WorldWideScience

Sample records for c100-3 hcr43 ns5

  1. NS5A Resistance: Clinical Implications and Treatment Possibilities.

    Science.gov (United States)

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

    2016-01-01

    Treatments with interferon-free direct-acting antiviral agents have high efficacy, with sustained virological response rates of more than 90%. Nevertheless, they fail to eliminate the infection in 1-7% of patients. The majority of virological failures are due to relapse following treatment discontinuation, while virological rebound during therapy is rare. Although not the only factor, the presence of resistance-associated variants is one of the major causes for said failure. Resistance-associated variants affect the sequence involved in protein synthesis on which different direct-acting antiviral agents act (NS3/4A, NS5A, NS5B). Of all these variants, the ones with the greatest impact are resistance-associated variants that affect the NS5A region due to their long-term persistence. In this article we will describe the most significant NS5A resistance-associated variants, the clinical relevance of their detection both before and after treatment, their persistence over time, and lastly, we will devote particular attention to discussing what approach to adopt when dealing with treatment failure to an antiviral regimen that includes NS5A inhibitors. PMID:26991826

  2. Thermodynamics of the near-extremal NS5-brane

    International Nuclear Information System (INIS)

    We consider the thermodynamics of the near-extremal NS5-brane in type IIA string theory. The central tool we use is to map phases of six-dimensional Kaluza-Klein black holes to phases of near-extremal M5-branes with a transverse circle in eleven-dimensional supergravity. By S-duality these phases correspond to phases of the near-extremal type IIA NS5-brane. One of our main results is that in the canonical ensemble the usual near-extremal NS5-brane background, dual to a uniformly smeared near-extremal M5-brane, is subdominant to a new background of near-extremal M5-branes localized on the transverse circle. This new stable phase has a limiting temperature, which lies above the Hagedorn temperature of the usual NS5-brane phase. We discuss the limiting temperature and compare the different behavior of the NS5-brane in the canonical and microcanonical ensembles. We also briefly comment on the thermodynamics of near-extremal Dp-branes on a transverse circle

  3. On NS5-brane instantons and volume stabilization

    NARCIS (Netherlands)

    Looyestijn, H.T.; Vandoren, S.

    2008-01-01

    We study general aspects of NS5-brane instantons in relation to the stabilization of the volume modulus in Calabi-Yau compactifications of type II strings with fluxes, and their orientifold versions. These instantons correct the Kahler potential and generically yield significant contributions to the

  4. Semiclassical Strings Probing NS5 Brane Wrapped on S^5

    OpenAIRE

    Ebrahim, Hajar

    2005-01-01

    We study little string theory on R^1 x S^5, defined by a theory which lives on type IIA N NS5 branes wrapped on S^5, using its supergravity dual. In particular we study semiclassical rotating closed strings in this background. We also consider Penrose limit of this background that leads to a plane wave on which string theory is exactly solvable.

  5. D3-branes in NS5-branes backgrounds

    CERN Document Server

    Ribault, S

    2003-01-01

    We study D3-branes in an NS5-branes background defined by an arbitrary 4d harmonic function. Using a gauge-invariant formulation of Born-Infeld dynamics as well as the supersymmetry condition, we find the general solution for the $\\omega$-field. We propose an interpretation in terms of the Myers effect.

  6. D3-branes in NS5-brane backgrounds

    International Nuclear Information System (INIS)

    We study D3-branes in an NS5-branes background defined by an arbitrary 4d harmonic function. Using a gauge-invariant formulation of Born-Infeld dynamics as well as the supersymmetry condition, we find the general solution for the ω-field. We propose an interpretation in terms of the Myers effect. (author)

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

    Science.gov (United States)

    Adiga, Rama

    2016-10-01

    ZIKV infection has become a global threat spreading across 31 countries in Central America, South America, and the Caribbean. However, little information is available about the molecular epidemiology of ZIKV. Shared mutation of a threonine residue to alanine at the same position in the C terminal of NS5 sequences was observed in sequences from Colombia, Mexico, Panama, and Martinique. The sequences in the phylogenetic tree fell within the same cluster. Based on shared mutation the presence of a Latin American genotype was proposed. Comparison of African and Asian lineages yielded R29N, N273S, H383Q, and P391S mutation. The study highlights that mutation of amino acids at NS5 may contribute to neutropism of ZIKV. J. Med. Virol. 88:1821-1826, 2016. © 2016 Wiley Periodicals, Inc. PMID:27335310

  8. Heterotic NS5-branes from closed string tachyon condensation

    Science.gov (United States)

    Garcia-Etxebarria, Iñaki; Montero, Miguel; Uranga, Angel

    2014-12-01

    We show how to construct the familiar heterotic NS5 brane as a topological soliton in a supercritical version of heterotic string theory. Closed string tachyon condensation removes the extra dimensions, leaving the NS5 in ten dimensions, in a process highly reminiscent of the K-theoretical description of type II D-branes, but linking nontrivial gauge bundles and geometry. This establishes a new kind of equivalence between gravitational and gauge configurations, reminiscent of the gauge/geometry correspondence. We also use the K-theory description to build other heterotic branes as solitons of closed string tachyons. The construction requires a modification of the anomalous Bianchi identity for H3 in supercritical heterotic string theory. We give various proofs for the existence of this modification.

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

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Gottwein, Judith M; Mikkelsen, Lotte S; Jensen, Tanja B; Bukh, Jens

    2011-01-01

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

  10. Inhibition of hepatitis C virus infection by NS5A-specific aptamer.

    Science.gov (United States)

    Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wang, Xiaohong; Yang, Darong; Qin, Yuwen; Yu, Rong; Liu, Nianli; Xu, Li; Fang, Xiaohong; Zhu, Haizhen

    2014-06-01

    To increase efficacy of hepatitis C treatment, future regiments will incorporate multiple direct-acting antiviral drugs. HCV NS5A protein was expressed and purified. Aptamers against NS5A were screened and obtained by the selective evolution of ligands by exponential enrichment approach and the antiviral actions of the aptamers were tested. The mechanisms through which the aptamers exert their antiviral activity were explored. The aptamers NS5A-4 and NS5A-5 inhibit HCV RNA replication and infectious virus production without causing cytotoxicity in human hepatocytes. The aptamers do not affect hepatitis B virus replication in HepG2.2.15 cells. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) are not induced by the aptamers in HCV-infected hepatocytes. Further study shows that domain I and domain III of NS5A protein are involved in the suppression of HCV RNA replication and infectious virus production by NS5A-4. Y2105H within NS5A is the major resistance mutation identified. NS5A aptamer disrupts the interaction of NS5A with core protein. The data suggest that the aptamers against NS5A protein may exert antiviral effects through inhibiting viral RNA replication, preventing the interaction of NS5A with core protein. Aptamers for NS5A may be used to understand the mechanisms of virus replication and assembly and served as potential therapeutic agents for hepatitis C. PMID:24713119

  11. Structure and Function of Flavivirus NS5 Methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Zhou,Y.; Ray, D.; Zhao, Y.; Dong, H.; Ren, S.; Li, Z.; Guo, Y.; Bernard, K.; Shi, P.; Li, H.

    2007-01-01

    The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

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

    Science.gov (United States)

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

    2016-02-19

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

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

    Science.gov (United States)

    De Maio, Federico A; Risso, Guillermo; Iglesias, Nestor G; Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G; Mammi, Pablo; Mancini, Estefania; Yanovsky, Marcelo J; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V

    2016-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  15. 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; Jensen, Sanne B; Gottwein, Judith M; Bukh, Jens

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xuesong Gao

    2013-05-01

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

  17. 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.

  18. Fundamental strings and NS5-branes from unstable D-branes in supergravity

    CERN Document Server

    Lu, J X

    2006-01-01

    By using the non-supersymmetric $p$-brane solutions delocalized in arbitrary number of transverse directions in type II supergravities, we show how they can be regarded as interpolating solutions between unstable D$p$-branes (a non-BPS D-brane or a pair of coincident D-brane-antiD-brane) and fundamental strings and also between unstable D$p$-branes and NS5-branes. We also show that some of these solutions can be regarded as interpolating solutions between NS5/$\\bar{\\rm NS}$5 and D$p$-branes (for $p \\leq 5$). This gives a closed string description of the tachyon condensation and lends support to the conjecture that the open string theory on unstable D-branes at the tachyonic vacuum has soliton solutions describing not only the lower dimensional BPS D-branes, but also the fundamental strings as well as the NS5-branes.

  19. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2000-01-01

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

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

    CERN Document Server

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

    2003-01-01

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

  2. 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....

  3. Thermodynamics of Superstring on Near-extremal NS5 and Effective Hagedorn Behavior

    CERN Document Server

    Sugawara, Yuji

    2012-01-01

    We study the thermodynamical torus partition function of superstring on the near-extremal black NS5-brane background. The exact partition function has been computed with the helps of our previous works:[arXiv:1012.5721 [hep-th

  4. Characterization of thiobarbituric acid derivatives as inhibitors of hepatitis C virus NS5B polymerase

    Directory of Open Access Journals (Sweden)

    Park Mi Young

    2011-01-01

    Full Text Available Abstract In an effort to find chemicals inhibiting the enzymatic activity of the hepatitis C virus (HCV NS5B polymerase, a series of thiobarbituric acid derivatives were selected from a library provided by Korea Research Institute of Chemical Technology and characterized. The selected compounds exhibited IC50 values ranging from 1.7 to 3.8 μM, and EC50 values ranging from 12.3 to 20.7 μM against NS5B polymerase of type 1b strain. They showed little effect against type 2a polymerase. One of the compounds, G05, was selected and further characterized. It inhibited the synthesis of RNA by recombinant HCV NS5B polymerase in a dose dependent manner. The CC50 value was 77 μM. The inhibition was in a noncompetitive manner with the substrate UTP. The compound did not inhibit the elongation step of RNA synthesis in a single-cycle processive polymerization assay. It inhibited the binding of NS5B polymerase to the template RNA in a dose-dependent manner.

  5. Induction of intrahepatic HCV NS4B, NS5A and NS5B-specific cellular immune responses following peripheral immunization.

    Directory of Open Access Journals (Sweden)

    Krystle A Lang Kuhs

    Full Text Available Numerous studies have suggested that an effective Hepatitis C Virus (HCV vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.

  6. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.

    Science.gov (United States)

    Tan, B H; Fu, J; Sugrue, R J; Yap, E H; Chan, Y C; Tan, Y H

    1996-02-15

    The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA. PMID:8607261

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

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

  9. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

    Directory of Open Access Journals (Sweden)

    Hongping Dong

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

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

    Science.gov (United States)

    Marcotrigiano, Joseph

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

  11. Analysis of mutant NS5B proteins encoded by isolates from chimpanzees chronically infected following clonal HCV RNA inoculation

    International Nuclear Information System (INIS)

    We hypothesized that mutations in the HCV NS5B polymerase, which occur during infection, may affect RNA-dependent RNA polymerase (RdRp) activity. NS5B proteins corresponding to a genotype 1a infectious clone and mutants identified in chimpanzees following inoculation with the clone were expressed and purified and their in vitro RdRp activity was compared to a NS5B genotype 1b control. A Gln-65-to-His mutation increased RdRp activity by 1.8-fold as compared to the infectious clone. Moreover, this NS5B1a protein had RdRp activity similar to the NS5B1b control. Three NS5B proteins representing mutations found in another animal had no in vitro RdRp activity. All mutations were maintained in the majority circulating virus for at least 216 weeks. The results demonstrate that some in vivo mutations of NS5B directly enhance in vitro RdRp activity. In addition, they suggest that the in vitro RdRp activity of NS5B may not always reflect in vivo activity within replication complexes

  12. Hepatitis C virus NS5A protein cooperates with phosphatidylinositol 4-kinase IIIα to induce mitochondrial fragmentation.

