19 weeks

  10. Assessment of two carrier materials for phosphate solubilizing biofertilizers and their effect on growth of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Mukhtar, Salma; Shahid, Izzah; Mehnaz, Samina; Malik, Kauser A

    2017-12-01

    Biofertilizers are usually carrier-based inoculants containing beneficial microorganisms. Incorporation of microorganisms in carrier material enables easy-handling, long-term storage and high effectiveness of biofertilizers. Objective of the present study was to assess enriched biogas sludge and soil as biofertilizer carriers on growth and yield of wheat. Six phosphate solubilizing strains were used in this study. Three phosphate solubilizing strains, 77-NS2 (Bacillus endophyticus), 77-CS-S1 (Bacillus sphaericus) and 77-NS5 (Enterobacter aerogenes) were isolated from the rhizosphere of sugarcane, two strains, PSB5 (Bacillus safensis) and PSB12 (Bacillus megaterium) from the rhizosphere of wheat and one halophilic phosphate solubilizing strain AT2RP3 (Virgibacillus sp.) from the rhizosphere of Atriplex amnicola, were used as bioinoculants. Phosphate solubilization ability of these strains was checked in vitro in Pikovskaya medium, containing rock phosphate (RP) as insoluble P source, individually supplemented with three different carbon sources, i.e., glucose, sucrose and maltose. Maximum phosphate solubilization; 305.6μg/ml, 217.2μg/ml and 148.1μg/ml was observed in Bacillus strain PSB12 in Pikovskaya medium containing sucrose, maltose and glucose respectively. A field experiment and pot experiments in climate control room were conducted to study the effects of biogas sludge and enriched soil based phosphorous biofertilizers on growth of wheat. Bacillus strain PSB12 significantly increased root and shoot dry weights and lengths using biogas sludge as carrier material in climate control room experiments. While in field conditions, significant increase in root and shoot dry weights, lengths and seed weights was seen by PSB12 and PSB5 (Bacillus) and Enterobacter strain 77-NS5 using biogas sludge as carrier. PSB12 also significantly increased both root and shoot dry weights and lengths in field conditions when used as enriched soil based inoculum. These results

  11. All-oral combination of ledipasvir, vedroprevir, tegobuvir, and ribavirin in treatment-naïve patients with genotype 1 HCV infection.

    Science.gov (United States)

    Wyles, David L; Rodriguez-Torres, Maribel; Lawitz, Eric; Shiffman, Mitchell L; Pol, Stanislas; Herring, Robert W; Massetto, Benedetta; Kanwar, Bittoo; Trenkle, James D; Pang, Phil S; Zhu, Yanni; Mo, Hongmei; Brainard, Diana M; Subramanian, G Mani; McHutchison, John G; Habersetzer, François; Sulkowski, Mark S

    2014-07-01

    This phase II trial assessed the efficacy and safety of a combination regimen of the nonstructural protein (NS)5A inhibitor ledipasvir (LDV), NS3 protease inhibitor vedroprevir (VDV), non-nucleoside NS5B inhibitor tegobuvir (TGV), and ribavirin (RBV) in treatment-naïve patients with chronic hepatitis C virus (HCV) genotype 1 without cirrhosis. Patients were randomized 1:2 to LDV 30 mg once daily (QD; Arm 1; n = 46) or LDV 90 mg QD (Arm 2; n = 94); patients in both arms also received VDV 200 mg QD, TGV 30 mg twice-daily, and RBV 1,000-1,200 mg/day. Patients in Arm 2 with vRVR, defined as HCV RNA below the lower limit of quantification (LLOQ) from treatment weeks 2 to 10, were randomized 1:1 to stop treatment at 12 weeks or continue for 24 weeks. Sustained virologic response 12 weeks after treatment (SVR12) was higher in patients receiving 90 mg of LDV for 24 weeks (63%), compared with LDV 90 mg for 12 weeks (54%) and LDV 30 mg for 24 weeks (48%). In patients with very rapid virologic response (vRVR) in Arm 2, SVR12 was achieved by 68% and 81% of patients treated for 12 and 24 weeks, respectively. Virologic breakthrough was more common in patients with HCV genotype 1a and was associated with resistance-associated variants for all three direct-acting antiviral agents (DAAs); however, in all but 1 patient who relapsed, resistance-associated variants directed against only one or two of the DAAs were detected. The most common adverse events were fatigue, headache, nausea, rash, and diarrhea. In patients with HCV genotype 1, an interferon-free regimen containing LDV/VDV/TGV/RBV was well tolerated and led to SVR12 in up to 63% of patients. LDV 90 mg is currently being investigated in combination with the nucleotide polymerase inhibitor, sofosbuvir. © 2014 by the American Association for the Study of Liver Diseases.

  12. Dasabuvir (ABT333) for the treatment of chronic HCV genotype I: a new face of cure, an expert review.

    Science.gov (United States)

    Mantry, Parvez S; Pathak, Lakshmikant

    2016-01-01

    Hepatitis C virus (HCV) affects nearly 1.3% of US population and around 2% of people worldwide. It is associated with serious complication of Cirrhosis and Hepatocellular carcinoma leading to significant morbidity and mortality. Until now the only treatment option for this serious disease was interferon based therapy which had poor tolerance and at best SVR (Sustained virological response) in only 50% of cases. With the introduction of other direct - acting antiviral agents the treatment of HCV has been revolutionized with significantly high rates of cure. Among novel Direct acting antivirals are non-nucleoside inhibitor NS5B which is highly effective in treatment of HCV genotype 1 a and 1b including those with compensated cirrhosis achieving high cure rates with SVR more than 97 % in pooled analysis from six different phase 3 trials. This review will discuss the DAA - Dasabuvir, a non - nucleoside NS5B inhibitor, its mechanism of action, efficacy, safety & tolerance, and drug resistance. Dasabuvir is approved by FDA in combination with other DAA agents called as the 3D(Viekira Pak) in various interferon free regimens achieving high cure rates (SVR >95%) with low adverse effects. In Europe, it is approved by European medicines agency for use in combination with Ombitasvir, Paritaprevir, and ritonavir with or without ribavirin. The drug is used in treatment naive as well as previously treated patient with high success rates. It is also approved in patients with compensated cirrhosis, patients with HIV co-infection and liver transplant recipients which were in the past were excluded from treatment with interferon based therapy. Dasabuvir is extensively evaluated in large clinical trials and shown excellent SVR among HCV genotype1 patient population in combination with other oral DAAs, with good safety profile and tolerance. Its drawback is its genotype restriction, need for ribavirin (RBV) for 1a genotype, low resistance barrier and high cost. It is well tolerated

  13. Ledipasvir + sofosbuvir (Harvoni). A therapeutic advance in genotype 1 hepatitis C virus infection, despite uncertainties.

    Science.gov (United States)

    2015-12-01

    Treatment for chronic hepatitis C depends on the hepatitis C virus (HCV) genotype and the patient's clinical characteristics. A fixed-dose combination of ledipasvir + sofosbuvir has been authorised in the European Union for adults with HCV genotype 1 (HCV-1), HCV-3 or HCV-4 infection. Ledipasvir targets the HCV protein NS5A, while sofosbuvir inhibits the HCV RNA polymerase NS5B. The ledipasvir+ sofosbuvircombination has not been compared directly with other antiviral drugs. No information is available on its ability to prevent hepatic complications, even in patients with cirrhosis. In four trials including over 1800 treatment-naive patients infected with HCV-1, a 12-week course of ledipasvir + sofosbuviryielded a sustained virological response in nearly every case. This is better than that reported with peginterferon alfa-based protocols. In four trials including more than 900 HCV-1-infected patients in whom treatments including peginterferon alfa had failed, a 24-week course of ledipasvir+ sofosbuvir yielded a sustained virological response in nearly every case, which is far better than reported with peginterferon alfa + ribavirin + protease inhibitor combinations, based on indirect comparison. In these trials, a 24-week course of the ledipasvir + sofosbuvir combination was effective in almost all patients with compensated cirrhosis. The same treatment also showed major efficacy in a non-comparative trial in 337 HCV-1-infected patients with decompensated cirrhosis or who had undergone liver transplantation. In mid-2015, very few data are available on the ledipasvir + sofosbuvir combination in HCV-1-infected patients in whom sofosbuvir combination therapy has failed, or in patients with HCV-3 or HCV-4 infection. Comparative data on the adverse effects of the ledipasvir + sofosbuvir combination are mainly based on a double-blind, placebo-controlled trial in 155 patients. Overall, serious adverse effects were infrequent in this and other trials. The main adverse

  14. Safety and efficacy of ledipasvir/sofosbuvir on hepatitis C eradication in hepatitis C virus/human immunodeficiency virus co-infected patients.