    Science.gov (United States)

    Siu, Gavin Ka Yu; Zhou, Fan; Yu, Mei Kuen; Zhang, Leiliang; Wang, Tuanlao; Liang, Yongheng; Chen, Yangchao; Chan, Hsiao Chang; Yu, Sidney

    2016-01-01

    Hepatitis C virus (HCV) has long been observed to take advantage of the host mitochondria to support viral replication and assembly. The HCV core protein has been implicated to fragment host mitochondria. In this report, we have discovered that the non-structural protein 5A (NS5A) plays an instructive role in attaching ER with mitochondria, causing mitochondrial fragmentation. Dynamin-related protein 1(Drp1), a host protein essential to mitochondrial membrane fission, does not play a role in NS5A-induced mitochondrial fragmentation. Instead, phosphatidylinositol 4-kinase IIIα (PI4KA), which has been demonstrated to bind to NS5A and is required to support HCV life cycle, is required for NS5A to induce mitochondrial fragmentation. Both NS5A and core are required by HCV to fragment the mitochondria, as inhibiting either of their respective downstream proteins, PI4KA or Drp1, resulted in lengthening of mitochondria tubules in HCVcc-infected cells. By fragmenting the mitochondria, NS5A renders the cells more resistant to mitochondria mediated apoptosis. This finding indicates previously-ignored contribution of NS5A in HCV-induced mitochondria dysfunction. PMID:27010100

  13. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

    Science.gov (United States)

    Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. PMID:27030586

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-07-10

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

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

    Science.gov (United States)

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

    2016-06-17

    The prevalence of nonalcoholic fatty liver disease (NAFLD) is rising strikingly in Western countries and China. The molecular biological mechanism of NAFLD remains unclear, with no effective therapies developed so far. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone, with safe lipid lowering effects. FGF21 analogs are being developed for clinical application. Here we demonstrated that a novel gene, NS5ATP6, modulated intracellular triglyceride (TG) content independently of sirtuin1 (SIRT1) and sterol regulatory element binding protein 1 (SREBP1) in HepG2 cells. Interestingly, NS5ATP6 regulated FGF21 expression both at the mRNA and protein levels. The modulatory effects of NS5ATP6 on intracellular TG content depended upon FGF21. Further studies revealed that NS5ATP6 decreased the promoter activity of FGF21. In addition, NS5ATP6 regulated the expression of miR-577, which directly targeted and regulated FGF21. Therefore, miR-577 might be involved in NS5ATP6 regulation of FGF21 at the post-transcriptional level. In conclusion, NS5ATP6 regulates the intracellular TG level via FGF21, and independently of SIRT1 and SREBP1. PMID:27179781

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  17. 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.

  18. 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. PMID:22720059

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

    Directory of Open Access Journals (Sweden)

    Simon Reiss

    2013-05-01

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

  20. 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.

  1. The dengue virus NS5 protein as a target for drug discovery.

    Science.gov (United States)

    Lim, Siew Pheng; Noble, Christian G; Shi, Pei-Yong

    2015-07-01

    The non-structural protein 5 (NS5) of flaviviruses is the most conserved amongst the viral proteins. It is about 900 kDa and bears enzymatic activities that play vital roles in virus replication. Its N-terminal domain encodes dual N7 and 2'-O methyltransferase activities (MTase), and possibly guanylyltransferase (GTase) involved in RNA cap formation. The C-terminal region comprises a RNA-dependent RNA polymerase (RdRp) required for viral RNA synthesis. Both MTase and RdRp activities of dengue virus NS5 are well characterized, structurally and functionally. Numerous crystal structures of the flavivirus MTase and RdRp domains have been solved. Inhibitors of both functions have been identified through screening activities using biochemical and cell-based assays, as well as via rational design approaches. This review summaries the current knowledge as well as prospective views on these aspects. This article forms part of a symposium on flavivirus drug discovery in Antiviral Research. PMID:25912817

  2. Lifshitz metric with hyperscaling violation from NS5-Dp states in string theory

    International Nuclear Information System (INIS)

    In previous papers Dey and Roy (2012) [1,2] we have shown how Lifshitz-like space–time (space–time having a Lifshitz scaling along with a hyperscaling violation) can arise by taking near horizon limits of certain intersecting solutions (F-string with Dp-branes and also with two D-branes) of string theory. In this Letter we construct intersecting bound state solutions in the form of NS5-Dp-branes (with 1⩽p⩽6) of type II string theories. These are 1/4 BPS and threshold bound states unlike the known NS5-Dp bound states which are 1/2 BPS and non-threshold. We show that the near horizon limits of these solutions also lead to Lifshitz-like space–time with the dynamical scaling exponent z=0 and the hyperscaling violation exponent θ=9−p. The spatial dimension of the boundary theory is d=7−p. The dilatons in these theories are not constant in general (except for p=5) and therefore produce RG flows. So, we also consider the strong coupling phases of these theories and find that these phases also have similar Lifshitz-like structures, except for p=2, where it has an AdS3 structure

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

    Science.gov (United States)

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

    2011-09-15

    SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 μM, replicon EC(50)>100 μM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 μM, replicon EC(50)=1.4 μM) and 7r (NS5B IC(50)=0.017 μM, replicon EC(50)=0.3 μM) with improved enzyme and replicon activity. PMID:21840715

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-03

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

  5. Hepatitis C virus NS5A mediated STAT3 activation requires co-operation of Jak1 kinase

    International Nuclear Information System (INIS)

    Hepatitis C virus (HCV) is a major etiologic agent for chronic hepatitis worldwide and often leads to cirrhosis and hepatocellular carcinoma. However, the mechanism for development of chronic hepatitis or hepatocarcinogenesis by HCV remains unclear. Signal transducers and activators of transcription (STATs) family proteins function as the downstream effectors of cytokine signaling and play a critical role in cell growth regulation. In many cancers including liver, STAT3 is often constitutively activated, although the mechanism of persistent activation of STAT3 is unknown. The nonstructural protein 5A (NS5A) encoded from the HCV genome has shown cell growth regulatory properties. In this study, we have observed that HCV NS5A activates STAT3 phosphorylation, which in turn translocates into the nucleus. In vivo activation of STAT3 was also observed in the liver of transgenic mice expressing HCV NS5A. Introduction of NS5A in hepatoma cells modulated STAT3 downstream molecules Bcl-xL and p21 expression. To determine if STAT3 activation by NS5A could induce STAT3 mediated gene expression, a luciferase reporter construct based on a synthetic promoter was used to transfect hepatoma cells. Activation of endogenous cellular STAT3 by HCV NS5A induced luciferase gene expression through STAT3 specific binding elements. Our subsequent studies suggested that NS5A forms a complex with Jak1 and recruits STAT3 for activation. Taken together, our results suggested that NS5A activates STAT3 through co-operation of Jak1 kinase and activated STAT3 may contribute to HCV-mediated pathogenesis

  6. The HCV Non-Nucleoside Inhibitor Tegobuvir Utilizes a Novel Mechanism of Action to Inhibit NS5B Polymerase Function

    OpenAIRE

    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

    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...

  7. Inhibitors of the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B

    Directory of Open Access Journals (Sweden)

    Megan H. Powdrill

    2010-09-01

    Full Text Available More than 20 years after the identification of the hepatitis C virus (HCV as a novel human pathogen, the only approved treatment remains a combination of pegylated interferon-α and ribavirin. This rather non-specific therapy is associated with severe side effects and by far not everyone benefits from treatment. Recently, progress has been made in the development of specifically targeted antiviral therapy for HCV (STAT-C. A major target for such direct acting antivirals (DAAs is the HCV RNA-dependent RNA polymerase or non-structural protein 5B (NS5B, which is essential for viral replication. This review will examine the current state of development of inhibitors targeting the polymerase and issues such as the emergence of antiviral resistance during treatment, as well as strategies to address this problem.

  8. 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.

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

    Institute of Scientific and Technical Information of China (English)

    王晓杰; 相久全; 韩乐强; 张锦前; 成军

    2011-01-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Li, Yan; Yang, Zexiao

    2015-12-01

    Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups. PMID:26485449

  13. A Comprehensive Insight into the Chemical Space and ADME Features of Small Molecule NS5A Inhibitors.

    Science.gov (United States)

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

    2016-01-01

    Non-structural 5A (NS5A) protein plays a crucial role in the replication of hepatitis C virus (HCV) and during the past decade has attracted increasing attention as a promising biological target for the treatment of viral infections and related disorders. Small-molecule NS5A inhibitors have shown significant antiviral activity in vitro and in vivo. Several lead molecules are reasonably regarded as novel highly potent drug candidates with favorable ADME features and tolerable side effects. The first-in-class daclatasvir has recently been launched into the market and 14 novel molecules are currently under evaluation in clinical trials. From this perspective, we provide an overview of the available chemical space of small-molecule NS5A inhibitors and their PK properties, mainly focusing on the diversity in structure and scaffold representation. PMID:26585933

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

    Directory of Open Access Journals (Sweden)

    Sandra Regina Maruyama

    2014-02-01

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

  15. A Rab-GAP TBC Domain Protein Binds Hepatitis C Virus NS5A and Mediates Viral Replication▿

    Science.gov (United States)

    Sklan, Ella H.; Staschke, Kirk; Oakes, Tina M.; Elazar, Menashe; Winters, Mark; Aroeti, Benjamin; Danieli, Tsafi; Glenn, Jeffrey S.

    2007-01-01

    Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population. PMID:17686842

  16. 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.

  17. Insight into ligand selectivity in HCV NS5B polymerase: molecular dynamics simulations, free energy decomposition and docking

    OpenAIRE

    Li, Tong; Froeyen, Matheus; Herdewijn, Piet

    2009-01-01

    Abstract Modeling studies were performed on HCV NS5B polymerase in an effort to design new inhibitors. The binding models of five different scaffold inhibitors were investigated and compared by using molecular dynamics simulations, free energy calculation and decomposition. Our results show Tyr448 plays the most critical role in the binding of most inhibitors. In addition, favorable contributions of residues Pro197, Arg200, Cys366, Met414 and Tyr448 in a deep hydrophobic pocket pro...

  18. Next generation sequencing of the hepatitis C virus NS5B gene reveals potential novel S282 drug resistance mutations.

    Science.gov (United States)

    Ji, Hezhao; Kozak, Robert A; Biondi, Mia J; Pilon, Richard; Vallee, Dominic; Liang, Ben Binhua; La, David; Kim, John; Van Domselaar, Gary; Leonard, Lynne; Sandstrom, Paul; Brooks, James

    2015-03-01

    Identifying HCV drug resistance mutations (DRMs) is increasingly important as new direct acting antiviral therapies (DAA) become available. Tagged pooled pyrosequencing (TPP) was originally developed as cost-effective approach for detecting low abundance HIV DRMs. Using 127 HCV-positive samples from a Canadian injection drug user cohort, we demonstrated the suitability and efficiency of TPP for evaluating DRMs in HCV NS5B gene. At a mutation identification threshold of 1%, no nucleoside inhibitor DRMs were detected among these DAA naïve subjects. Clinical NS5B resistance to non-nucleoside inhibitors and interferon/ribavirin was predicted to be low within this cohort. S282T mutation, the primary mutation selected by sofosbuvir in vitro, was not identified while S282G/C/R variants were detected in 9 subjects. Further characterization on these new S282 variants using in silico molecular modeling implied their potential association with resistance. Combining TPP with in silico analysis detects NS5B polymorphisms that may explain differences in treatment outcomes. PMID:25600207

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

    Science.gov (United States)

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

    2016-02-01

    The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling and host cellular responses via to its ability to interact with various cellular proteins. FKBP8 is also reported to promote virus replication. Here, we show that NS5A specifically interacts with FKBP8 through coimmunoprecipitation and GST-pulldown studies. Additionally, confocal microscopy study showed that NS5A and FKBP8 colocalized in the cytoplasm. Overexpression of FKBP8 via the eukaryotic expression plasmid pDsRED N1 significantly promoted viral RNA synthesis. The cells knockdown of FKBP8 by lentivirus-mediated shRNA markedly decreased the virus replication when infected with CSFV. These data suggest that FKBP8 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of FKBP8 protein functions may be beneficial for developing new strategies to treat CSFV infection. PMID:26748656

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

    Directory of Open Access Journals (Sweden)

    Troels K H Scheel

    Full Text Available 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 directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1-7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a and QC69(7a recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a. Reduced virus production was linked to attenuated replication in all cases, 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 changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important

  1. 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.

  2. 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.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1-7 cell culture systems, and through reverse genetics demonstrated a universal importance of the...... amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had...... introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic...

  4. Understanding the drug resistance mechanism of hepatitis C virus NS5B to PF-00868554 due to mutations of the 423 site: a computational study.

    Science.gov (United States)

    Jiao, Pingzu; Xue, Weiwei; Shen, Yulin; Jin, Nengzhi; Liu, Huanxiang

    2014-04-01

    NS5B, a hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) that plays a key role in viral replication, is an important target in the discovery of antiviral agents. PF-00868554 is a potent non-nucleoside inhibitor (NNI) that binds to the Thumb II allosteric pocket of NS5B polymerase and has shown significant promise in phase II clinical trials. Unfortunately, several PF-00868554 resistant mutants have been identified. M423 variants were the most common NS5B mutations that occurred after PF-00868554 monotherapy. In this study, we used molecular dynamics (MD) simulations, binding free energy calculations and free energy decomposition to explore the drug resistance mechanism of HCV to PF-00868554 resulting from three representative mutations (M423T/V/I) in NS5B polymerase. Free energy decomposition analysis reveals that the loss of binding affinity mainly comes from the reduction of both van der Waals (ΔE(vdw)) and electrostatic interaction contributions in the gas phase (ΔE(ele)). Further structural analysis indicates that the location of PF-00868554 and the binding mode changed due to mutation of the residue at the 423 site of NS5B polymerase from methionine to threonine, isoleucine or valine, which further resulted in the loss of binding ability of PF-00868554 to NS5B polymerase. The obtained computational results will have important value for the rational design of novel non-nucleoside inhibitors targeting HCV NS5B polymerase. PMID:24452008

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

    Science.gov (United States)

    Li, Su; Feng, Shuo; Wang, Jing-Han; He, Wen-Rui; Qin, Hua-Yang; Dong, Hong; Li, Lian-Feng; Yu, Shao-Xiong; Li, Yongfeng; Qiu, Hua-Ji

    2015-08-01

    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. PMID:26266418

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Multiple mutations in hepatitis C virus NS5A domain II are required to confer a significant level of resistance to alisporivir.