    Science.gov (United States)

    He, Xiaoping; Hopkins, Lynne; Everett, George; Carter, Willie M; SchroppDyce, Cynthia; Abusaada, Khalid; Hsu, Vincent

    2017-10-28

    To evaluate the safety and efficacy of ledipasvir/sofosbuvir on hepatitis C eradication in patients with hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infection in an urban HIV clinic. A retrospective cohort study of 40 subjects co-infected with HIV-1 and HCV treated with the fixed-dose combination of ledipasvir and sofosbuvir for 12 wk from 2014 to 2016. All patients included were receiving antiretroviral therapy (ART) with HIV RNA values of 100 copies/mL or fewer regardless of baseline HCV RNA level. The primary end point was a sustained virologic response of HCV at 12 wk (SVR12) after the end of therapy. Of the 40 patients enrolled, 55% were black, 22.5% had been previously treated for HCV, and 25% had cirrhosis. The patients were on a wide range of ART. Overall, 39 patients (97.5%) had a SVR 12 after the end of therapy, including rates of 97.1% in patients with HCV genotype 1a and 100% in those with HCV genotype 1b. One patient with HCV genotype 3a was included and achieved SVR12. Rates of SVR12 were similar regardless of previous treatment or the presence of compensated cirrhosis. Only 1 patient experienced relapse at week 12 following treatment and deep sequencing didn't reveal any resistance associated mutation in the NS5A or NS5B region. Interestingly, 7 (17.5%) patients who were adherent to ART experienced HIV viral breakthrough which resolved after continuing the same ART regimen. Two (5%) patients experienced HIV-1 virologic rebound due to noncompliance with HIV therapy, which resolved after resuming the same ART regimen. No severe adverse events were observed and no patient discontinued treatment because of adverse events. The most common adverse events included headache (12.5%), fatigue (10%), and diarrhea (2.5%). This retrospective study demonstrated the high rates of SVR12 of ledipasvir/sofosbuvir on HCV eradication in patients co-infected with HCV and HIV, regardless of HCV baseline levels, HCV treatment history or cirrhosis

  15. 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

  16. Cost-utility analysis of ledipasvir/sofosbuvir for the treatment of genotype 1 chronic hepatitis C in Japan.

    Science.gov (United States)

    Igarashi, Ataru; Tang, Wentao; Guerra, Ines; Marié, Lucile; Cure, Sandrine; Lopresti, Michael

    2017-01-01

    Hepatitis C is the result of a ribonucleic acid (RNA) virus (hepatitis C virus; HCV). The Japan Society of Hepatology (JSH) estimated that 1.5-2 million people in Japan carry HCV. Six major HCV genotypes (GT) and a large number of subtypes have been described in the literature. In Japan, around 70% to 80% of people are infected with HCV genotype 1b. The progress of the disease primarily affects the liver and may lead to liver cirrhosis, hepatocellular carcinoma (HCC) and death. Sofosbuvir (SOF) is a nucleotide analogue NS5B inhibitor and ledipasvir (LDV) is an inhibitor of the HCV NS5A protein. They are combined in a single tablet regimen for the treatment of GT1 patients and resulted in sustained virological response (SVR) above 94% in large phase III trials. This analysis assesses the cost-utility of LDV/SOF in GT1 patients in Japan. A cohort of 10,000 patients was followed through a Markov model until they reached 100 years of age. GT1 treatment-naïve and experienced, non-cirrhotic and cirrhotic patients were studied separately. LDV/SOF was compared to several treatment regimens containing pegylated interferon (PEGIFN), telaprevir (TVR), simeprevir (SMV), daclatasvir (DCV), asunaprevir (ASV) and ribavirin (RBV). Discount rates of 2% were applied to costs and outcomes according to the Japanese guidelines. LDV/SOF was cost-effective against most comparators with incremental cost-effectiveness ratios (ICERs) below JPY 5,000,000. By applying a societal perspective, LDV/SOF was the dominant treatment strategy in all cases. Moreover, LDV/SOF reduced the number of cases of advanced liver disease. These results were robust to sensitivity analyses. LDV/SOF was cost-effective compared to most of the currently recommended treatments. Furthermore, LDV/SOF extends treatments to HCV-infected patients who are ineligible for interferon and RBV-based regimens. LDV/SOF thus has the potential to help reduce the burden of HCV in Japan.

  17. Safety and efficacy of ledipasvir-sofosbuvir in black patients with hepatitis C virus infection: A retrospective analysis of phase 3 data.

    Science.gov (United States)

    Wilder, Julius M; 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

    2016-02-01

    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. 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. © 2015 The Authors. HEPATOLOGY published by Wiley

  18. The Novel Cyclophilin Inhibitor CPI-431-32 Concurrently Blocks HCV and HIV-1 Infections via a Similar Mechanism of Action.

    Directory of Open Access Journals (Sweden)

    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.

  19. Generation and Characterization of a Hepatitis C Virus NS3 Protease-Dependent Bovine Viral Diarrhea Virus

    Science.gov (United States)

    Lai, Vicky C. H.; Zhong, Weidong; Skelton, Angela; Ingravallo, Paul; Vassilev, Venteislav; Donis, Ruben O.; Hong, Zhi; Lau, Johnson Y. N.

    2000-01-01

    Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, PH/B (wild-type HCV NS3 protease) and PH/B(S139A) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the PH/B clone yielded viable viruses, whereas the mutant clone, PH/B(S139A), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV−Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV−Npro was highly defective in viral replication and growth, a

  20. 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

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

    Science.gov (United States)

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

    2018-01-01

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

  2. Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives.

    Science.gov (United States)

    Coutard, Bruno; Barral, Karine; Lichière, Julie; Selisko, Barbara; Martin, Baptiste; Aouadi, Wahiba; Lombardia, Miguel Ortiz; Debart, Françoise; Vasseur, Jean-Jacques; Guillemot, Jean Claude; Canard, Bruno; Decroly, Etienne

    2017-03-01

    The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2'-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2'-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor ( S -adenosyl-l-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the αX α-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs. IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns, and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune

  3. Hepatitis C virus infection in Brazilian long-distance truck drivers

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    Freitas Nara R

    2010-08-01

    Full Text Available Abstract Hepatitis C virus (HCV infection is a global public health problem. Long-distance truck drivers live apart from their family for long periods of time, a lifestyle that favors at-risk behaviors such as unprotected sex with multiple partners and illicit drug use. As data concerning HCV infection in this population are still rare, this paper aims to investigate the prevalence, genotypes/subtypes, and the factors associated with HCV infection in long-distance truck drivers in Brazil. A cross-sectional survey was carried out with 641 Brazilian long-truck drivers who were recruited at a major truck stop located at kilometer 1,296 of the BR-153 highway, which is considered to be one of the longest roads in Brazil. All individuals were interviewed, and their serum samples were tested for the presence of antibodies to HCV (anti-HCV by ELISA and immunoblot. Anti-HCV positive samples were tested for HCV RNA by PCR amplification of the 5' NC and NS5B regions and were genotyped using the LiPA assay and nucleotide sequencing, respectively. Factors associated with HCV infection were identified with logistic regression. The prevalence of HCV infection was 1.4% (95% CI: 0.7-2.8. History of blood transfusion, sharing of personal hygiene tools, illicit drug use and HBV status were factors independently associated with HCV infection in the study population. HCV RNA was detected in 8/9 anti-HCV positive samples, in which genotypes 1 (n = 3, 2 (n = 2, and 3 (n = 3 were determined by LiPA. Using phylogenetic tree analysis of the NS5B region, subtypes 1a (n = 1, 1b (n = 2, 2b (n = 2 and 3a (n = 3 were identified. These data show that the prevalence of HCV infection among Brazilian truck drivers was similar to that observed for the general population. History of blood transfusion, sharing of personal hygiene tools, illicit drug use and HBV status were predictors of HCV infection. The HCV genotypes/subtypes identified in the study population are consistent with

  4. Multiple Introduction and Naturally Occuring Drug Resistance of HCV among HIV-Infected Intravenous Drug Users in Yunnan: An Origin of China's HIV/HCV Epidemics.

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    Min Chen

    Full Text Available The human immunodeficiency virus 1 (HIV-1 epidemic in China historically stemmed from intravenous drug users (IDUs in Yunnan. Due to a shared transmission route, hepatitis C virus (HCV/HIV-1 co-infection is common. Here, we investigated HCV genetic characteristics and baseline drug resistance among HIV-infected IDUs in Yunnan.Blood samples of 432 HIV-1/HCV co-infected IDUs were collected from January to June 2014 in six prefectures of Yunnan Province. Partial E1E2 and NS5B genes were sequenced. Phylogenetic, evolutionary and genotypic drug resistance analyses were performed.Among the 293 specimens successfully genotyped, seven subtypes were identified, including subtypes 3b (37.9%, 111/293, 3a (21.8%, 64/293, 6n (14.0%, 41/293, 1b (10.6%, 31/293, 1a (8.2%, 24/293, 6a (5.1%, 15/293 and 6u (2.4%, 7/293. The distribution of HCV subtypes was mostly related to geographic location. Subtypes 3b, 3a, and 6n were detected in all six prefectures, however, the other four subtypes were detected only in parts of the six prefectures. Phylogeographic analyses indicated that 6n, 1a and 6u originated in the western prefecture (Dehong and spread eastward and showed genetic relatedness with those detected in Burmese. However, 6a originated in the southeast prefectures (Honghe and Wenshan bordering Vietnam and was transmitted westward. These subtypes exhibited different evolutionary rates (between 4.35×10-4 and 2.38×10-3 substitutions site-1 year-1 and times of most recent common ancestor (tMRCA, between 1790.3 and 1994.6, suggesting that HCV was multiply introduced into Yunnan. Naturally occurring resistance-associated mutations (C316N, A421V, C445F, I482L, V494A, and V499A to NS5B polymerase inhibitors were detected in direct-acting antivirals (DAAs-naïve IDUs.This work reveals the temporal-spatial distribution of HCV subtypes and baseline HCV drug resistance among HIV-infected IDUs in Yunnan. The findings enhance our understanding of the characteristics and

  5. 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.)

  6. Hepatitis C virus prevalence and genetic diversity among pregnant women in Gabon, central Africa

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    Mahé Antoine

    2008-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV infection is a major global public health problem in both developed and developing countries. The prevalence and genetic diversity of HCV in pregnant women in Gabon, central Africa, is not known. We therefore evaluated the prevalence and the circulating genotypes of HCV in a large population cohort of pregnant women. Methods Blood samples (947 were collected from pregnant women in the five main cities of the country. The prevalence was evaluated by two ELISA tests, and the circulating genotypes were characterized by sequencing and phylogenetic analysis. Results Twenty pregnant women (2.1% were infected with HCV. The seroprevalence differed significantly by region (p = 0.004 and increased significantly with age (p = 0.05, being 1.3% at 14–20 years, 1.1% at 21–25 years, 1.9% at 26–30 years, 4.1% at 31–35 years and 6.0% at > 35 years. Sequencing in the 5'-UTR and NS5B regions showed that the circulating strains belonged to genotypes 4 (4e and 4c. Conclusion We found that the HCV seroprevalence in pregnant women in Gabon is almost as high as that in other African countries and increases with age. Furthermore, only genotype 4 (4e and 4c was found. More extensive studies aiming to evaluate the prevalence and heterogeneity of HCV genotypes circulating in the general population of the country are needed.

  7. Exact half-BPS type IIB interface solutions I: local solution and supersymmetric Janus

    Energy Technology Data Exchange (ETDEWEB)

    D' Hoker, Eric; Estes, John; Gutperle, Michael [Department of Physics and Astronomy, University of California, Los Angeles, CA 90095 (United States)

    2007-06-15

    The complete Type IIB supergravity solutions with 16 supersymmetries are obtained on the manifold AdS{sub 4} x S{sup 2} x S{sup 2} x {sigma} with SO(2, 3) x SO(3) x SO(3) symmetry in terms of two holomorphic functions on a Riemann surface {sigma}, which generally has a boundary. This is achieved by reducing the BPS equations using the above symmetry requirements, proving that all solutions of the BPS equations solve the full Type IIB supergravity field equations, mapping the BPS equations onto a new integrable system akin to the Liouville and Sine-Gordon theories, and mapping this integrable system to a linear equation which can be solved exactly. Amongst the infinite class of solutions, a non-singular Janus solution is identified which provides the AdS/CFT dual of the maximally supersymmetric Yang-Mills interface theory discovered recently. The construction of general classes of globally non-singular solutions, including fully back-reacted AdS{sub 5} x S{sup 5} and supersymmetric Janus doped with D5 and/or NS5 branes, is deferred to a companion paper.

  8. 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

    2017-03-07

    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.

  9. Alcohol, TLR4-TGF-β Antagonism, and Liver Cancer

    Science.gov (United States)

    Tsukamoto, Hidekazu; Mishra, Lopa; Machida, Keigo

    2016-01-01

    Alcohol abuse and obesity are two known risk factors for hepatocellular carcinoma (HCC) that also synergistically promote HBV/HCV-related carcinogenesis. TLR4, the PAMP for endotoxin participates in inflammatory processes such as M1 activation of hepatic macrophages in alcoholic liver disease. However its role in liver carcinogenesis via ectopic expression and activation, has only recently been revealed in alcohol/HCV-associated HCC models. Alcohol feeding to mice expressing the HCV Ns5a in a hepatocyte specific manner, aggravates liver inflammation via activation of overexpressed TLR4 in the parenchymal cells. Long-term alcohol feeding produces liver tumors in these transgenic mice in a manner dependent on TLR4. From these mice, CD133+/CD49f+ tumor initiating stem cell-like cells (TICs) have been isolated. These TICs exhibit self-renewal and tumorigenic activities driven by TLR4-dependent upregulation of the stem cell factor NANOG. Defective TGF-β tumor suppressor pathway is identified in the TICs and mediated by NANOG target genes Igf2bp3 and Yap1. This TGF-β pathway antagonism is responsible in part for TIC’s tumorigenic activity and chemoresistance. Conversely, mice with attenuated TGF-β pathway due to haploinsufficiency of β2-Spectrin, spontaneously develop liver tumors and alcohol-feeding increases tumor incidence in a TLR4 dependent manner. This reciprocal antagonism between TLR4 and TGF-β pathways may serve as a novel therapeutic target for HCC. PMID:26201318

  10. Daclatasvir: a review of its use in adult patients with chronic hepatitis C virus infection.

    Science.gov (United States)

    McCormack, Paul L

    2015-04-01

    Daclatasvir (Daklinza®) is an inhibitor of hepatitis C virus (HCV) NS5A protein. It is a new, oral, direct-acting antiviral with potent pangenotypic activity. This article provides a narrative review of the efficacy and tolerability of daclatasvir in combination with other agents in the treatment of patients with chronic HCV infection and summarizes its pharmacological properties. Since daclatasvir has a different mechanism of action to other current direct-acting antivirals, it provides additive or synergistic antiviral activity when used in combination. It produces high sustained virological response rates when used in combination with peginterferon-α plus ribavirin in patients chronically infected with HCV genotypes 1-4, and provides even higher response rates when used in an interferon-free, all-oral combination with sofosbuvir, with or without ribavirin. Daclatasvir has a moderately high genetic barrier to resistance, is effective during short-term treatment over 12 weeks and has a tolerability profile similar to that of placebo. In conclusion, daclatasvir is a highly effective and well tolerated, oral, once-daily, direct-acting antiviral for use in combination therapy in adult patients chronically infected with HCV.

  11. Effect of the coadministration of daclatasvir on the pharmacokinetics of a combined oral contraceptive containing ethinyl estradiol and norgestimate.

    Science.gov (United States)

    Bifano, Marc; Sevinsky, Heather; Hwang, Carey; Kandoussi, Hamza; Jiang, Hao; Grasela, Dennis; Bertz, Richard

    2014-01-01

    Daclatasvir is a highly selective NS5A replication complex inhibitor currently in development for the treatment of chronic hepatitis C infection. Daclatasvir is active at picomolar concentrations and demonstrates in vitro activity against a broad range of HCV genotypes. The primary objective of this study was to assess the effect of daclatasvir on the pharmacokinetics of a combined oral contraceptive containing ethinyl estradiol and norgestimate (Ortho Tri-Cyclen(®)). In this open-label single-sequence study, 20 healthy female subjects received ethinyl estradiol and norgestimate for three cycles, with coadministration of daclatasvir in cycle 3. Pharmacokinetics of ethinyl estradiol and the active metabolites of norgestimate (norelgestromin and norgestrel) were assessed in cycles 2 and 3. Adjusted ratios of geometric means and 90% CIs were estimated for the maximum observed plasma concentration (ethinyl estradiol 1.11 [1.02, 1.20], norelgestromin 1.06 [0.99, 1.14] and norgestrel 1.07 [0.99, 1.16]) and area under the plasma concentration-time curve in one dosing interval (ethinyl estradiol 1.01 [0.95, 1.07], norelgestromin 1.12 [1.06, 1.17] and norgestrel 1.12 [1.02, 1.23]). Coadministration of daclatasvir resulted in no clinically relevant effects on exposure to ethinyl estradiol, norelgestromin or norgestrel.

  12. Current Status in the Therapy of Liver Diseases

    Directory of Open Access Journals (Sweden)

    Philipp Uhl

    2014-04-01

    Full Text Available Hepatic diseases, like viral hepatitis, autoimmune hepatitis, hereditary hemochromatosis, non-alcoholic fatty liver disease (NAFLD and Wilson’s disease, play an important role in the development of liver cirrhosis and, hence, hepatocellular carcinoma. In this review, the current treatment options and the molecular mechanisms of action of the drugs are summarized. Unfortunately, the treatment options for most of these hepatic diseases are limited. Since hepatitis B (HBV and C (HCV infections are the most common causes of liver cirrhosis and hepatocellular carcinoma, they are the focus of the development of new drugs. The current treatment of choice for HBV/HCV infection is an interferon-based combination therapy with oral antiviral drugs, like nucleos(tide analogues, which is associated with improving the therapeutic success and also preventing the development of resistances. Currently, two new protease inhibitors for HCV treatment are expected (deleobuvir, faldaprevir and together with the promising drug, daclatasvir (NS5A-inhibitor, currently in clinical trials, adequate therapy is to be expected in due course (circumventing the requirement of interferon with its side-effects, while in contrast, efficient HBV therapeutics are still lacking. In this respect, entry inhibitors, like Myrcludex B, the lead substance of the first entry inhibitor for HBV/HDV (hepatitis D infection, provide immense potential. The pharmacokinetics and the mechanism of action of Myrcludex B are described in detail.