    Science.gov (United States)

    Garcia-Rivera, Jose A; Bobardt, Michael; Chatterji, Udayan; Hopkins, Sam; Gregory, Matthew A; Wilkinson, Barrie; Lin, Kai; Gallay, Philippe A

    2012-10-01

    Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance. PMID:22802259

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  12. Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

    Directory of Open Access Journals (Sweden)

    Lappalainen Maija

    2009-06-01

    Full Text Available Abstract Background The viral genome of hepatitis C virus constitutes a 9.6-kb single-stranded positive-sense RNA which encodes altogether 11 viral proteins. In order to study the humoral immune responses against different HCV proteins in patients suffering from chronic HCV infection, we produced three structural (core, E1 and E2 and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B in Sf9 insect cells by using the baculovirus expression system. Results The recombinant HCV core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins were purified and used in Western blot analysis to determine antibody responses against individual HCV protein in 68 HCV RNA and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. These sera were also analysed with INNO-LIA Score test for HCV antibodies against core, NS3, NS4AB and NS5A, and the results were similar to the ones obtained by Western blot method. Based on our Western blot analyses we found that the major immunogenic HCV antigens were the core, NS4B, NS3 and NS5A proteins which were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. There were no major genotype specific differences in antibody responses to individual HCV proteins. A common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized NS2 protein and only three sera (from genotype 3 recognised NS5B. Conclusion The data shows significant variation in the specificity in humoral immunity in chronic HCV patients.

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

    Directory of Open Access Journals (Sweden)

    Kristen M Bullard

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

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

    Science.gov (United States)

    Bullard, Kristen M; Gullberg, Rebekah C; Soltani, Elnaz; Steel, J Jordan; Geiss, Brian J; Keenan, Susan M

    2015-01-01

    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 may be less

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

    Directory of Open Access Journals (Sweden)

    Sevil Aydın

    2013-01-01

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

  16. Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes

    OpenAIRE

    Hanley, Kathryn A.; Lee, Jay J.; Blaney, Joseph E.; Murphy, Brian R.; Whitehead, Stephen S

    2002-01-01

    Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication ...

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

    Directory of Open Access Journals (Sweden)

    Jun Itakura

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

  18. On decoupled theories in (5+1) dimensions from (F, D1, NS5, D5) supergravity configuration

    International Nuclear Information System (INIS)

    It is well-known that (N, M) 5-branes of type IIB supergravity form a non-threshold bound state with (N', M') strings called the (F, D1, NS5, D5) bound state where the strings lie along one of the spatial directions of the 5-branes (hep-th/9905056). By taking low energy limits in appropriate ways on this supergravity configuration, we obtain the supergravity duals of various decoupled theories in (5+1) dimensions corresponding to noncommutative open string (NCOS) theory, open D-string (OD1) theory and open D5-brane (OD5) theory. We then study the SL(2, Z) transformation properties of these theories. We show that when the asymptotic value of the axion (χ0) is rational (for which χ0 can be put to zero), NCOS theory is always related to OD1 theory by strong-weak or S-duality symmetry. We also discuss the self-duality conjecture (hep-th/0006062) of both NCOS and OD1 theories. On the other hand, when chi0 is irrational, we find that SL(2, Z) duality on NCOS theory gives another NCOS theory with different values of the parameters, but for OD1 theory SL(2,Z) duality always gives an NCOS theory. SL(2, Z) transformation on OD5 theory reveals that it gives rise to Little String Theory (LST) when chi0 rational, but it gives another OD5 theory with different values of the parameters when χ0 is irrational. (author)

  19. Metabolism and Disposition of Pan-Genotypic Inhibitor of Hepatitis C Virus NS5A Ombitasvir in Humans.

    Science.gov (United States)

    Shen, Jianwei; Serby, Michael; Surber, Bruce; Lee, Anthony J; Ma, Junli; Badri, Prajakta; Menon, Rajeev; Kavetskaia, Olga; de Morais, Sonia M; Sydor, Jens; Fischer, Volker

    2016-08-01

    Ombitasvir (also known as ABT-267) is a potent inhibitor of hepatitis C virus (HCV) nonstructural protein 5A (NS5A), which has been developed in combination with paritaprevir/ritonavir and dasabuvir in a three direct-acting antiviral oral regimens for the treatment of patients infected with HCV genotype 1. This article describes the mass balance, metabolism, and disposition of ombitasvir in humans without coadministration of paritaprevir/ritonavir and dasabuvir. Following the administration of a single 25-mg oral dose of [(14)C]ombitasvir to four healthy male volunteers, the mean total percentage of the administered radioactive dose recovered was 92.1% over the 192-hour sample collection in the study. The recovery from the individual subjects ranged from 91.4 to 93.1%. Ombitasvir and corresponding metabolites were primarily eliminated in feces (90.2% of dose), mainly as unchanged parent drug (87.8% of dose), but minimally through renal excretion (1.9% of dose). Biotransformation of ombitasvir in human involves enzymatic amide hydrolysis to form M23 (dianiline), which is further metabolized through cytochrome P450-mediated oxidative metabolism (primarily by CYP2C8) at the tert-butyl group to generate oxidative and/or C-desmethyl metabolites. [(14)C]Ombitasvir, M23, M29, M36, and M37 are the main components in plasma, representing about 93% of total plasma radioactivity. The steady-state concentration measurement of ombitasvir metabolites by liquid chromatography-mass spectrometry analysis in human plasma following multiple doses of ombitasvir, in combination with paritaprevir/ritonavir and dasabuvir, confirmed that ombitasvir is the main component (51.9% of all measured drug-related components), whereas M29 (19.9%) and M36 (13.1%) are the major circulating metabolites. In summary, the study characterized ombitasvir metabolites in circulation, the metabolic pathways, and the elimination routes of the drug. PMID:27179128

  20. Hepatitis C Virus (HCV) NS5B Nonnucleoside Inhibitors Specifically Block Single-Stranded Viral RNA Synthesis Catalyzed by HCV Replication Complexes In Vitro▿

    OpenAIRE

    Yang, Wengang; Sun, Yongnian; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2006-01-01

    Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

  1. 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.

  2. Structure-based optimization and derivatization of 2-substituted quinolone-based non-nucleoside HCV NS5B inhibitors with submicromolar cellular replicon potency.

    Science.gov (United States)

    Cheng, Yu; Shen, Jian; Peng, Run-Ze; Wang, Gui-Feng; Zuo, Jian-Ping; Long, Ya-Qiu

    2016-06-15

    HCV NS5B polymerase is an attractive and validated target for anti-HCV therapy. Starting from our previously identified 2-aryl quinolones as novel non-nucleoside NS5B polymerase inhibitors, structure-based optimization furnished 2-alkyl-N-benzyl quinolones with improved antiviral potency by employing privileged fragment hybridization strategy. The N-(4-chlorobenzyl)-2-(methoxymethyl)quinolone derivative 5f proved to be the best compound of this series, exhibiting a selective sub-micromolar antiviral effect (EC50=0.4μM, SI=10.8) in Huh7.5.1 cells carrying a HCV genotype 2a. Considering the undesirable pharmacokinetic property of the highly substituted quinolones, a novel chemotype of 1,6-naphthyridine-4,5-diones were evolved via scaffold hopping, affording brand new structure HCV inhibitors with compound 6h (EC50 (gt2a)=2.5μM, SI=7.2) as a promising hit. Molecular modeling studies suggest that both of 2-alkyl quinolones and 1,6-naphthyridine-4,5-diones function as HCV NS5B thumb pocket II inhibitors. PMID:27133482

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

    Institute of Scientific and Technical Information of China (English)

    Jin-ling Dong; Shun-ai Liu; Qi Wang; Jin-qian Zhang; and Jun Cheng

    2013-01-01

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

  4. Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases.

    Science.gov (United States)

    Vincetti, Paolo; Caporuscio, Fabiana; Kaptein, Suzanne; Gioiello, Antimo; Mancino, Valentina; Suzuki, Youichi; Yamamoto, Naoki; Crespan, Emmanuele; Lossani, Andrea; Maga, Giovanni; Rastelli, Giulio; Castagnolo, Daniele; Neyts, Johan; Leyssen, Pieter; Costantino, Gabriele; Radi, Marco

    2015-06-25

    This study describes the discovery of novel dengue virus inhibitors targeting both a crucial viral protein-protein interaction and an essential host cell factor as a strategy to reduce the emergence of drug resistance. Starting from known c-Src inhibitors, a virtual screening was performed to identify molecules able to interact with a recently discovered allosteric pocket on the dengue virus NS5 polymerase. The selection of cheap-to-produce scaffolds and the exploration of the biologically relevant chemical space around them suggested promising candidates for chemical synthesis. A series of purines emerged as the most interesting candidates able to inhibit virus replication at low micromolar concentrations with no significant toxicity to the host cell. Among the identified antivirals, compound 16i proved to be 10 times more potent than ribavirin, showed a better selectivity index and represents the first-in-class DENV-NS5 allosteric inhibitor able to target both the virus NS5-NS3 interaction and the host kinases c-Src/Fyn. PMID:26039671

  5. 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. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle...... 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...

  6. An updated evolutionary study of Flaviviridae NS3 helicase and NS5 RNA-dependent RNA polymerase reveals novel invariable motifs as potential pharmacological targets.

    Science.gov (United States)

    Papageorgiou, Louis; Loukatou, Styliani; Sofia, Kossida; Maroulis, Dimitrios; Vlachakis, Dimitrios

    2016-06-21

    The rate of Flaviviridae family virus infections worldwide has increased dramatically in the last few years. In addition, infections caused by arthropod vector viruses including Hepatitis C, West Nile, Dengue fever, Yellow fever and Japanese encephalitis are emerging throughout the world. Based on a recent taxon update, the Flaviviridae family comprises four main genera; Flavivirus, Hepacivirus, Pestivirus and a recent genus Pegivirus. Although the new scientific classification plays a key role in providing useful information about the relationships between viruses, many new documented viruses remain unclassified. Furthermore, based on the different results of several studies the classification is unclear. In an effort to provide more insights into the classification of viruses, a holistic evolutionary study of the two viral enzymes NS3 helicase and NS5 RNA-dependent RNA polymerase (RdRp) has been conducted in this study. These two viral enzymes are very crucial for the inhibition of viruses due to the fact that they are involved in the survival, proliferation and transmission of viruses. The main goal of this study is the presentation of two novel updated phylogenetic trees of the enzymes NS3 helicase and NS5 RdRp as a reliable phylogeny "map" to correlate the information of the closely related viruses and identify new possible targets for the Flaviviridae family virus inhibition. Despite the earliest trials for drugs against Flaviviridae related viruses, no antiviral drug vaccine has been available to date. Therefore there is an urgent need for research towards the development of efficient antiviral agents. PMID:26864387

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

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

    Directory of Open Access Journals (Sweden)

    Karolina Olejniczak

    2008-01-01

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

  10. Isolation and structural characterization of Coryxin, a novel cyclic lipopeptide from Corynebacterium xerosis NS5 having emulsifying and anti-biofilm activity.