  13. Development of a New Structural Class of Broadly Acting HCV Non-Nucleoside Inhibitors Leading to the Discovery of MK-8876

    Energy Technology Data Exchange (ETDEWEB)

    McComas, Casey C.; Palani, Anandan; Chang, Wei; Holloway, M. Katharine; Lesburg, Charles A.; Li, Peng; Liverton, Nigel; Meinke, Peter T.; Olsen, David B.; Peng, Xuanjia; Soll, Richard M.; Ummat, Ajay; Wu, Jie; Wu, Jin; Zorn, Nicolas; Ludmerer, Steven W. (Merck); (WuXi App Tec)

    2017-07-25

    Studies directed at developing a broadly acting non-nucleoside inhibitor of HCV NS5B led to the discovery of a novel structural class of 5-aryl benzofurans that simultaneously interact with both the palm I and palm II binding regions. An initial candidate was potent in vitro against HCV GT1a and GT1b replicons, and induced multi-log reductions in HCV viral load when orally dosed to chronic GT1 infected chimpanzees. However, in vitro potency losses against clinically relevant GT1a variants prompted a further effort to develop compounds with sustained potency across a broader array of HCV genotypes and mutants. Ultimately, a biology and medicinal chemistry collaboration led to the discovery of the development candidate MK-8876. MK-8876 demonstrated a pan-genotypic potency profile and maintained potency against clinically relevant mutants. It demonstrated moderate bioavailability in rats and dogs, but showed low plasma clearance characteristics consistent with once-daily dosing. Herein we describe the efforts which led to the discovery of MK-8876, which advanced into Phase 1 monotherapy studies for evaluation and characterization as a component of an all-oral direct-acting drug regimen for the treatment of chronic HCV infection.

  14. The GB virus C (GBV-C NS3 serine protease inhibits HIV-1 replication in a CD4+ T lymphocyte cell line without decreasing HIV receptor expression.

    Directory of Open Access Journals (Sweden)

    Sarah L George

    Full Text Available INTRODUCTION: Persistent infection with GBV-C (GB Virus C, a non-pathogenic virus related to hepatitis C virus (HCV, prolongs survival in HIV infection. Two GBV-C proteins, NS5A and E2, have been shown previously to inhibit HIV replication in vitro. We investigated whether the GBV-C NS3 serine protease affects HIV replication. RESULTS: GBV-C NS3 protease expressed in a human CD4+ T lymphocyte cell line significantly inhibited HIV replication. Addition of NS4A or NS4A/4B coding sequence to GBV-C NS3 increased the effect on HIV replication. Inhibition of HIV replication was dose-dependent and was not mediated by increased cell toxicity. Mutation of the NS3 catalytic serine to alanine resulted in loss of both HIV inhibition and protease activity. GBV-C NS3 expression did not measurably decrease CD4 or CXCR4 expression. CONCLUSION: GBV-C NS3 serine protease significantly inhibited HIV replication without decreasing HIV receptor expression. The requirement for an intact catalytic serine at the active site indicates that inhibition was mediated by proteolytic cleavage of an unidentified target(s.

  15. Retinoids and rexinoids inhibit hepatitis C virus independently of retinoid receptor signaling.

    Science.gov (United States)

    Murakami, Yuko; Fukasawa, Masayoshi; Kaneko, Yukihiro; Suzuki, Tetsuro; Wakita, Takaji; Fukazawa, Hidesuke

    2014-02-01

    Using a high-throughput screening system involving HCV JFH-1-Huh 7.5.1 cells, we determined that the ligands of class II nuclear receptors, retinoids and rexinoids inhibit HCV infection. Retinoids, ligands of retinoic acid receptor (RAR), and rexinoids, ligands of retinoid X receptor (RXR), reduced extracellular HCV RNA of HCV infected cells in a dose-dependent manner. The 50% effective concentrations were below 10 nM, and the 50% cytotoxic concentrations were over 10 μM. Both agonists and antagonists demonstrated inhibition, which indicates that the effect is not dependent on retinoic acid signaling. These chemicals reduced HCV RNA and NS5A protein levels in cells harboring the subgenomic HCV replicon RNA, which suggests that the chemicals affect HCV RNA replication. These compounds were also effective against persistently infected cells, although the reduction in the intracellular HCV RNA was smaller than that of the extracellular HCV RNA, suggesting that viral post-replication step is also inhibited. In combination with interferon (IFN), retinoid exhibited a synergistic effect. Retinoids did not enhance expression of the IFN effector molecule PKR. These series of compounds warrant further investigation as new class of HCV drugs, for the clinical translation of our observation may lead to increased anti-HCV efficacy. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. 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.

  17. Single-walled carbon nanotubes oust graphene and semiconductor saturable absorbers in Q-switched solid-state lasers at 2 µm

    Science.gov (United States)

    Mateos, Xavier; Loiko, Pavel; Choi, Sun Young; Rotermund, Fabian; Aguiló, Magdalena; Díaz, Francesc; Griebner, Uwe; Petrov, Valentin

    2017-09-01

    The superior performance of a saturable absorber (SA) based on randomly-oriented single-walled carbon nanotubes (SWCNTs) operating in the spectral region of their first fundamental transition E 11 over a graphene-SA containing several (n  =  3) carbon layers and a commercial semiconductor SA (SESA) for passive Q-switching of bulk thulium solid-state lasers is demonstrated. Using SWCNT-SA in a compact diode-pumped Tm:KLuW laser, a maximum average output power of 1.36 W is achieved at 1932-1944 nm corresponding to a slope efficiency of 41% and pulse characteristics of 48 ns/5.5 µJ which are superior to that for graphene-SA (123 ns/3.1 µJ) and SESA (61 ns/1.1 µJ). SWCNT-SAs are promising for eye-safe miniature lasers at ~2 µm generating nanosecond pulses in the MHz repetition frequency range.

  18. Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method for diagnosis of Turkey Meningoencephalitis Virus.

    Science.gov (United States)

    Davidson, Irit; Raibstein, Israel; Altory-Natour, Amira; Simanov, Michael; Khinich, Yevgeny

    2017-06-01

    The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, drooping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA has now been upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the envelope and the non-structural NS5 gene, increasing the sensitivity by 10-100-fold compared to the previously existing assays. Based on the recent complete sequences of five TMEV isolates we have now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA assay was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009 and 2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least in experimentally infected turkeys and in three cases of disease encountered in commercial flocks.

  19. 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.

  20. Flavivirus Antagonism of Type I Interferon Signaling Reveals Prolidase as a Regulator of IFNAR1 Surface Expression.

    Science.gov (United States)

    Lubick, Kirk J; Robertson, Shelly J; McNally, Kristin L; Freedman, Brett A; Rasmussen, Angela L; Taylor, R Travis; Walts, Avram D; Tsuruda, Seitaro; Sakai, Mizuki; Ishizuka, Mariko; Boer, Elena F; Foster, Erin C; Chiramel, Abhilash I; Addison, Conrad B; Green, Richard; Kastner, Daniel L; Katze, Michael G; Holland, Steven M; Forlino, Antonella; Freeman, Alexandra F; Boehm, Manfred; Yoshii, Kentaro; Best, Sonja M

    2015-07-08

    Type I interferon (IFN-α/β or IFN-I) signals through two receptor subunits, IFNAR1 and IFNAR2, to orchestrate sterile and infectious immunity. Cellular pathways that regulate IFNAR1 are often targeted by viruses to suppress the antiviral effects of IFN-I. Here we report that encephalitic flaviviruses, including tick-borne encephalitis virus and West Nile virus, antagonize IFN-I signaling by inhibiting IFNAR1 surface expression. Loss of IFNAR1 was associated with binding of the viral IFN-I antagonist, NS5, to prolidase (PEPD), a cellular dipeptidase implicated in primary immune deficiencies in humans. Prolidase was required for IFNAR1 maturation and accumulation, activation of IFNβ-stimulated gene induction, and IFN-I-dependent viral control. Human fibroblasts derived from patients with genetic prolidase deficiency exhibited decreased IFNAR1 surface expression and reduced IFNβ-stimulated signaling. Thus, by understanding flavivirus IFN-I antagonism, prolidase is revealed as a central regulator of IFN-I responses. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Detection of Dengue Virus in Bat Flies (Diptera: Streblidae) of Common Vampire Bats, Desmodus rotundus, in Progreso, Hidalgo, Mexico.

    Science.gov (United States)

    Abundes-Gallegos, Judith; Salas-Rojas, Monica; Galvez-Romero, Guillermo; Perea-Martínez, Leonardo; Obregón-Morales, Cirani Y; Morales-Malacara, Juan B; Chomel, Bruno B; Stuckey, Matthew J; Moreno-Sandoval, Hayde; García-Baltazar, Anahi; Nogueda-Torres, Benjamin; Zuñiga, Gerardo; Aguilar-Setién, Alvaro

    2017-12-12

    Blood-feeding arthropods play a major role in the transmission of several flaviviruses, which represent an important problem for human health. Currently, dengue is one of the most important arboviral emerging diseases worldwide. Furthermore, some previous studies have reported the presence of viral nucleic acids and antibodies against dengue virus (DENV) in wild animals. Our knowledge of the role played by wildlife reservoirs in the sylvatic transmission and maintenance of DENV remains limited. Our objective was to screen blood-feeding ectoparasites (bat flies) and their common vampire bat (Desmodus rotundus) hosts, for flaviviruses in Hidalgo, Mexico. We detected Flavivirus sequences in 38 pools of ectoparasites (Diptera: Streblidae, Strebla wiedemanni and Trichobius parasiticus) and 8 tissue samples of D. rotundus by RT-PCR and semi-nested PCR using FlaviPF1S, FlaviPR2bis, and FlaviPF3S primers specific for NS5, a gene highly conserved among flaviviruses. Phylogenetic inference analysis performed using the maximum likelihood algorithm implemented in PhyML showed that six sequences clustered with DENV (bootstrap value = 53.5%). Although this study supports other reports of DENV detection in bats and arthropods other than Aedes mosquitoes, the role of these ectoparasitic flies and of hematophagous bats in the epidemiology of DENV still warrants further investigation.

  2. Entangled de Sitter from stringy axionic Bell pair I: an analysis using Bunch-Davies vacuum

    Science.gov (United States)

    Choudhury, Sayantan; Panda, Sudhakar

    2018-01-01

    In this work, we study the quantum entanglement and compute entanglement entropy in de Sitter space for a bipartite quantum field theory driven by an axion originating from Type IIB string compactification on a Calabi-Yau three fold (CY^3) and in the presence of an NS5 brane. For this computation, we consider a spherical surface S^2, which divides the spatial slice of de Sitter (dS_4) into exterior and interior sub-regions. We also consider the initial choice of vacuum to be Bunch-Davies state. First we derive the solution of the wave function of the axion in a hyperbolic open chart by constructing a suitable basis for Bunch-Davies vacuum state using Bogoliubov transformation. We then derive the expression for density matrix by tracing over the exterior region. This allows us to compute the entanglement entropy and Rényi entropy in 3+1 dimension. Furthermore, we quantify the UV-finite contribution of the entanglement entropy which contain the physics of long range quantum correlations of our expanding universe. Finally, our analysis complements the necessary condition for generating non-vanishing entanglement entropy in primordial cosmology due to the axion.

  3. The heme oxygenase 1 product biliverdin interferes with hepatitis C virus replication by increasing antiviral interferon response.

    Science.gov (United States)

    Lehmann, Elisabeth; El-Tantawy, Walid Hamdy; Ocker, Matthias; Bartenschlager, Ralf; Lohmann, Volker; Hashemolhosseini, Said; Tiegs, Gisa; Sass, Gabriele

    2010-02-01

    The anti-inflammatory and antiapoptotic heme degrading enzyme heme oxygenase-1 (HO-1) has been shown recently to interfere with replication of hepatitis C virus (HCV). We investigated the effect of HO-1 products carbon monoxide (CO), iron and biliverdin on HCV replication using the replicon cell lines Huh-5-15 and LucUbiNeo-ET, stably expressing HCV proteins NS3 through NS5B. Incubation of these cell lines in the presence of the CO donor methylene chloride transiently reduced HCV replication, whereas an increase of iron in cell culture by administration of FeCl(3) or iron-saturated lactoferrin did not interfere with HCV replication. Likewise, depletion of iron by deferoxamine during induction of HO-1 by cobalt-protoporphyrin IX did not restore HCV replication. The most prominent effect was observed after incubation of replicon cell lines in the presence of biliverdin. Biliverdin seems to interfere with HCV replication-mediated oxidative stress by inducing expression of antiviral interferons, such as interferon alpha2 and alpha17. The antioxidant biliverdin reduces HCV replication in vitro by triggering the antiviral interferon response and might improve HCV therapy in the future.

  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

    requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens. IMPORTANCE: Hepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing......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. West Nile virus circulation in South-Eastern Romania, 2011 to 2013.

    Science.gov (United States)

    Dinu, S; Cotar, A I; Pănculescu-Gătej, I R; Fălcuţă, E; Prioteasa, F L; Sîrbu, A; Oprişan, G; Bădescu, D; Reiter, P; Ceianu, C S

    2015-05-21

    Lineage 2 West Nile virus (WNV), previously found only in sub-Saharan Africa and Madagascar, was identified in Hungary in 2004 and has rapidly expanded in Europe in the past decade. Following a significant outbreak of West Nile fever with neurological cases caused by lineage 1 WNV in Romania in 1996, scattered cases have been recorded in the south-east of the country in each transmission season. Another outbreak, affecting a larger area and caused by lineage 2 WNV, was recorded in 2010. We analysed human sera from neuroinvasive West Nile fever cases and mosquitoes, sampled in south-eastern Romania between 2011 and 2013, for the presence of WNV genome, and obtained partial NS5 and envelope glycoprotein sequences. Human- and mosquito-derived WNV sequences were highly similar (99%) to Volgograd 2007 lineage 2 WNV and differed from isolates previously detected in central and southern Europe. WNV was detected in one pool of Culex pipiens s.l. males, documenting vertical transmission. Lineage 4 WNV, of unknown pathogenicity to mammals, was found in the amphibian-feeding mosquito Uranotaenia unguiculata from the Danube Delta. Our results present molecular evidence for the maintenance of the same isolates of Volgograd 2007-like lineage 2 WNV in south-eastern Romania between 2011 and 2013.

  6. Structure of a murine norovirus NS6 protease-product complex revealed by adventitious crystallisation.

    Directory of Open Access Journals (Sweden)

    Eoin N Leen

    Full Text Available Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro, NS7(pol by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6(pro, which has been determined to a resolution of 1.6 Å. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6(pro within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6(pro C-terminus is formed in vivo by NS6(pro processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro specificity.

  7. Interferon-based combination treatment for chronic hepatitis C in the era of direct acting antivirals.

    Science.gov (United States)

    Alexopoulou, Alexandra; Karayiannis, Peter

    2015-01-01

    The development of protease inhibitors (PIs) such as telaprevir and boceprevir constitutes a milestone in chronic hepatitis C antiviral treatment since it has achieved sustained virological response (SVR) rates of up to 75% in naïve and 29-88% in treatment-experienced patients with genotype 1 infection. Both require combination treatment with pegylated interferon (PEG-IFN) plus ribavirin (RBV) as PI monotherapy results in resistant mutations. New direct acting antiviral agents (DAAs) have recently been approved or their approval is imminent. Simeprevir administered orally as one pill per day in combination with PEG-IFN/RBV will be the next PI to be approved. The SVR rates at about 72-80% for treatment-naïve patients are not a major improvement over telaprevir or boceprevir. However, this treble combination has fewer side effects and drug-drug interactions and most patients undergo shorter treatment duration (24 months) due to earlier treatment responses. Sofosbuvir is the first available once-daily NS5B polymerase inhibitor which has been approved in combination with PEG-IFN/RBV for just 12 weeks with 89% SVR in treatment-naïve patients with genotype 1 infection and 83-100% in treatment-experienced patients with genotypes 2/3. The current review focuses on the recent rapid and continuous developments in the management of chronic HCV infection with DAAs in combination with PEG-IFN/RBV.

  8. Usutu virus, Austria and Hungary, 2010–2016

    Science.gov (United States)

    Bakonyi, Tamás; Erdélyi, Károly; Brunthaler, René; Dán, Ádám; Weissenböck, Herbert; Nowotny, Norbert

    2017-01-01

    Usutu virus (USUV, Flaviviridae) was first reported in Europe in Austria in 2001, where it caused wild bird (mainly blackbird) mortality until 2005. Since 2006 no further USUV cases were diagnosed in the country. However, the virus emerged in other European countries (Hungary, Italy, Switzerland, Spain, Germany and the Czech Republic) between 2005 and 2011. In 2016, widespread USUV-associated wild bird mortality was observed in Germany, France, Belgium and the Netherlands. In this study, we report the results of passive monitoring for USUV in Austria and Hungary between 2010 and 2016. In Hungary, USUV caused sporadic cases of wild bird mortality between 2010 and 2015 (altogether 18 diagnosed cases), whereas in summer and autumn 2016 the number of cases considerably increased to 12 (ten blackbirds, one Eurasian jay and one starling). In Austria, USUV was identified in two blackbirds in 2016. Phylogenetic analyses of coding-complete genomes and partial regions of the NS5 protein gene revealed that USUVs from Hungary between 2010 and 2015 are closely related to the virus that emerged in Austria in 2001 and in Hungary in 2005, while one Hungarian sequence from 2015 and all sequences from Hungary and Austria from 2016 clustered together with USUV sequences reported from Italy between 2009 and 2010. The results of the study indicate continuous USUV circulation in the region and exchange of USUV strains between Italy, Austria and Hungary. PMID:29018253

  9. Usutu virus, Austria and Hungary, 2010-2016.

    Science.gov (United States)

    Bakonyi, Tamás; Erdélyi, Károly; Brunthaler, René; Dán, Ádám; Weissenböck, Herbert; Nowotny, Norbert

    2017-10-11

    Usutu virus (USUV, Flaviviridae) was first reported in Europe in Austria in 2001, where it caused wild bird (mainly blackbird) mortality until 2005. Since 2006 no further USUV cases were diagnosed in the country. However, the virus emerged in other European countries (Hungary, Italy, Switzerland, Spain, Germany and the Czech Republic) between 2005 and 2011. In 2016, widespread USUV-associated wild bird mortality was observed in Germany, France, Belgium and the Netherlands. In this study, we report the results of passive monitoring for USUV in Austria and Hungary between 2010 and 2016. In Hungary, USUV caused sporadic cases of wild bird mortality between 2010 and 2015 (altogether 18 diagnosed cases), whereas in summer and autumn 2016 the number of cases considerably increased to 12 (ten blackbirds, one Eurasian jay and one starling). In Austria, USUV was identified in two blackbirds in 2016. Phylogenetic analyses of coding-complete genomes and partial regions of the NS5 protein gene revealed that USUVs from Hungary between 2010 and 2015 are closely related to the virus that emerged in Austria in 2001 and in Hungary in 2005, while one Hungarian sequence from 2015 and all sequences from Hungary and Austria from 2016 clustered together with USUV sequences reported from Italy between 2009 and 2010. The results of the study indicate continuous USUV circulation in the region and exchange of USUV strains between Italy, Austria and Hungary.Emerging Microbes &Infections (2017) 6, e85; doi:10.1038/emi.2017.72; published online 11 October 2017.