    Science.gov (United States)

    Dalili, Dina; Amini, Mohsen; Faramarzi, Mohammad Ali; Fazeli, Mohammad Reza; Khoshayand, Mohammad Reza; Samadi, Nasrin

    2015-11-01

    Herein we reported the structure and several properties of a new biosurfactants produced by Corynebacterium xerosis strain NS5. This strain was capable of producing a novel lipopeptide biosurfactant that we have named coryxin. The biosurfactant structure was characterized by using Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance spectroscopy (NMR), and Liquid chromatography-mass spectrometry (LC-MS). It contained a hydrophobic moiety of 3-hydroxydecanoic acid and a peptide part predicted as a sequence of seven amino acids including Asn-Arg-Asn-Gln-Pro-Asn-Ser. Coryxin lowered the surface tension of water to 31.4 mN/m, with a critical micelle concentration of 25mg/l. It was a strong emulsifier with an emulsification index of 61% against n-hexane. Coryxin showed antibacterial activity against test organisms belonging to Gram-positive and Gram-negative bacteria and disrupted preformed biofilms of Staphylococcus aureus (82.5%), Streptococcus mutans (80%), Escherichia coli (66%) and Pseudomonas aeruginosa (30%). In conclusion, microbial surfactant from C. xerosis exhibited inhibitory and disruptive activities against biofilm formation that could be of use in biofilm-related menace. PMID:26280817

  11. Mutations in nonstructural 5A gene of hepatitis C virus and its response to interferon alfa%丙型肝炎病毒NS5A基因变异与干扰素疗效的关系

    Institute of Scientific and Technical Information of China (English)

    张琳; 赵桂珍; 石理兰; 曹丽

    2003-01-01

    目的:探讨HCV1b型慢性丙型肝炎患者HCV NS5A基因变异与干扰素(IFN)疗效的关系,NS5A2209-2248片断是否存在干扰素敏感决定区(ISDR).方法:留取慢性丙型肝炎患者干扰素治疗前血清,应用RT-PCR法扩增NS5A基因片段,用直接测序法进行核苷酸及氨基酸序列测定.结果:11例HCV1b型患者中1例为中间型,其余为野生型.2例表现为完全应答,均为野生型,9例为无应答.两组之间核苷酸及氨基酸序列无显著差异.对1例患者的动态观察发现,干扰素治疗后其核苷酸及氨基酸序列均有所变化,改变了其在基因树中的位置.结论:HCVlb型NS5A基因变异与干扰素疗效无关,NS5A2209-2248区高度保守.未证实存在ISDR.干扰素治疗可引起HCV准种改变.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...... 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...

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

    Science.gov (United States)

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

    2016-09-01

    Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. It constitutes a major public health around the world. There is no vaccine available against HCV, and current therapies are effective in only small percentage of patients. HCV has wide population-specific genotype variability. Genotype knowledge and viral load assessment are equally important for designing therapeutic strategies. Taking into account that the molecular epidemiology of HCV variants circulating in Tunisia is not yet well elucidated, and that, at present, little is known about the distribution pattern of HCV in Monastir region (Tunisia), we aimed, herein, to evaluate the prevalence of HCV genotypes in Monastir and to identify risk-related factors. For this purpose, 50 anti-HCV antibody-positive cases were diagnosed and subjected to viral RNA extraction, amplification, genotyping, and viral load quantification. Molecular epidemiology was studied by 5' untranslated region (5' UTR) sequencing as compared with the non-structural 5B (NS5B) and core region sequences. Overall concordance between 5' UTR, core, and NS5B sequencing was 100 %. The highest prevalent genotype was 1b (50 %) followed by genotypes 1a (16 %), 4a (12 %), 2a (10 %), 2c (8 %), and 3a (4 %). Interestingly, the subtype 1b had a statistically significant higher viral load than the other genotypes followed by subtype 1a. Based on these data, this study revealed a high prevalence of HCV genotype 1 (subtypes 1b and 1a) compared to other genotypes. A continued monitoring of HCV and knowledge of circulating genotypes could impact on future vaccine formulations. PMID:27189386

  14. The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

    International Nuclear Information System (INIS)

    Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E315) and NS5 (NS5654,655) proteins, and into the 3' non-coding region (Δ30) of TBEV/DEN4. The variant that contained all three mutations (vΔ30/E315/NS5654,655) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vΔ30/E315/NS5654,655 should be further evaluated as a TBEV vaccine.

  15. 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.

  16. Metallization of the {\\beta}-SiC(100) 3\\times2 Surface: a DFT Investigation

    OpenAIRE

    Westover, James; Oughaddou, Hamid; Enriquez, Hanna; Kara, Abdelkader

    2011-01-01

    Using density functional theory (DFT) we report results for the electronic structure and vibrational dynamics of hydrogenated {\\beta} reconstructed Silicon Carbide (001) (3x2) surfaces with various levels of hydrogenation. These results were obtained using density functional theory with a generalized gradient exchange correlation function. The calculations reveal that metallization can be achieved via hydrogen atoms occupying the second silicon layer. Further increases of hydrogen occupation ...

  17. Hepatitis C virus expressing reporter tagged NS5A protein

    DEFF Research Database (Denmark)

    2014-01-01

    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...

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

    Science.gov (United States)

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

    2016-02-20

    Sofosbuvir is a direct acting antiviral medication used to treat Hepatitis C viral infection. The present study focuses on the degradation behavior of the drug under various stress conditions (hydrolysis, oxidative, thermal and photolytic) as per International Conference on Harmonization (ICH Q1A (R2)) guidelines. A high performance liquid chromatographic system (HPLC) was used to develop a selective, precise and accurate method for separating all the degradation products. The separation was achieved on a Sunfire™ C18 (150mm×4.6mm×5μm) stationary phase with a mobile phase of 10mM ammonium acetate (pH 5.0) buffer and acetonitrile in gradient elution mode. A quadrupole-time of flight mass analyzer equipped with an electrospray ionization technique was used to propose the structural information based on the MS/MS and accurate mass measurements. Seven degradation products were identified and characterised by LC-ESI-QTOF-MS/MS. In silico toxicity of the drug and its degradation products was determined using TOPKAT and DEREK toxicity prediction softwares. The proposed method was validated as per the ICH Q2 guidelines. PMID:26771133

  19. Intra-host variation structure of classical swine fever virus NS5B in relation to antiviral therapy.

    Science.gov (United States)

    Haegeman, Andy; Vrancken, Robert; Neyts, Johan; Koenen, Frank

    2013-05-01

    Classical swine fever (CSF) is one of most important diseases of the Suidea with severe social economic consequences in case of outbreaks. Antivirals have been demonstrated, in recent publications, to be an interesting alternative method of fighting the disease. However, classical swine fever virus is an RNA virus which presents a challenge as intra-host variation and the error prone RNA dependent RNA polymerase (RdRp) could lead to the emergence/selection of resistant variants hampering further treatment. Therefore, it was the purpose of this study to investigate the intra-host variation of the RdRp gene, targeted by antivirals, in respect to antiviral treatment. Using the non-unique nucleotide changes, a limited intra-host variation was found in the wild type virus with 2 silent and 2 non-synonymous sites. This number shifted significantly when an antiviral resistant variant was analyzed. In total 22nt changes were found resulting in 14 amino acid changes whereby each genome copy contained at least 2 amino-acid changes in the RdRp. Interestingly, the frequency of the mutations situated in close proximity to a region involved in antiviral resistance in CSFV and bovine viral diarrhea virus (BVDV) was elevated compared to the other mutations. None of the identified mutations in the resistant variant and which could potentially result in antiviral resistance was present in the wild type virus as a non-unique mutation. In view of the spectrum of mutations identified in the resistance associated region and that none of the resistance associated mutations reported for another strain of classical swine fever for the same antiviral were observed in the study, it can be suggested that multiple mutations confer resistance to some degree. Although the followed classical approach allowed the analysis the RdRp as a whole, the contribution of unique mutations to the intra-host variation could not be completely resolved. There was a significant difference in de number of unique mutations found between: 1/wild type virus and the antiviral resistant variant and 2/between both and the number to be expected from the error rate of the RT-PCR process. This indicates that the some of the unique mutations contributed to the intra-host variation and that the antiviral pressure also shifted this pattern. This is important as one of the non-synonymous mutations found in the resistant variant and which was located in the antiviral resistance associated region, was present in the wild type virus as a unique mutation. The findings presented in this study not only show the importance of intra-host variation analysis but also warrants further research certainly in view of the potential inclusion of antivirals in a control/eradication strategy. PMID:23511203

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

    Science.gov (United States)

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

    2016-02-15

    Using a functional high-throughput screening (HTS) and subsequent SAR studies, we have discovered a novel series of non-nucleoside dengue viral polymerase inhibitors. We report the elaboration of SAR around hit compound 1 as well as the synthesis and antiviral evaluation of 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole and 5-phenyl-2-[2-(2-thienyl)ethenyl]-1,3,4-oxadiazole analogues derived from a rapid and easily accessible chemical pathway. A large number of compounds prepared by this method were shown to possess in vitro activity against the polymerase of dengue virus. The most potent inhibitors were tested against Dengue virus clinical isolates on infected cells model and exhibit submicromolar activity on the four dengue virus serotypes. PMID:26774922

  1. Probing Maldacena-Nunez in IR with ${\\bar D3}$ branes

    OpenAIRE

    Pal, Shesansu Sekhar

    2004-01-01

    We probed Maldacena-Nunez solution in IR with p coincident anti D3 branes and found that these probe branes become a fuzzy NS5 brane. Doing the dual analysis i.e. from the NS5 brane point of view with the charge of p anti D3 brane on the world-volume of NS5 brane, we showed that to leading order this potential matches with that of p anti D3 branes and the potential on the NS5 brane has a stable minima and have also calculated the potential, from the NS5 brane point of view, for a small fluctu...

  2. Characterization of a HV Nais protein derived from a patient with hepatoma

    International Nuclear Information System (INIS)

    A cDNA encoding hepatitis C virus NS5A protein was isolated from the serum of a patient with hepatocellular carcinoma. The NS5AHCC was localized in both the cytoplasmic and nuclear fractions of Huh-7 cells. Immunoprecipitation and electrophoresis experiments showed four major phosphorylated species of NS5AHCC, p58, p56, p53, and p50. Two mutants (NS5AHCC-NLSmt and NS5AHCC-TSmt) carrying mutations on the putative nuclear localization signal were engineered. NS5AHCC-NLSmt was localized exclusively in the cytoplasm, whereas some forms of NS5AHCC-TSmt can be transported into the nucleus. These NS5AHCC mutant proteins were capable of transactivating c-fos and SV40 promoters. However, the transactivation efficiency was not dependent on its capability of nuclear localization. Subsequently, interaction between NS5AHCC mutants and Grb2 was studied. While capable of transactivating oncogenic promoters, NS5AHCC-TSmt could not interact with Grb2. Our results suggested that other cytosolic pathways independent of Grb2-mediated mechanisms were involved in the transactivation activity of HCV NS5A

  3. Affinity-Based Screening Technology and HCV Drug Discovery

    Institute of Scientific and Technical Information of China (English)

    LI Bin

    2003-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    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 t...

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

    Science.gov (United States)

    Su, Chan-I; Tseng, Chung-Hsin

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    Highlights: ► For the first time how DENV NS5 increases RANTES production. ► DENV NS5 physically interacts with human Daxx. ► 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.

  8. 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.

  9. 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.

  10. Penrose Limits of Branes and Marginal Intersecting Branes

    CERN Document Server

    Ryang, S

    2003-01-01

    We construct the Penrose limit backgrounds in closed forms along the generic null geodesics for the near-horizon geometries of D1, D3, D5, NS1 and NS5 branes. The Penrose limit metrics of D1, D5 and NS1 have non-trivial dependence of the light-cone time coordinate, while those of D3 and NS5 have no its dependence. We study the Penrose limits on the marginal 1/4 supersymmetric configurations of standard intersecting branes, such as the NS-NS intersection of NS1 and NS5, the R-R intersections of Dp and Dq over some spatial dimensions and the mix intersections of NS5 and Dp over (p -1)-dimensional spaces. They are classified into three types that correspond to the Penrose limits of D1, D3 and D5 backgrounds.

  11. Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus.

    Science.gov (United States)

    Odreman-Macchioli, María; Vielma, Silvana; Atchley, Daniel; Comach, Guillermo; Ramirez, Alvaro; Pérez, Saberio; Téllez, Luis; Quintero, Beatriz; Hernández, Erick; Muñoz, Maritza; Mendoza, José

    2013-03-01

    Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3'-noncoding region (3'NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3'NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3'NC regions showed a weak (kappa = 0.109; CI 95%) and a moderate (kappa = 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan format was much more sensitive than the C-prM/SYBR Green I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples. PMID:23781709

  12. Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1α and PKR

    Directory of Open Access Journals (Sweden)

    Sudha Govindarajan

    2012-11-01

    Full Text Available Abstract Background Interaction of non-structural protein 5A (NS5A of Hepatitis C virus (HCV with human kinases namely, casein kinase 1α (ck1α and protein kinase R (PKR have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. Results Serine 232 of NS5A is known to be phosphorylated by human ck1α. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1α has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1α has been identified from the model and these are found to be conserved well in the ck1 family. ck1α – substrate peptide complex has also been used to understand the structural basis of association between ck1α and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available. Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. Conclusions The substrate interacting residues in ck1α have been identified using the structural model of kinase - substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional

  13. 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

  14. 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.