  10. Exploring Genomic, Geographic and Virulence Interactions among Epidemic and Non-Epidemic St. Louis Encephalitis Virus (Flavivirus Strains.

    Directory of Open Access Journals (Sweden)

    Luis A Diaz

    Full Text Available St. Louis encephalitis virus (SLEV is a re-emerging arbovirus in South America. In 2005, an encephalitis outbreak caused by SLEV was reported in Argentina. The reason for the outbreak remains unknown, but may have been related to virological factors, changes in vectors populations, avian amplifying hosts, and/or environmental conditions. The main goal of this study was to characterize the complete genome of epidemic and non-epidemic SLEV strains from Argentina. Seventeen amino acid changes were detected; ten were non-conservative and located in proteins E, NS1, NS3 and NS5. Phylogenetic analysis showed two major clades based on geography: the North America and northern Central America (NAnCA clade and the South America and southern Central America (SAsCA clade. Interestingly, the presence of SAsCA genotype V SLEV strains in the NAnCA clade was reported in California, Florida and Texas, overlapping with known bird migration flyways. This work represents the first step in understanding the molecular mechanisms underlying virulence and biological variation among SLEV strains.

  11. Moving beyond interferon alfa: investigational drugs for hepatitis C virus infection.

    Science.gov (United States)

    Wyles, David L

    2010-01-01

    Numerous direct-acting drugs to treat hepatitis C virus (HCV) infection are in development, offering the potential for substantial improvement over current interferon alfa-based therapy and the possibility of effective interferon alfa-sparing regimens in achieving cure of HCV infection. Drugs furthest along in clinical development include HCV nonstructural protein 3 (NS3) protease inhibitors (eg, telaprevir, boceprevir), which have potent anti-HCV activity but low barriers to resistance and considerable likelihood of cross-resistance. Nucleoside analogue nonstructural protein 5B (NS5B) polymerase inhibitors exhibit a high barrier to resistance and cross-HCV genotype and subtype activity. Nonnucleoside analogue polymerase inhibitors have a low barrier to resistance and are characterized by a substantial frequency of preexisting resistance mutations. The initial use of direct-acting drugs will be as add-on treatment to interferon alfa and ribavirin regimens. The success of interferon alfa-sparing regimens will depend on presenting a sufficiently high barrier to resistance with direct-acting drugs and whether the immunomodulatory effects of interferon alfa are needed for cure of HCV infection.

  12. 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

  13. The hepatitis C virus replicon presents a higher barrier to resistance to nucleoside analogs than to nonnucleoside polymerase or protease inhibitors.

    Science.gov (United States)

    McCown, Matthew F; Rajyaguru, Sonal; Le Pogam, Sophie; Ali, Samir; Jiang, Wen-Rong; Kang, Hyunsoon; Symons, Julian; Cammack, Nick; Najera, Isabel

    2008-05-01

    Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.

  14. Inflation from supergravity with gauged R-symmetry in de Sitter vacuum

    Energy Technology Data Exchange (ETDEWEB)

    Antoniadis, I. [LPTHE, UMR CNRS 7589 Sorbonne Universites, UPMC Paris 6, Paris (France); University of Bern, Albert Einstein Center, Institute for Theoretical Physics, Bern (Switzerland); Chatrabhuti, A.; Isono, H. [Chulalongkorn University, Department of Physics, Faculty of Science, Bangkok (Thailand); Knoops, R. [Universite de Geneve, Section de Mathematiques, Geneva (Switzerland); KU Leuven, Instituut voor Theoretische Fysica, Leuven (Belgium)

    2016-12-15

    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 Kaehler 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. (orig.)

  15. Ring relations and mirror map from branes

    Energy Technology Data Exchange (ETDEWEB)

    Assel, Benjamin [Theoretical Physics Department, CERN,CH-1211 Geneva 23 (Switzerland)

    2017-03-29

    We study the space of vacua of three-dimensional 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 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 interpretations in terms of operator insertions. This provides a new derivation of the Coulomb branch quantum relations. Secondly the map between the Higgs and Coulomb operators of mirror dual theories can be derived in a trivial way from IIB S-duality.

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

    Science.gov (United States)

    Brinton, Margo A.

    2013-01-01

    West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. Flaviviruses replicate in the cytoplasm of infected cells and modify the host cell environment. Although much has been learned about virion structure and virion-endosomal membrane fusion, the cell receptor(s) used have not been definitively identified and little is known about the early stages of the virus replication cycle. Members of the genus Flavivirus differ from members of the two other genera of the family by the lack of a genomic internal ribosomal entry sequence and the creation of invaginations in the ER membrane rather than double-membrane vesicles that are used as the sites of exponential genome synthesis. The WNV genome 3' and 5' sequences that form the long distance RNA-RNA interaction required for minus strand initiation have been identified and contact sites on the 5' RNA stem loop for NS5 have been mapped. Structures obtained for many of the viral proteins have provided information relevant to their functions. Viral nonstructural protein interactions are complex and some may occur only in infected cells. Although interactions between many cellular proteins and virus components have been identified, the functions of most of these interactions have not been delineated. PMID:24378320

  17. Semi-doubled sigma models for five-branes

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, Tetsuji [Research and Education Center for Natural Sciences, Keio University,Hiyoshi 4-1-1, Yokohama, Kanagawa 223-8521 (Japan); Department of Physics, Tokyo Institute of Technology,Tokyo 152-8551 (Japan)

    2016-02-01

    We study two-dimensional 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 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{sub 2}{sup 3}-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{sub 2}{sup 3}-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 smearing procedure. This is nothing but the signal which indicates that the nongeometric feature emerges in the considering model.

  18. Complete genome sequence of T'Ho virus, a novel putative flavivirus from the Yucatan Peninsula of Mexico.

    Science.gov (United States)

    Briese, Thomas; Loroño-Pino, Maria A; Garcia-Rejon, Julian E; Farfan-Ale, Jose A; Machain-Williams, Carlos; Dorman, Karin S; Lipkin, W Ian; Blitvich, Bradley J

    2017-06-12

    We previously reported the discovery of a novel, putative flavivirus designated T'Ho virus in Culex quinquefasciatus mosquitoes in the Yucatan Peninsula of Mexico. A 1358-nt region of the NS5 gene was amplified and sequenced but an isolate was not recovered. The complete genome of T'Ho virus was sequenced using a combination of unbiased high-throughput sequencing, 5' and 3' rapid amplification of cDNA ends, reverse transcription-polymerase chain reaction and Sanger sequencing. The genome contains a single open reading frame of 10,284 nt which is flanked by 5' and 3' untranslated regions of 97 and 556-nt, respectively. Genome sequence alignments revealed that T'Ho virus is most closely related to Rocio virus (67.4% nucleotide identity) and Ilheus virus (65.9%), both of which belong to the Ntaya group, followed by other Ntaya group viruses (58.8-63.3%) and Japanese encephalitis group viruses (62.0-63.7%). Phylogenetic inference is in agreement with these findings. This study furthers our understanding of flavivirus genetics, phylogeny and diagnostics. Because the two closest known relatives of T'Ho virus are human pathogens, T'Ho virus could be an unrecognized cause of human disease. It is therefore important that future studies investigate the public health significance of this virus.

  19. The discovery of IDX21437: Design, synthesis and antiviral evaluation of 2'-α-chloro-2'-β-C-methyl branched uridine pronucleotides as potent liver-targeted HCV polymerase inhibitors.