  15. Solution Conformations of the substrates and Inhibitor of Hepatitis C Virus NS3 Protease

    International Nuclear Information System (INIS)

    Hepatitis C virus (HCV) has been known to be an enveloped virus with a positive strand RNA genome and the major agent of the vast majority of transfusion associated cases of hepatitis. For viral replication, HCV structural proteins are first processed by host cell signal peptidases and NS2/NS3 site of the nonstructural protein is cleaved by a zinc-dependent protease NS2 with N-terminal NS3. The four remaining junctions are cleaved by a separate NS3 protease. The solution conformations of NS4B/5A, NS5A/5B substrates and NS5A/5B inhibitor have been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data suggested that the both NS5A/5B substrate and inhibitor appeared to have a folded turn-like conformation not only between P1 and P6 position but also C-terminal region, whereas the NS4B/5A substrate exhibited mostly extended conformation. In addition, we have found that the conformation of the NS5A/5B inhibitor slightly differs from that of NS5A/5B substrate peptide, suggesting different binding mode for NS3 protease. These findings will be of importance for designing efficient inhibitor to suppress HCV processing

  16. Zika Virus Targets Human STAT2 to Inhibit Type I Interferon Signaling.

    Science.gov (United States)

    Grant, Alesha; Ponia, Sanket S; Tripathi, Shashank; Balasubramaniam, Vinod; Miorin, Lisa; Sourisseau, Marion; Schwarz, Megan C; Sánchez-Seco, Mari Paz; Evans, Matthew J; Best, Sonja M; García-Sastre, Adolfo

    2016-06-01

    The ongoing epidemic of Zika virus (ZIKV) illustrates the importance of flaviviruses as emerging human pathogens. All vector-borne flaviviruses studied thus far have to overcome type I interferon (IFN) to replicate and cause disease in vertebrates. The mechanism(s) by which ZIKV antagonizes IFN signaling is unknown. Here, we report that the nonstructural protein NS5 of ZIKV and other flaviviruses examined could suppress IFN signaling, but through different mechanisms. ZIKV NS5 expression resulted in proteasomal degradation of the IFN-regulated transcriptional activator STAT2 from humans, but not mice, which may explain the requirement for IFN deficiency to observe ZIKV-induced disease in mice. The mechanism of ZIKV NS5 resembles dengue virus (DENV) NS5 and not its closer relative, Spondweni virus (SPOV). However, unlike DENV, ZIKV did not require the E3 ubiquitin ligase UBR4 to induce STAT2 degradation. Hence, flavivirus NS5 proteins exhibit a remarkable functional convergence in IFN antagonism, albeit by virus-specific mechanisms. PMID:27212660

  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

    replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5' end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent...... 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...

  18. Flaviviral Replication Complex: Coordination between RNA Synthesis and 5’-RNA Capping

    Directory of Open Access Journals (Sweden)

    Valerie J. Klema

    2015-08-01

    Full Text Available Genome replication in flavivirus requires (— strand RNA synthesis, (+ strand RNA synthesis, and 5’-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER membrane. Two major components of the replication complex are the viral non-structural (NS proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (— RNA synthesis, (+ RNA synthesis, and 5’-RNA capping and methylation.

  19. Rotating and orbiting strings in the near-horizon brane backgrounds

    CERN Document Server

    Ryang, S

    2003-01-01

    Using the Schwarzschild-type coordinates in stead of the global ones we reconstruct the classical rotating closed string solutions in the AdS*5 x S*5 backgrounds. They are explicitly described by the Jacobi elliptic and trigonometrical functions of worldsheet coordinates. We study the orbiting closed string configurations in the near-horizon geometries of Dp, NS1 and NS5 branes, and derive the energy and spin of them, whose relation takes a simple form for short strings. Specially in the D5 and NS5 backgrounds we have a linear relation that the energy of the point-like string is proportional to the spin, which is associated with the spectrum of strings in the pp-wave geometries obtained by taking a special Penrose limit on the D5 and NS5 backgrounds.

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

    Directory of Open Access Journals (Sweden)

    Eliana F Castro

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

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

    Science.gov (United States)

    Castro, Eliana F; Campos, Rodolfo H; Cavallaro, Lucía V

    2014-01-01

    Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs. PMID:24950191

  2. HLA-B27 Selects for Rare Escape Mutations that Significantly Impair Hepatitis C Virus Replication and Require Compensatory Mutations

    OpenAIRE

    Neumann-Haefelin, Christoph; Oniangue-Ndza, Cesar; Kuntzen, Thomas; Schmidt, Julia; Nitschke, Katja; Sidney, John; Caillet-Saguy, Célia; Binder, Marco; Kersting, Nadine; Kemper, Michael W.; Power, Karen A.; Ingber, Susan; Reyor, Laura L.; Hills-Evans, Kelsey; Kim, Arthur Y.

    2011-01-01

    HLA-B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant HLA-B27 restricted HCV-specific CD8+ T cell epitope NS5B2841-2849 (ARMILMTHF) has been shown to be limited by viral fitness costs as well as broad T cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27 restricted epitope NS5B2936-2944 (GRAAICGKY) in order to further define the mechanisms of pr...

  3. The Heterotic Enhancon

    CERN Document Server

    Natsuume, M

    2002-01-01

    The enhancon mechanism is studied in the heterotic string theory. We consider the N_L=0 winding strings with momentum (NS1-W*) and the Kaluza-Klein dyons (KK5-NS5*). The NS1-W* and KK5-NS5* systems are dualized to the D4-D0* and D6-D2* systems respectively under the d=6 heterotic/IIA S-duality. The heterotic form has a number of advantages over the type IIA form. We study these backgrounds and obtain the enhancon radii by brane probe analysis. The results are consistent with the S-duality.

  4. D-Brane solutions in a light-like linear dilaton background

    CERN Document Server

    Nayak, R R; Nayak, Rashmi R.; Panigrahi, Kamal L.

    2006-01-01

    The light-like linear dilaton background presents a simple time dependent solution of type II supergravity equations of motion that preserves 1/2 supersymmetry in ten dimensions. We construct supergravity D-brane solutions in a linear dilaton background starting from the known intersecting brane solutions in string theory. By applying a Penrose limit on the intersecting (NS1-NS5-NS5')- brane solution, we find out a D5-brane in a linear dilaton background. We solve the Killing spinor equations for the brane solutions explicitly, and show that they preserve 1/4 supersymmetry. We also find a M5-brane solution in eleven dimensional supergravity.

  5. Drug: D08951 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available Hepatitis C [DS:H00413] nonnucleoside polymerase inhibitor HCV NS5B is RNA-dependent RNA polymerase(RdRP). N...D08951 Drug Nesbuvir (USAN) C22H23FN2O5S 446.1312 446.4918 D08951.gif Treatment of

  6. The juxtamembrane sequence of the Hepatitis C virus polymerase can affect RNA synthesis and inhibition by allosteric polymerase inhibitors.

    Science.gov (United States)

    Wen, Y; Lin, X; Fan, B; Ranjith-Kumar, C T; Kao, C C

    2015-08-01

    The Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), nonstructural protein 5B (NS5B), is anchored in the membrane through a C-terminal helix. A sequence of ca. 12 residues that connects the catalytically competent portion of the RdRp and the C-terminal helix, the juxtamembrane sequence (JMS), has a poorly defined role in RdRp function in a large part since it is translated from a cis-acting RNA element (CRE) that is essential for HCV replication. Using a HCV replicon that transposed a second copy of CRE to the 3' UTR of the HCV replicon, we demonstrate that amino acid substitutions in the JMS were detrimental for HCV replicon replication. Substitutions in the JMS also resulted in a defect in de novo-initiated RNAs synthesis in vitro and in a cell-based reporter assay. A nonnucleoside inhibitor of the NS5B that binds to the catalytic pocket was less potent in inhibiting NS5B in the presence of JMS mutations. The JMS mutants exhibit reduced stability in thermodenaturation assays, suggesting that the JMS helps confer a more stable conformation to NS5B that could impact RNA synthesis. PMID:25895103

  7. Restricted Isotypic Antibody Reactivity to Hepatitis C Virus Synthetic Peptides in Immunocompromised Patients

    Science.gov (United States)

    Devesa, Marisol; de Saez, Arlette; León, Graciela; Sirit, Firelei; Cosson, Clarisa; Bermúdez, Henry; Liprandi, Ferdinando; Noya, Oscar; Pujol, Flor H.

    1999-01-01

    An enzyme immunoassay based on three synthetic peptides from the core, NS4, and NS5 regions of hepatitis C virus allowed the detection of antibodies in 100% of immunocompetent infected patients and in 91% of immunocompromised patients (hemodialysis and hemophiliac patients). Immune impairment seemed to restrict the spectrum of antibody isotypes reacting to the core peptide. PMID:10066669

  8. Discovery of conjugated thiazolidinone-thiadiazole scaffold as anti-dengue virus polymerase inhibitors.

    Science.gov (United States)

    Manvar, Dinesh; Küçükgüzel, İlkay; Erensoy, Gizem; Tatar, Esra; Deryabaşoğulları, Gökhan; Reddy, Haarika; Talele, Tanaji T; Cevik, Ozge; Kaushik-Basu, Neerja

    2016-01-15

    Dengue virus (DENV) infection is a significant health threat to the global population with no therapeutic option. DENV NS5 RNA-dependent RNA polymerase (RdRp) is the key replicating protein of the virus and thus an attractive target for drug development. Herein, we report on the synthesis and biological evaluation of a series of hybrid thiazolidinone-thiadiazole derivatives as a new class of DENV-2 NS5 RdRp inhibitors. This yielded compounds 12 and 21 with IC50 values of 2.3 μM and 2.1 μM, respectively, as promising leads. Limited SAR analysis indicated 3-fluorobenzylidene as the optimal substituent at C5-position of the thiazolidinone core, whereas both 2-chlorophenyl and 3-fluorophenyl substituents were equally effective at C5-position of the 1,3,4-thiadiazole core. Biophysical characterization and molecular docking studies conferred the binding site of this scaffold on DENV NS5 polymerase. Binding mode of compound 21 in Thumb pocket-II of DENV-2 NS5 polymerase will form the basis for future structure-activity relationship optimization. PMID:26697747

  9. UniProt search blastx result: AK288325 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288325 J090022F22 P27915|POLG_DEN3P Genome polyprotein [Contains: Capsid protein C (Core prote ... (EC 2.7.7.48) (NS5)] - Dengue virus type 3 (strain Philippines /H87/1956) (DENV-3) 0 ...

  10. Hot little string correlators: a view from supergravity

    International Nuclear Information System (INIS)

    We study the propagation of a massless minimally coupled scalar in the near horizon geometry of non-extremal NS5-branes. Using the holographic principle for dilatonic backgrounds we compute the two-point function of an operator in little string theory at the Hagedorn temperature. We then comment on relations with correlation functions in two dimensional string theory. (author)

  11. Metastable Supersymmetry Breaking Vacua on Abelian Brane Models

    CERN Document Server

    Halyo, Edi

    2009-01-01

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

  12. Resistance analysis of baseline and treatment-emergent variants in hepatitis C virus genotype 1 in the AVIATOR study with paritaprevir-ritonavir, ombitasvir, and dasabuvir.

    Science.gov (United States)

    Krishnan, Preethi; Tripathi, Rakesh; Schnell, Gretja; Reisch, Thomas; Beyer, Jill; Irvin, Michelle; Xie, Wangang; Larsen, Lois; Cohen, Daniel; Podsadecki, Thomas; Pilot-Matias, Tami; Collins, Christine

    2015-09-01

    AVIATOR, a phase 2 clinical trial, evaluated ritonavir-boosted paritaprevir (a protease inhibitor), ombitasvir (an NS5A inhibitor), and dasabuvir (a nonnucleoside polymerase inhibitor) (the three-drug [3D] regimen) with or without ribavirin (RBV) for 8, 12, or 24 weeks in 406 HCV genotype 1 (GT1)-infected patients. The rate of sustained virologic response 24 weeks after treatment ranged from 88% to 100% across the arms of the 3D regimen with or without RBV; 20 GT1a-infected patients and 1 GT1b-infected patient experienced virologic failure (5.2%). Baseline resistance-conferring variants in NS3 were rare. M28V in GT1a and Y93H in GT1b were the most prevalent preexisting variants in NS5A, and C316N in GT1b and S556G in both GT1a and GT1b were the most prevalent variants in NS5B. Interestingly, all the GT1a sequences encoding M28V in NS5A were from the United States, while GT1b sequences encoding C316N and S556G in NS5B were predominant in the European Union. Variants preexisting at baseline had no significant impact on treatment outcome. The most prevalent treatment-emergent resistance-associated variants (RAVs) in GT1a were R155K and D168V in NS3, M28T and Q30R in NS5A, and S556G in NS5B. The single GT1b-infected patient experiencing virologic failure had no RAVs in any target. A paritaprevir-ritonavir dose of 150/100 mg was more efficacious in suppressing R155K in NS3 than a 100/100-mg dose. In patients who failed after receiving 12 or more weeks of treatment, RAVs were selected in all 3 targets, while most patients who relapsed after 8 weeks of treatment did so without any detectable RAVs. Results from this study guided the selection of the optimal treatment regimen, treatment duration, and paritaprevir dose for further development of the 3D regimen. (This study has been registered at ClinicalTrials.gov under registration number NCT01464827.). PMID:26100711

  13. Function of nonstructural 5A protein of genotype 2a in replication and infection of HCV with gene substitution

    Institute of Scientific and Technical Information of China (English)

    Yong-Zhi Wang; Wen-Bo Wang; Ming-Mei Cao; Wen Wang; Lan-Juan Zhao; Gang Xu; Hao Ren; Zhong-Tian Qi

    2011-01-01

    AIM: To explore the function of Nonstructural 5A (NS5A) protein of genotype 2a (JFH1) in the replication and infection of hepatitis C virus (HCV).METHODS: Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PPCR) method and the restriction enzyme reaction.In vitro RNA transcripts of chimera, prototype J6JFH and negative control J6JFH1 (GNDD) were prepared and transfected into Huh-7.5 cells with liposomes. Immunofluorescence assay (IFA), fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.RESULTS: The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point (2.58± 5.97×106 vs 4.27 ± 1.72×104, PP = 00.0032). The maximal level of HCV RNA in chimera was 5.6±1.8×104 GE/μg RNA at day 34 after transfection, while the wild type reached a peak level at day 13 which was 126 folds higher (70.65 ± 14.11×105 vs 0.56 ± 0.90×105, = 0.028). HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level. Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles, ranging from 10 ± 5 ffu/mL to 78.3 ± 23.6 ffu/mL, while that of FL-J6JFH1 ranged from 5.8 ± 1.5×102 ffu/mL to 2.5 ± 1.4×104 ffu/mL.CONCLUSION: JFH1 NS5A might play an important role in the robust replication of J6JFH1. The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.