    Science.gov (United States)

    Alexandre, François-René; Badaroux, Eric; Bilello, John P; Bot, Stéphanie; Bouisset, Tony; Brandt, Guillaume; Cappelle, Sylvie; Chapron, Christopher; Chaves, Dominique; Convard, Thierry; Counor, Clément; Da Costa, Daniel; Dukhan, David; Gay, Marion; Gosselin, Gilles; Griffon, Jean-François; Gupta, Kusum; Hernandez-Santiago, Brenda; La Colla, Massimiliano; Lioure, Marie-Pierre; Milhau, Julien; Paparin, Jean-Laurent; Peyronnet, Jérôme; Parsy, Christophe; Pierra Rouvière, Claire; Rahali, Houcine; Rahali, Rachid; Salanson, Aurélien; Seifer, Maria; Serra, Ilaria; Standring, David; Surleraux, Dominique; Dousson, Cyril B

    2017-09-15

    Herein we describe the discovery of IDX21437 35b, a novel RPd-aminoacid-based phosphoramidate prodrug of 2'-α-chloro-2'-β-C-methyluridine monophosphate. Its corresponding triphosphate 6 is a potent inhibitor of the HCV NS5B RNA-dependent RNA polymerase (RdRp). Despite showing very weak activity in the in vitro Huh-7 cell based HCV replicon assay, 35b demonstrated high levels of active triphosphate 6 in mouse liver and human hepatocytes. A biochemical study revealed that the metabolism of 35b was mainly attributed to carboxyesterase 1 (CES1), an enzyme which is underexpressed in HCV Huh-7-derived replicon cells. Furthermore, due to its metabolic activation, 35b was efficiently processed in liver cells compared to other cell types, including human cardiomyocytes. The selected RP diastereoisomeric configuration of 35b was assigned by X-ray structural determination. 35b is currently in Phase II clinical trials for the treatment of HCV infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. A Hepatitis C Virus-Associated Chronic Hepatitis Patient Developing Various Adverse Events Including Severe Gingivitis, Gingival Bleeding, and Inflammation of Genital Vulva during the Course of Antiviral Therapy with Elbasvir/Grazoprevir

    Directory of Open Access Journals (Sweden)

    Kazuo Tarao

    2017-11-01

    Full Text Available Oral direct-acting antivirals comprise the main therapy for hepatitis C virus (HCV-associated liver disease in Japan. Daclatasvir/asunaprevir is the primary agent and sofosbuvir/ledipasvir is the secondary agent for HCV genotype 1b. Ombitasvir/paritaprevir/ritonavir was also recommended as a therapy for HCV genotype 1b. More recently, elbasvir (NS5A inhibitor/grazoprevir (NS3/4A protease inhibitor was also recommended as a potent therapy for HCV genotype 1b infection. This agent achieved an SVR12 as high as 96.5% for HCV virus-associated chronic hepatitis. We recently encountered a case treated with this agent and the female patient showed various adverse events, such as severe gingivitis, gingival bleeding, severe tonsillitis, inflammation of the genital vulva, and the sustained sensation of being hungry. In spite of the gingival bleeding, there was no depletion of the platelet count, nor elongation of the prothrombin time. She tolerated these adverse events and finally completed the therapy and achieved SVR12.

  1. Interferon-Free Hepatitis C Treatment before and after Liver Transplantation: The Role of HCV Drug Resistance

    Directory of Open Access Journals (Sweden)

    Bruno Roche

    2015-09-01

    Full Text Available Hepatitis C virus (HCV infection is one of the leading causes of end-stage liver disease and the main indication for liver transplantation (LT in most countries. All patients who undergo LT with detectable serum HCV RNA experience graft reinfection progressing to cirrhosis within five years in 20% to 30% of them. Obtaining a sustained virological response (SVR greatly improves overall and graft survival. Until 2011, standard antiviral therapy using PEGylated interferon (PEG-IFN and ribavirin (RBV was the only effective therapy, with an SVR rate around 30% in this setting. For patients infected with genotype 1, first generation NS3/4A protease inhibitors (PIs, boceprevir (BOC or telaprevir (TVR, associated with PEG-IFN and RBV for 48 weeks have increased the SVR rates to 60% in non-transplant patients. However, tolerability and drug-drug interactions with calcineurin inhibitors (CNI are both limiting factors of their use in the liver transplant setting. Over recent years, the efficacy of antiviral C therapy has improved dramatically using new direct-acting antiviral (DAA agents without PEG-IFN and/or RBV, leading to SVR rates over 90% in non-transplant patients. Results available for transplant patients showed a better efficacy and tolerability and less drug-drug interactions than with first wave PIs. However, some infrequent cases of viral resistance have been reported using PIs or NS5A inhibitors pre- or post-LT that can lead to difficulties in the management of these patients.

  2. Study on the forced degradation behaviour of ledipasvir: Identification of major degradation products using LC-QTOF-MS/MS and NMR.

    Science.gov (United States)

    Swain, Debasish; Samanthula, Gananadhamu

    2017-05-10

    Ledipasvir, a novel NS5A inhibitor is used in the management of hepatitis C virus infections. The drug was subjected to forced degradation studies as per the conditions prescribed in ICH Q1 (R2) guideline. Ledipasvir degraded in hydrolytic (acid, alkaline and neutral) and oxidative stress conditions. The drug was found to be stable in thermal and photolytic conditions. Eight novel degradation products were obtained and were well separated using an HPLC C18 stationary phase (150×4.6mm, 5μm) and mobile phase composed of formic acid/acetonitrile in gradient elution mode. All the degradation products were characterised using tandem mass spectrometry with a time-of-flight analyser and the major degradation products of hydrolytic and oxidative stress were isolated and their structural confirmation was studied using 1 H and 13 C NMR. A well resolved chromatographic method proposed in this study suggests that the proposed analytical method finds its application as a stability indicating assay method for the drug and can be used in routine analysis. Copyright © 2017. Published by Elsevier B.V.

  3. The GB virus C (GBV-C) NS3 serine protease inhibits HIV-1 replication in a CD4+ T lymphocyte cell line without decreasing HIV receptor expression.

    Science.gov (United States)

    George, Sarah L; Varmaz, Dino; Tavis, John E; Chowdhury, Adnan

    2012-01-01

    Persistent infection with GBV-C (GB Virus C), a non-pathogenic virus related to hepatitis C virus (HCV), prolongs survival in HIV infection. Two GBV-C proteins, NS5A and E2, have been shown previously to inhibit HIV replication in vitro. We investigated whether the GBV-C NS3 serine protease affects HIV replication. GBV-C NS3 protease expressed in a human CD4+ T lymphocyte cell line significantly inhibited HIV replication. Addition of NS4A or NS4A/4B coding sequence to GBV-C NS3 increased the effect on HIV replication. Inhibition of HIV replication was dose-dependent and was not mediated by increased cell toxicity. Mutation of the NS3 catalytic serine to alanine resulted in loss of both HIV inhibition and protease activity. GBV-C NS3 expression did not measurably decrease CD4 or CXCR4 expression. GBV-C NS3 serine protease significantly inhibited HIV replication without decreasing HIV receptor expression. The requirement for an intact catalytic serine at the active site indicates that inhibition was mediated by proteolytic cleavage of an unidentified target(s).

  4. 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.

  5. 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.

  6. Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening.

    Science.gov (United States)

    Qin, Shanshan; Ren, Yiran; Fu, Xu; Shen, Jie; Chen, Xin; Wang, Quan; Bi, Xin; Liu, Wenjing; Li, Lixin; Liang, Guangxin; Yang, Cheng; Shui, Wenqing

    2015-07-30

    Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K(d) determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K(d) calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Molecular characterization of HCV in a Swedish county over 8 years (2002–2009 reveals distinct transmission patterns

    Directory of Open Access Journals (Sweden)

    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.

  8. Hepatitis C virus prevalence and genetic diversity among pregnant women in Gabon, central Africa

    Science.gov (United States)

    Ndong-Atome, Guy-Roger; Makuwa, Maria; Njouom, Richard; Branger, Michel; Brun-Vézinet, Francoise; Mahé, Antoine; Rousset, Dominique; Kazanji, Mirdad

    2008-01-01

    Background Hepatitis C virus (HCV) infection is a major global public health problem in both developed and developing countries. The prevalence and genetic diversity of HCV in pregnant women in Gabon, central Africa, is not known. We therefore evaluated the prevalence and the circulating genotypes of HCV in a large population cohort of pregnant women. Methods Blood samples (947) were collected from pregnant women in the five main cities of the country. The prevalence was evaluated by two ELISA tests, and the circulating genotypes were characterized by sequencing and phylogenetic analysis. Results Twenty pregnant women (2.1%) were infected with HCV. The seroprevalence differed significantly by region (p = 0.004) and increased significantly with age (p = 0.05), being 1.3% at 14–20 years, 1.1% at 21–25 years, 1.9% at 26–30 years, 4.1% at 31–35 years and 6.0% at > 35 years. Sequencing in the 5'-UTR and NS5B regions showed that the circulating strains belonged to genotypes 4 (4e and 4c). Conclusion We found that the HCV seroprevalence in pregnant women in Gabon is almost as high as that in other African countries and increases with age. Furthermore, only genotype 4 (4e and 4c) was found. More extensive studies aiming to evaluate the prevalence and heterogeneity of HCV genotypes circulating in the general population of the country are needed. PMID:18559087

  9. 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.

  10. Phylogenetic analysis revealed the central roles of two African countries in the evolution and worldwide spread of Zika virus.

    Science.gov (United States)

    Shen, Shu; Shi, Junming; Wang, Jun; Tang, Shuang; Wang, Hualin; Hu, Zhihong; Deng, Fei

    2016-04-01

    Recent outbreaks of Zika virus (ZIKV) infections in Oceania's islands and the Americas were characterized by high numbers of cases and the spread of the virus to new areas. To better understand the origin of ZIKV, its epidemic history was reviewed. Although the available records and information are limited, two major genetic lineages of ZIKV were identified in previous studies. However, in this study, three lineages were identified based on a phylogenetic analysis of all virus sequences from GenBank, including those of the envelope protein (E) and non-structural protein 5 (NS5) coding regions. The spatial and temporal distributions of the three identified ZIKV lineages and the recombination events and mechanisms underlying their divergence and evolution were further elaborated. The potential migration pathway of ZIKV was also characterized. Our findings revealed the central roles of two African countries, Senegal and Cote d'Ivoire, in ZIKV evolution and genotypic divergence. Furthermore, our results suggested that the outbreaks in Asia and the Pacific islands originated from Africa. The results provide insights into the geographic origins of ZIKV outbreaks and the spread of the virus, and also contribute to a better understanding of ZIKV evolution, which is important for the prevention and control of ZIKV infections.