  14. 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.

  15. 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.

  16. Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein.

    OpenAIRE

    Hosein, B; Fang, C T; Popovsky, M A; J. Ye; Zhang, M; WANG, C. Y.

    1991-01-01

    Cloning and expression of hepatitis C virus have allowed the development of immunoassays to detect hepatitis C virus infection. However, currently available recombinant fusion protein C100-3 assays, based on a nonstructural protein of the virus, are limited in sensitivity, particularly for detecting acute infection. In this report seroconversion panels showed that an assay based on synthetic peptides, derived from immunodominant regions of both capsid and nonstructural proteins, accelerated h...

  17. Observations on fluxes near anti-branes

    Science.gov (United States)

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

    2016-01-01

    We revisit necessary conditions for gluing local (anti-)D3 throats into flux throats with opposite charge. These consistency conditions typically reveal singularities in the 3-form fluxes whose meaning is being debated. In this note we prove, under well-motivated assumptions, that unphysical singularities can potentially be avoided when the anti-branes polarise into spherical NS5 branes, with a specific radius. If a consistent solution can then indeed be found, our analysis seems to suggests a rather large correction to the radius of the polarization sphere compared to the probe result. We furthermore comment on the gluing conditions at finite temperature and point out that one specific assumption of a recent no-go theorem can be broken if anti-branes are indeed to polarise into spherical NS5 branes at zero temperature.

  18. Neveu-Schwarz 5-branes in type-IIA supergravity and gravitational anomalies

    International Nuclear Information System (INIS)

    We construct a gravitational-anomaly-free effective action for the coupled system of type-IIA D=10 dynamical supergravity interacting with a NS5-brane. The NS5-brane is considered as elementary in that the associated current is a δ function supported on its world volume. Our approach is based on a Chern kernel which encodes the singularities of the three-form field strength near the brane in an SO(4)-invariant way and provides a solution for its Bianchi identity in terms of a two-form potential. A dimensional reduction of the recently constructed anomaly-free effective action for an elementary M5-brane in D=11 is seen to reproduce our ten-dimensional action. The Chern-kernel approach provides in particular a concrete realization of the anomaly cancellation mechanism envisaged by Witten

  19. Observations on fluxes near anti-branes

    CERN Document Server

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

    2015-01-01

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

  20. Genetic clustering of recent classical swine fever virus isolates from Karnataka, India revealed the emergence of subtype 2.2 replacing subtype 1.1.

    Science.gov (United States)

    Shivaraj, D B; Patil, S S; Rathnamma, D; Hemadri, D; Isloor, S; Geetha, S; Manjunathareddy, G B; Gajendragad, M R; Rahman, H

    2015-09-01

    The phylogenetic analysis of 11 CSFV isolates from Karnataka, India obtained during the year 2012-13 was undertaken to obtain the most reliable genetic typing of the CSFV isolates based on E2, NS5B and 5'UTR genomic regions. The study indicated that all the 11 CSFV isolates belonged to subgroup 2.2. The most reliable classification was obtained with sequence data from the NS5B region which separated all the isolates based on the history of outbreak and geographic origin. Analysis of full length E2 amino acid sequences revealed different genetic makeup of Indian 2.2 isolates compared to 2.2 isolates from different countries. The group 2.2 viruses are gradually spreading as confirmed by frequent detection/ isolation of group 2.2 viruses in the recent years and replacing the subgroup 1.1 viruses, which were hitherto predominantly involved in CSF outbreaks in India. PMID:26396984

  1. Evidence of intratypic recombination in natural populations of hepatitis C virus

    International Nuclear Information System (INIS)

    Hepatitis C virus (HCV) has high genomic variability and, since its discovery, at least six different types and an increasing number of subtypes have been reported. Genotype 1 is the most prevalent genotype found in South America. In the present study, three different genomic regions (5 UTR, core and NS5B) of four HCV strains isolated from Peruvian patients were sequenced in order to investigate the congruence of HCV genotyping for these three genomic regions. Phylogenetic analysis using 5 UTR-core sequences found strain PE22 to be related to subtype 1a. To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossover event had taken place in the NS5B protein. We discuss the consequences of this observation on HCV genotype classification, laboratory diagnosis and treatment of HCV infection

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-11-25

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

  3. Heterotic string plus five-brane systems with asymptotic AdS3

    CERN Document Server

    Gemmer, Karl-Philip; Lechtenfeld, Olaf; Nölle, Christoph; Popov, Alexander D

    2012-01-01

    We present NS1+NS5-brane solutions of heterotic supergravity on curved geometries. They interpolate between a near horizon AdS3 x X^k x T^{7-k} region and R^{1,1} x c(X^k) x T^{7-k}, where X^k (with k = 3,5,6,7) is a k-dimensional geometric Killing spinor manifold, c(X^k) its Ricci-flat cone and T^{7-k} a (7-k)-torus. The solutions require first order alpha'-corrections to the field equations, and special point-like instantons play an important role, whose singular support is a calibrated submanifold wrapped by the NS5-brane. It is also possible to add a gauge anti-5-brane. We determine the super isometries of the near horizon geometry which are supposed to appear as symmetries of the holographically dual two-dimensional conformal field theory.

  4. Phytochemical Study of Myrtopsis corymbosa, Perspectives for Anti-dengue Natural Compound Research

    Directory of Open Access Journals (Sweden)

    Paul Coulerie

    2013-05-01

    Full Text Available In order to find new molecules for anti-viral drug design, we screened 102 ethyl acetate extracts obtained from 51 plants native from New-Caledonia for an antiviral activity against the dengue 2 virus RNA dependant RNA polymerase (DENV-NS5 RdRp. Leaf and bark extracts of Myrtopsis corymbosa which strongly inhibited the DENV-NS5 were selected for chemical investigation. We present here the first chemical study of M. corymbosa which led us to isolate three coumarins, namely ramosin (1, myrsellinol (1 and myrsellin (3, and three alkaloids, namely skimmianine (4, γ-fagarin (4 and haplopin (6. These compounds were identified as major compounds from the active extracts of the plant. However, they demonstrated only weak antiviral activity on the dengue virus. Further studies are necessary to know if the antiviral activity is due to a synergy between several compounds or due to the presence of other minor compounds.

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

    International Nuclear Information System (INIS)

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

  6. Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1α and PKR

    OpenAIRE

    Sudha Govindarajan; Yamunadevi Subburaj; Tyagi Nidhi; Das Saumitra; Srinivasan Narayanaswamy

    2012-01-01

    Abstract Background Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1α (ck1α) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against H...

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

    CERN Document Server

    Kluson, J

    2016-01-01

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

  8. 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

  9. Observations on fluxes near anti-branes

    OpenAIRE

    Cohen-Maldonado, Diego; Diaz Dorronsoro, Juan; Van Riet, Thomas; Vercnocke, Bert

    2016-01-01

    We revisit necessary conditions for gluing local (anti-)D3 throats into flux throats with opposite charge. These consistency conditions typically reveal singularities in the three-form fluxes whose meaning is being debated. In this note we prove, under well-motivated assumptions, that unphysical singularities can potentially be avoided when the anti-branes polarise into spherical NS5 branes with a specific radius. If a consistent solution can then indeed be found, our analysis seems to sugges...

  10. Meta-Stable Brane Configurations by Quartic Superpotential for Bifundamentals

    Science.gov (United States)

    Ahn, Changhyun

    The type IIA nonsupersymmetric meta-stable brane configuration consisting of three NS5-branes, D4-branes and anti-D4-branes where the electric gauge theory superpotential has a quartic term for the bifundamentals besides a mass term is constructed. By adding the orientifold 4-plane and 6-plane to this brane configuration, we also describe the intersecting brane configurations of type IIA string theory corresponding to the meta-stable nonsupersymmetric vacua of corresponding gauge theories.

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

    Science.gov (United States)

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

    2016-06-23

    Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease. PMID:27337340

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

    Indian Academy of Sciences (India)

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

    2014-03-01

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

  13. Molecular genotyping of HCV infection in seropositive blood donor

    Science.gov (United States)

    Zarin, Siti Noraziah Abu; Ibrahim, Nazlina

    2013-11-01

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

  14. Drug: D10105 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D10105 Drug Daclatasvir hydrochloride (JAN); Daclatasvir dihydrochloride (USAN) C40...H50N8O6. 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

  15. Drug: D10165 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D10165 Drug Setrobuvir (USAN) C25H25FN4O6S2 560.12 560.6176 D10165.gif Treatment of...Hepatitis C Antiinfectives [BR:br08307] Antivirals Anti-HCV agent Polymerase inhibitor NS5B polymerase inhibitor Setrobuvir D101...65 Setrobuvir (USAN) CAS: 1071517-39-9 PubChem: 135626883 LigandBox: D101

  16. ( p, q)-five brane and ( p, q)-string solutions, their bound state and its near horizon limit

    Science.gov (United States)

    Klusoň, Josef

    2016-06-01

    We determine ( p, q)-string and ( p, q)-five brane solutions of type IIB supergravity using SL (2 , ℤ)-symmetry of the full type IIB superstring theory. We also determine SL (2 , ℤ)-transformed solution corresponding to the bound state of NS5-branes and fundamental strings. Then we analyze its near horizon limit and we show that it leads to the AdS3 × S 3 with mixed fluxes.

  17. 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

  18. Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn2+ concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp

  19. The black hole interior and a curious sum rule

    International Nuclear Information System (INIS)

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

  20. HCV genotyping using statistical classification approach

    Directory of Open Access Journals (Sweden)

    Norris Ellie D

    2009-07-01

    Full Text Available Abstract The genotype of Hepatitis C Virus (HCV strains is an important determinant of the severity and aggressiveness of liver infection as well as patient response to antiviral therapy. Fast and accurate determination of viral genotype could provide direction in the clinical management of patients with chronic HCV infections. Using publicly available HCV nucleotide sequences, we built a global Position Weight Matrix (PWM for the HCV genome. Based on the PWM, a set of genotype specific nucleotide sequence "signatures" were selected from the 5' NCR, CORE, E1, and NS5B regions of the HCV genome. We evaluated the predictive power of these signatures for predicting the most common HCV genotypes and subtypes. We observed that nucleotide sequence signatures selected from NS5B and E1 regions generally demonstrated stronger discriminant power in differentiating major HCV genotypes and subtypes than that from 5' NCR and CORE regions. Two discriminant methods were used to build predictive models. Through 10 fold cross validation, over 99% prediction accuracy was achieved using both support vector machine (SVM and random forest based classification methods in a dataset of 1134 sequences for NS5B and 947 sequences for E1. Prediction accuracy for each genotype is also reported.

  1. Braneworld localisation in hyperbolic spacetime

    CERN Document Server

    Crampton, B; Stelle, K S

    2014-01-01

    We present a construction employing a type IIA supergravity and 3-form flux background together with an NS5-brane that localises massless gravity near the 5-brane worldvolume. The nonsingular underlying type IIA solution is a lift to 10D of the vacuum solution of the 6D Salam-Sezgin model and has a hyperbolic ${\\cal H}^{(2,2)}\\times S^1$ structure in the lifting dimensions. A fully back-reacted solution including the NS5-brane is constructed by recognising the 10D Salam-Sezgin vacuum solution as a "brane resolved through transgression." The background hyperbolic structure plays a key r\\^ole in generating a mass gap in the spectrum of the transverse-space wave operator, which gives rise to the localisation of gravity on the 6D NS5-brane worldvolume, or, equally, in a further compactification to 4D. Also key to the successful localisation of gravity is the specific form of the corresponding transverse wavefunction Schr\\"odinger problem, which asymptotically involves a $V=-1/(4\\rho^2)$ potential, where $\\rho$ is...