  11. Zika Virus: Immune Evasion Mechanisms, Currently Available Therapeutic Regimens, and Vaccines.

    Science.gov (United States)

    Asif, Arun; Manzoor, Sobia; Tuz-Zahra, Fatima; Saalim, Muhammad; Ashraf, Maliha; Ishtiyaq, Javeria; Khalid, Madiha

    2017-12-01

    The sudden emergence of infectious pathogens such as Zika virus (ZIKV) holds global health concerns. Recent dissemination of ZIKV from Pacific to Americas with an upsurge of congenital anomalies and Guillain Barre Syndrome (GBS) in adults has created an alarming situation. High-throughput studies are in progress to understand ZIKV's mode of pathogenesis and mechanism of immune escape, yet the pathogenesis remains obscure. Mainly ZIKV's envelope (E) protein and nonstructural proteins (mainly NS1 and NS5) manipulate host cell to support viral immune escape by modulation of the interferon pathway and complement antagonism. The development of direct therapeutics for ZIKV infection is required to overcome the rapidly evolving viral threat. Currently, the existing strategies for ZIKV treatment are only supportive. Although, there is no prophylactic or therapeutic vaccine presently available, however, recent efforts have brought up ZIKV vaccines into clinical trial phase 1. This review presents the highlights of recent advances in understanding immune evasion strategies adapted by ZIKV and existing therapies against the virus.

  12. Improved detection of Zika virus RNA in human and animal specimens by a novel, highly sensitive and specific real-time RT-PCR assay targeting the 5'-untranslated region of Zika virus.

    Science.gov (United States)

    Chan, Jasper Fuk-Woo; Yip, Cyril Chik-Yan; Tee, Kah-Meng; Zhu, Zheng; Tsang, Jessica Oi-Ling; Chik, Kenn Ka-Heng; Tsang, Terance Gi-Wai; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Sridhar, Siddharth; Yin, Feifei; Hung, Ivan Fan-Ngai; Chau, Sandy Ka-Yee; Zhang, Anna Jinxia; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2017-05-01

    We developed and evaluated five novel real-time RT-PCR assays targeting conserved regions in the 5'-untranslated region (5'-UTR), envelope (E'), non-structural protein 2A (NS2A), NS5 and 3'-UTR of the ZIKV genome. The ZIKV-5'-UTR assay exhibited the lowest in vitro limit of detection (5-10 RNA copies/reaction and 3.0 × 10-1 plaque-forming units/ml). Compared to the modified version of a widely adopted RT-PCR assay targeting the ZIKV-E gene, the ZIKV-5'-UTR assay showed better sensitivity in human clinical specimens, and representative mouse specimens, including many organs which are known to be involved in human ZIKV infection but difficult to obtain in clinical settings. The ZIKV-5'-UTR assay detected ZIKV RNA in 84/84 (100.0%) ZIKV-E'-positive and an additional 30/296 (10.1%, P ZIKV-E'-negative mouse specimens. The higher sensitivity of the ZIKV-5'-UTR assay was most significant in kidney and testis/epididymis specimens (P ZIKV-5'-UTR assay and dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, hepatitis C virus and Chikungunya virus. The highly sensitive and specific ZIKV-5'-UTR assay may help to improve the laboratory diagnosis of ZIKV infection. © 2017 John Wiley & Sons Ltd.

  13. Zika Virus spreading in South America: Evolutionary analysis of emerging neutralizing resistant Phe279Ser strains.

    Science.gov (United States)

    Giovanetti, Marta; Milano, Teresa; Alcantara, Luiz Carlos; Carcangiu, Laura; Cella, Eleonora; Lai, Alessia; Lo Presti, Alessandra; Pascarella, Stefano; Zehender, Gianguglielmo; Angeletti, Silvia; Ciccozzi, Massimo

    2016-05-01

    To investigate the genetic diversity of Zika Virus (ZIKV) and the relationships existing among these circulating viruses worldwide. To evaluate the genetic polymorphisms harbored from ZIKV that can have an influence on the virus circulation. Three different ZIKV dataset were built. The first dataset included 63 E gene sequences, the second one 22 NS3 sequences and the third dataset was composed of 108 NS5 gene sequences. Phylogenetic and selective pressure analysis was performed. The edited nucleic acid alignment from the Envelope dataset was used to generate a conceptual translation to the corresponding peptide sequences through UGene software. The phylogeographic reconstruction was able to discriminate unambiguously that the Brazilian strains are belonged to the Asian lineage. The structural analysis reveals instead the presence of the Ser residue in the Brazilian sequences (however already observed in other previously reported ZIKV infections) that could suggest the presence of a neutralization-resistant population of viruses. Phylogenetic, evolutionary and selective pressure analysis contributed to improve the knowledge on the circulation of ZIKV. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

  14. Non-Abelian supertubes

    Science.gov (United States)

    Fernández-Melgarejo, José J.; Park, Minkyu; Shigemori, Masaki

    2017-12-01

    A supertube is a supersymmetric configuration in string theory which occurs when a pair of branes spontaneously polarizes and generates a new dipole charge extended along a closed curve. The dipole charge of a codimension-2 supertube is characterized by the U-duality monodromy as one goes around the supertube. For multiple codimension-2 supertubes, their monodromies do not commute in general. In this paper, we construct a supersymmetric solution of five-dimensional supergravity that describes two supertubes with such non-Abelian monodromies, in a certain perturbative expansion. In supergravity, the monodromies are realized as the multi-valuedness of the scalar fields, while in higher dimensions they correspond to non-geometric duality twists of the internal space. The supertubes in our solution carry NS5 and 5 2 2 dipole charges and exhibit the same monodromy structure as the SU(2) Seiberg-Witten geometry. The perturbative solution has AdS2 × S 2 asymptotics and vanishing four-dimensional angular momentum. We argue that this solution represents a microstate of four-dimensional black holes with a finite horizon and that it provides a clue for the gravity realization of a pure-Higgs branch state in the dual quiver quantum mechanics.

  15. 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

    2016-08-27

    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.

  16. Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Naïve Patients

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Genetic History of Hepatitis C Virus in East Asia ▿

    Science.gov (United States)

    Pybus, Oliver G.; Barnes, Eleanor; Taggart, Rachel; Lemey, Philippe; Markov, Peter V.; Rasachak, Bouachan; Syhavong, Bounkong; Phetsouvanah, Rattanaphone; Sheridan, Isabelle; Humphreys, Isla S.; Lu, Ling; Newton, Paul N.; Klenerman, Paul

    2009-01-01

    The hepatitis C virus (HCV), which currently infects an estimated 3% of people worldwide, has been present in some human populations for several centuries, notably HCV genotypes 1 and 2 in West Africa and genotype 6 in Southeast Asia. Here we use newly developed methods of sequence analysis to conduct the first comprehensive investigation of the epidemic and evolutionary history of HCV in Asia. Our analysis includes new HCV core (n = 16) and NS5B (n = 14) gene sequences, obtained from serum samples of jaundiced patients from Laos. These exceptionally diverse isolates were analyzed in conjunction with all available reference strains using phylogenetic and Bayesian coalescent methods. We performed statistical tests of phylogeographic structure and applied a recently developed “relaxed molecular clock” approach to HCV for the first time, which indicated an unexpectedly high degree of rate variation. Our results reveal a >1,000-year-long development of genotype 6 in Asia, characterized by substantial phylogeographic structure and two distinct phases of epidemic history, before and during the 20th century. We conclude that HCV lineages representing preexisting and spatially restricted strains were involved in multiple, independent local epidemics during the 20th century. Our analysis explains the generation and maintenance of HCV diversity in Asia and could provide a template for further investigations of HCV spread in other regions. PMID:18971279

  19. PDB-UF: database of predicted enzymatic functions for unannotated protein structures from structural genomics

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    Rychlewski Leszek

    2006-02-01

    Full Text Available Abstract Background The number of protein structures from structural genomics centers dramatically increases in the Protein Data Bank (PDB. Many of these structures are functionally unannotated because they have no sequence similarity to proteins of known function. However, it is possible to successfully infer function using only structural similarity. Results Here we present the PDB-UF database, a web-accessible collection of predictions of enzymatic properties using structure-function relationship. The assignments were conducted for three-dimensional protein structures of unknown function that come from structural genomics initiatives. We show that 4 hypothetical proteins (with PDB accession codes: 1VH0, 1NS5, 1O6D, and 1TO0, for which standard BLAST tools such as PSI-BLAST or RPS-BLAST failed to assign any function, are probably methyltransferase enzymes. Conclusion We suggest that the structure-based prediction of an EC number should be conducted having the different similarity score cutoff for different protein folds. Moreover, performing the annotation using two different algorithms can reduce the rate of false positive assignments. We believe, that the presented web-based repository will help to decrease the number of protein structures that have functions marked as "unknown" in the PDB file. Availability http://paradox.harvard.edu/PDB-UF and http://bioinfo.pl/PDB-UF

  20. 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.