  2. Clinical impact of the hepatitis C virus mutations in the era of directly acting antivirals.

    Science.gov (United States)

    Coppola, Nicola; Minichini, Carmine; Starace, Mario; Sagnelli, Caterina; Sagnelli, Evangelista

    2016-10-01

    Introduced in 2013-2014, the second- and third-wave directly acting antivirals (DAAs) have strongly enhanced the efficacy and tolerability of anti-HCV treatment, with a sustained virological response (SVR) in 90-95% of cases treated. The majority of patients who did not achieve an SVR were found to be infected with HCV strains with a reduced susceptibility to these drugs. Indeed, the high error rate of the viral polymerase and a fast virion production (100-fold higher than the human immunodeficiency virus) result in a mixture of viral genetic populations (quasi-species) pre-existing treatment initiation. These mutants occur frequently in the NS5A region, with a moderate frequency in the NS3/4A region and rarely in the NS5B region. Treatment-induced resistant mutants to NS5A DAAs persist for years after treatment discontinuation, whereas those resistant to the NS3 DAAs have a shorter duration. This review focuses on the type and prevalence of viral strains with a reduced sensitivity to DAAs, their clinical impact and influence on the response to treatment and, consequently, on treatment choice for DAA-experienced patients. J. Med. Virol. 88:1659-1671, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991255

  3. 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.

  4. Cloning and Prokaryotic Expression of α-Gliadin Gene from Psathyrostachys huashanica%华山新麦草α-醇溶蛋白基因的克隆及原核表达

    Institute of Scientific and Technical Information of China (English)

    王玉卿; 赵继新; 庞玉辉; 降彦苗; 陈新宏; 武军; 刘淑会

    2011-01-01

    [Objective] The present study aimed at cloning and analyzing the α-gliadin genes from Psathyrostachys huashanica and expressing it in E. coli. [Method] The α-gliadin genes were amplified from P. huashanica by AS-PCR and then the cloned gene Gli-Ns-5 was inserted into pET-28a(+). The recombinant plasmids pET28a-Gli-Ns were expressed in a prokaryotic expression system after its transformation into BL21(DE3) pLysS host strain. [Result] Four new α-glidain genes, Gli-Ns-2 (FJ713595), Gli-Ns-3 (GQ139525), Gli-Ns-4 (GQ139526) and Gli-Ns-5 (GQ139527), were isolated and characterized from the genomic DNA of Psathyrostachys huashanica. Molecular structure analysis revealed that these four genes had the typical structure of α-gliadin and contained 8 or 9 cysteine residues, respectively. Two internal stop codons were identified in coding region of FJ713595,indicating that it was a pseudogene. The results of SDS-PAGE and Western-blot demonstrated that the fusion protein could express normally in the prokaryotic expression system. [Conclusion] The four α-gliadin genes ofPsathyrostachys huashanica were cloned and the gene Gli-Ns-5 (GQ139527) was successfully expressed in E.coli. This study could provide new candidate genes for wheat quality improvement.%[目的]克隆华山新麦草(Psathyrostachys huashanica)的α-醇溶蛋白基因,并对其进行生物信息学分析,构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白.[方法]采用同源克隆法从华山新麦草基因组DNA中分离克隆出α-醇溶蛋白基因并进行序列分析,将克隆的华山新麦草仅α-醇溶蛋白基因Gli-Ns-5克隆到表达载体pET-28a(+)上,获得重组质粒pET28a-Gli-Ns转化大肠杆菌BL21(DE3)并诱导表达.[结果]从华山新麦草基因组DNA中克隆了4个α-醇溶蛋白基因:Gli-Ns-2(FJ713595)、Gli-Ns-3(GQ139525)、Gli-Ns-4(G0139526)和Gli-Ns-5(GQ139527).序列分析表明,4条序列具有α-醇溶蛋白的典型结构特征,含有8个或9个

  5. Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094.

    Science.gov (United States)

    Ehteshami, Maryam; Tao, Sijia; Ozturk, Tugba; Zhou, Longhu; Cho, Jong Hyun; Zhang, Hongwang; Amiralaei, Sheida; Shelton, Jadd R; Lu, Xiao; Khalil, Ahmed; Domaoal, Robert A; Stanton, Richard A; Suesserman, Justin E; Lin, Biing; Lee, Sam S; Amblard, Franck; Whitaker, Tony; Coats, Steven J; Schinazi, Raymond F

    2016-08-01

    Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on β-d-2'-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2'-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2'-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo Finally, we found that although both 2'-C-methyl-GTP and 2'-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2'-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2'-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites. PMID:27216050

  6. Phylogeographic characterization of tick-borne encephalitis virus from patients, rodents and ticks in Slovenia.

    Directory of Open Access Journals (Sweden)

    Luka Fajs

    Full Text Available Tick-borne encephalitis virus (TBEV is the most important arboviral agent causing infections of the central nervous system in central Europe. Previous studies have shown that TBEV exhibits pronounced genetic variability, which is often correlated to the geographical origin of TBEV. Genetic variability of TBEV has previously been studied predominantly in rodents and ticks, while information about the variability in patients is scarce. In order to understand the molecular relationships of TBEV between natural hosts, vectors and humans, as well as correlation between phylogenetic and geographical clustering, sequences of TBEV E and NS5 protein genes, were obtained by direct sequencing of RT-PCR products from TBE-confirmed patients as well as from rodents and ticks collected from TBE-endemic regions in Slovenia. A total of 27 partial E protein gene sequences representing 15 human, 4 rodent and 8 tick samples and 30 partial NS5 protein gene sequences representing 17 human, 5 rodent and 8 tick samples were obtained. The complete genome sequence of TBEV strain Ljubljana I was simultaneously obtained. Phylogenetic analysis of the E and NS5 protein gene sequences revealed a high degree of TBEV variability in patients, ticks and rodents. Furthermore, an evident correlation between geographical and phylogenetic clustering was shown that was independent of the TBEV host. Moreover, we show the presence of a possible recombination event in the TBEV genome obtained from a patient sample, which was supported with multiple recombination event detection methods. This is the first study that simultaneously analyzed the genetic relationships of directly sequenced TBEV samples from patients, ticks and rodents and provides the largest set of patient-derived TBEV sequences up to date. In addition, we have confirmed the geographical clustering of TBEV sequences in Slovenia and have provided evidence of a possible recombination event in the TBEV genome, obtained from a

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kenny-Walsh Elizabeth

    2006-11-01

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

  9. Are metabolic factors still important in the era of direct antiviral agents in patients with chronic hepatitis C?

    Directory of Open Access Journals (Sweden)

    Alessandro Grasso

    2013-01-01

    Full Text Available The high rate of sustained viral response (SVR to boceprevir or telaprevir-based triple therapy in hepatitis C (HCV-related, non-cirrhotic naïve patients or relapsers to previous antiviral treatment leads clinicians to believe that the impact of metabolic host factors on SVR is minimal when triple therapy is used, unlike what is observed with the peginterferon and ribavirin schedules. This concept is strongly expressed by some opinion leaders on the basis of the data derived from sub-analyses of registrative trials as well as from a post-hoc analysis of the phase II C208 clinical trial. The perception of unrestrainable therapeutic success with the use of newer, more powerful antivirals is now reinforced by the brilliant results obtained with sofosbuvir, an HCV NS5B polymerase inhibitor, as well as by the data from the phase II and III studies on the various combinations of second-generation NS3/4A inhibitors and NS5A and/or NS5B inhibitors. However, a great deal of concern has emerged from the real world scenario in which patients are often older and have more comorbidities than patients in the “world of trials”. Furthermore, many of them have advanced fibrosis and previous failure with peginterferon and ribavirin treatment. Some data from the recent literature suggest that the host metabolic factors may play a minor but non-negligible role in these difficult-to-treat patients, an issue that will hopefully be investigated in further studies. This editorial aims to provide a detailed analysis of the role that host metabolic factors played in the past and what role they may play in the era of direct antiviral agents.

  10. Daclatasvir-containing all-oral regimens for the treatment of hepatitis C virus infection.

    Science.gov (United States)

    Yang, Sheng-Shun; Kao, Jia-Horng

    2016-03-01

    The treatment of chronic hepatitis C is revolutionizing rapidly. The aim of this study is to review the efficacy and safety of daclatasvir (DCV)-containing all-oral regimens in clinical studies for chronic hepatitis C treatment. Using PubMed and search terms of 'DCV,' 'hepatitis C virus (HCV) treatment,' and 'HCV NS5A inhibitors,' literature on the clinical development of DCV, as well as abstracts presented at the April 2015 annual meeting of the European Association for the Study of the Liver (EASL) and November 2014 annual meeting of the American Association for the Study of Liver Diseases were reviewed. The final search was undertaken on 14 July 2015. With its potent antiviral activity to all HCV genotypes (GT) demonstrated in preclinical, phases 1-3 studies, DCV has been acting as a very competent team player in clinical trials of all-oral regimens. It is generally safe and well tolerated with a low genetic barrier to resistance and low potential for drug-drug interaction. Administered with a non-structural protein 3 (NS3) protease inhibitor (asunaprevir, ASV) with or without a non-nucleoside NS5B polymerase inhibitor (beclabuvir, BCV), or a nucleotide NS5B polymerase inhibitor (sofosbuvir, SOF), DCV is able to achieve greater than a 90-% HCV eradication rate in both treatment-naïve and treatment-experienced patients with GT 1. A triple combination regimen with DCV/ASV/BCV results in 100% sustained virologic response (SVR) rates in HCV GT 4 treatment-naïve subjects. DCV/SOF combination also had demonstrated up to 90-% SVR rates in GT 3-infected non-cirrhotic patients. The efficacy and safety of DCV-containing all-oral regimens highlight a new era of interferon-free therapy for chronic hepatitis C. PMID:26542068

  11. Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes.

    Science.gov (United States)

    Fournier, Carole; Helle, François; Descamps, Véronique; Morel, Virginie; François, Catherine; Dedeurwaerder, Sarah; Wychowski, Czeslaw; Duverlie, Gilles; Castelain, Sandrine

    2013-05-01

    A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines. PMID:23288424

  12. Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

    Directory of Open Access Journals (Sweden)

    Vlastimil Jirasko

    Full Text Available Non-structural protein 2 (NS2 plays an important role in hepatitis C virus (HCV assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

  13. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

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

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

    International Nuclear Information System (INIS)

    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

  16. Stability of linear dilaton black holes at the Hagedorn temperature

    CERN Document Server

    Bertoldi, Gaetano

    2009-01-01

    We study the thermodynamics and the small fluctuations of a linear dilaton black hole, in an S-dual version of the near-horizon limit of type IIB NS5 branes. The thermodynamical analysis shows that the black hole is in a Hagedorn phase, with marginal stability. The dynamical analysis confirms that the speed of sound of the dual theory vanishes and that there are no evident instabilities at the level of supergravity. We clarify the physical meaning of some singularities of the retarded correlator that were thought to lead to instabilities.

  17. Stability of linear dilaton black holes at the Hagedorn temperature

    International Nuclear Information System (INIS)

    We study the thermodynamics and the small fluctuations of a linear dilaton black hole, in an S-dual version of the near-horizon limit of type IIB NS5 branes. The thermodynamical analysis shows that the black hole is in a Hagedorn phase, with marginal stability. The dynamical analysis confirms that the speed of sound of the dual theory vanishes and that there are no evident instabilities at the level of supergravity. We clarify the physical meaning of some singularities of the retarded correlator that were thought to lead to instabilities.

  18. Brane brick models, toric Calabi-Yau 4-folds and 2d (0,2) quivers

    Science.gov (United States)

    Franco, Sebastián; Lee, Sangmin; Seong, Rak-Kyeong

    2016-02-01

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

  19. Twistor approach to string compactifications: A review

    International Nuclear Information System (INIS)

    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.

  20. Human Dengue Antibodies against Structural and Nonstructural Proteins

    OpenAIRE

    Valdés, Katia; Alvarez, Mayling; Pupo, Maritza; Vázquez, Susana; Rodríguez, Rayner; Guzmán, María G.

    2000-01-01

    Antibodies against dengue virus type 2 and 4 proteins in acute-phase sera of 10 primary and 10 secondary dengue fever and dengue hemorrhagic fever patients were studied by Western blotting. In the first group the immune response was barely detectable, while in the second group more proteins were detected, with a very strong reaction. Anti-NS1 and -NS3 antibodies were detected mainly in secondary cases. Anti-E, -NS3, and -NS5 antibodies were detected in a high number of cases. The possibility ...

  1. Comparative Amino Acid Sequences of Dengue Viruses

    OpenAIRE

    Haishi, Shozo; TANAKA Mariko; Igarashi, Akira

    1990-01-01

    Amino acid (AA) sequences of 4 serotype of dengue viruses deduced from their nucleotide (nt) sequences of genomic RNA were analyzed for each genome segment and each stretch of 10 AA residues. Precursor of membrane protein (pM), and 4 nonstructural proteins (NS1, NS3, NS4B, NS5) were highly conserved, while another nonstructural protein (NS2A) was least conserved among 5 strains of dengue viruses. When homology was compared among heterotypic viruses, type 1 and type 3 dengue viruses showed clo...

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

    OpenAIRE

    Benvenuti, Sergio; Pasquetti, Sara

    2016-01-01

    D3 branes stretching between webs of (p,q) 5branes provide an interesting class of 3d N=2 theories. For generic pq-webs however the low energy field theory is not known. We use 3d mirror symmetry and Type IIB S-duality to construct Abelian gauge theories corresponding to D3 branes ending on both sides of a pq-web made of many coincident NS5's intersecting one D5. These theories contain chiral monopole operators in the superpotential and enjoy a non trivial pattern of global symmetry enhanceme...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    transfusions several years before. One of her blood donors was identified and tested negative for anti-HCV and HCV RNA, as were her husband and offspring. Phylogenetic analysis of the E1 and NS5B regions confirmed that this HCV strain belonged to genotype 5a. However, the E1 region analysis indicates that our......Genotype 5 of hepatitis C virus (HCV) has been rarely identified in South America. A female of African descent who never left Brazil was found to be infected by this genotype in Mato Grosso state, Central Brazil. The patient denied drug injections and revealed that she had received blood...

  4. Elliptic Genus of E-strings

    OpenAIRE

    Kim, Joonho; Kim, Seok; Lee, Kimyeong; Park, Jaemo; Vafa, Cumrun

    2014-01-01

    We study a family of 2d N=(0,4) gauge theories which describes at low energy the dynamics of E-strings, the M2-branes suspended between a pair of M5 and M9 branes. The gauge theory is engineered using a duality with type IIA theory, leading to the D2-branes suspended between an NS5-brane and 8 D8-branes on an O8-plane. We compute the elliptic genus of this family of theories, and find agreement with the known results for single and two E-strings. The partition function can in principle be com...

  5. Novel Branches of (0,2) Theories

    CERN Document Server

    Quigley, Callum; Stern, Mark

    2012-01-01

    We show that recently proposed linear sigma models with torsion can be obtained from unconventional branches of conventional gauge theories. This observation puts models with log interactions on firm footing. If non-anomalous multiplets are integrated out, the resulting low-energy theory involves log interactions of neutral fields. For these cases, we find a sigma model geometry which is both non-toric and includes brane sources. These are heterotic sigma models with branes. Surprisingly, there are massive models with compact complex non-Kahler target spaces, which include brane/anti-brane sources. The simplest conformal models describe wrapped heterotic NS5-branes. We present examples of both types.

  6. Comments on s-rule violating configurations in field theory

    CERN Document Server

    Cottrell, William; Pillai, Mohandas

    2015-01-01

    We explicitly construct a configuration of ${\\cal N}=4$ supersymmetry Yang-Mills theory with gauge group $U(N)$ on an interval on length $L$ with a D5-like boundary condition on one end and an NS5-like boundary condition on the other. For $N>1$, such a configuration violates the $s$-rule and is non-supersymmetric. We compute the energy relative to the BPS bound of these configurations and find that it is proportional to $N(N^2-1) g_{YM4}^{-2} L^{-3}$.

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

    Science.gov (United States)

    Alexandrov, Sergei; Banerjee, Sibasish

    2013-10-01

    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.

  8. Modularity, Quaternion-Kahler spaces and Mirror Symmetry

    CERN Document Server

    Alexandrov, Sergei

    2013-01-01

    We provide an explicit twistorial construction of quaternion-Kahler 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.

  9. Core as a Novel Viral Target for Hepatitis C Drugs

    OpenAIRE

    Guillaume Mousseau; Snyder, John K.; Arthur Donny Strosberg; Virginia Takahashi; Smitha Kota

    2010-01-01

    Hepatitis C virus (HCV) infects over 130 million people worldwide and is a major cause of liver disease. No vaccine is available. Novel specific drugs for HCV are urgently required, since the standard-of-care treatment of pegylated interferon combined with ribavirin is poorly tolerated and cures less than half of the treated patients. Promising, effective direct-acting drugs currently in the clinic have been described for three of the ten potential HCV target proteins: NS3/NS4A protease, NS5B...

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

    CERN Document Server

    Franco, Sebastian; Seong, Rak-Kyeong

    2015-01-01

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

  11. Sofosbuvir treatment and hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

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

  12. 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.

  13. 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.

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

    OpenAIRE

    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. Seq...

  15. Discovery of Metal Ions Chelator Quercetin Derivatives with Potent Anti-HCV Activities

    Directory of Open Access Journals (Sweden)

    Dongwei Zhong

    2015-04-01

    Full Text Available Analogues or isosteres of α,γ-diketoacid (DKA 1a show potent inhibition of hepatitis C virus (HCV NS5B polymerase through chelation of the two magnesium ions at the active site. The anti-HCV activity of the flavonoid quercetin (2 could partly be attributed to it being a structural mimic of DKAs. In order to delineate the structural features required for the inhibitory effect and improve the anti-HCV potency, two novel types of quercetin analogues, 7-O-arylmethylquercetins and quercetin-3-O-benzoic acid esters, were designed, synthesized and evaluated for their anti-HCV properties in cell-based assays. Among the 38 newly synthesized compounds, 7-O-substituted derivative 3i and 3-O-substituted derivative 4f were found to be the most active in the corresponding series (EC50 = 3.8 μM and 9.0 μΜ, respectively. Docking studies suggested that the quercetin analogues are capable of establishing key coordination with the two magnesium ions as well as interactions with residues at the active site of HCV NS5B.

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

    International Nuclear Information System (INIS)

    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

  17. Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors.

    Science.gov (United States)

    De Luca, Andrea; Di Giambenedetto, Simona; Lo Presti, Alessandra; Sierra, Saleta; Prosperi, Mattia; Cella, Eleonora; Giovanetti, Marta; Torti, Carlo; Caudai, Cinzia; Vicenti, Ilaria; Saladini, Francesco; Almi, Paolo; Grima, Pierfrancesco; Blanc, Pierluigi; Fabbiani, Massimiliano; Rossetti, Barbara; Gagliardini, Roberta; Kaiser, Rolf; Ciccozzi, Massimo; Zazzi, Maurizio

    2015-04-01

    Background.  Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods.  Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results.  All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions.  Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I. PMID:26213689

  18. Approved Antiviral Drugs over the Past 50 Years.

    Science.gov (United States)

    De Clercq, Erik; Li, Guangdi

    2016-07-01

    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

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

    Directory of Open Access Journals (Sweden)

    Jong-Woo Kim

    2013-01-01

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

  20. First Description of Hepacivirus and Pegivirus Infection in Domestic Horses in China: A Study in Guangdong Province, Heilongjiang Province and Hong Kong District.

    Science.gov (United States)

    Lu, Gang; Sun, Lingshuang; Xu, Tao; He, Dong; Wang, Zengchao; Ou, Shudan; Jia, Kun; Yuan, Liguo; Li, Shoujun

    2016-01-01

    Since 2012, three viruses, known as equine hepacivirus (EqHV), equine pegivirus (EPgV) and Theiler's disease-associated virus (TDAV), have been discovered in equines. Given that these viruses are the newest members of the Flaviviridae family, genomic information concerning circulating EqHV, EPgV and TDAV strains around the world is limited. To date, no genetic surveillance studies have been performed on these three viruses in the equine population of China. Here, a total of 177 serum samples were collected from equines across China between 2014 and 2015. Using PCR, we detected viral RNA in the serum samples, six of which were EqHV positive and two of which were EPgV positive. Co-infection with the two viruses was not observed among the Chinese equines studied, and TDAV RNA was not detected in the equine serum samples collected for this study. Phylogenetic analysis of partial NS5B open reading frame (ORF), NS3 ORF, and 5' untranslated region nucleotide sequences from EqHV as well as partial NS3 ORF sequence from EPgV indicated that EqHV and EPgV have evolved into two main clades by themselves, both of which are circulating in China. Based on the partial NS5B and NS3 ORF sequences of EqHV, the sequences of one clade were also split into two subclades. This study enriches our knowledge of the geographic distribution of these three equine viruses. PMID:27182887

  1. Core as a Novel Viral Target for Hepatitis C Drugs

    Directory of Open Access Journals (Sweden)

    Guillaume Mousseau

    2010-08-01

    Full Text Available Hepatitis C virus (HCV infects over 130 million people worldwide and is a major cause of liver disease. No vaccine is available. Novel specific drugs for HCV are urgently required, since the standard-of-care treatment of pegylated interferon combined with ribavirin is poorly tolerated and cures less than half of the treated patients. Promising, effective direct-acting drugs currently in the clinic have been described for three of the ten potential HCV target proteins: NS3/NS4A protease, NS5B polymerase and NS5A, a regulatory phosphoprotein. We here present core, the viral capsid protein, as another attractive, non-enzymatic target, against which a new class of anti-HCV drugs can be raised. Core plays a major role in the virion’s formation, and interacts with several cellular proteins, some of which are involved in host defense mechanisms against the virus. This most conserved of all HCV proteins requires oligomerization to function as the organizer of viral particle assembly. Using core dimerization as the basis of transfer-of-energy screening assays, peptides and small molecules were identified which not only inhibit core-core interaction, but also block viral production in cell culture. Initial chemical optimization resulted in compounds active in single digit micromolar concentrations. Core inhibitors could be used in combination with other HCV drugs in order to provide novel treatments of Hepatitis C.

  2. Dengue Virus Control of Type I IFN Responses: A History of Manipulation and Control.

    Science.gov (United States)

    Castillo Ramirez, Jorge Andrés; Urcuqui-Inchima, Silvio

    2015-06-01

    The arthropod-borne diseases caused by dengue virus (DENV) are a major and emerging problem of public health worldwide. Infection with DENV causes a series of clinical manifestations ranging from mild flu syndrome to severe diseases that include hemorrhage and shock. It has been demonstrated that the innate immune response plays a key role in DENV pathogenesis. However, in recent years, it was shown that DENV evades the innate immune response by blocking type I interferon (IFN-I). It has been demonstrated that DENV can inhibit both the production and the signaling of IFN-I. The viral proteins, NS2A and NS3, inhibit IFN-I production by degrading cellular signaling molecules. In addition, the viral proteins, NS2A, NS4A, NS4B, and NS5, can inhibit IFN-I signaling by blocking the phosphorylation of the STAT1 and STAT2 molecules. Finally, NS5 mediates the degradation of STAT2 using the proteasome machinery. In this study, we briefly review the most recent insights regarding the IFN-I response to DENV infection and its implication for pathogenesis. PMID:25629430

  3. 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

  4. 3d N=2 mirror symmetry, pq-webs and monopole superpotentials

    CERN Document Server

    Benvenuti, Sergio

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Samian, R.

    2005-01-01

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

  6. 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

  7. Sofosbuvir/velpatasvir: A promising combination.

    Science.gov (United States)

    Bonaventura, Aldo; Montecucco, Fabrizio

    2016-07-01

    Hepatitis C virus (HCV) affects 3% of the world population. It represents the main cause of chronic liver disease and is responsible for extra-hepatic complications, such as type 2 diabetes and cardiovascular diseases. HCV includes 7 genotypes differing in the nucleotide sequence variability, the geographic distribution, the rates of viral clearance, the risk of progression to liver fibrosis and to hepatocellular carcinoma, and the response to therapy. Last years have seen remarkable advances in the field of HCV infection with the approval of direct antiviral agents (DAAs) targeting key viral proteins involved in the HCV replication. Several oral regimens combining DAAs from different families have been developed and these regimens showed increased and sustained virological response rates to above 90% reducing the treatment duration to 12 wk or less. In particular, sofosbuvir, a nucleotide analogue nonstructural (NS)5B polymerase inhibitor, and velpatasvir, a NS5A inhibitor, have been tested in two phase 3 trials, the ASTRAL-2 (against HCV genotype 2) and the ASTRAL-3 (against HCV genotype 3), demonstrating to be effective, safe, and well tolerated in patients who were 18 years of age or older and had at least a 6-mo history of HCV infection with a compensated liver disease. PMID:27429714

  8. 2001 spring school on superstrings and related matters

    International Nuclear Information System (INIS)

    This proceedings contains the lectures given at the 2001 Trieste Spring School on String Theory. Several important and active areas of research in string theory related topics were covered in this school. One of the main topics of the School was the recently conjectured duality between gauge theory living on D-branes and and gravity (or more precisely string theory) living in the near horizon geometry around the D-branes. J. Maldacena gave a set of lectures on the gauge theory/gravity duality in different examples. M. Strassler's lectures dealt with a very interesting generalization of the gauge theory/gravity duality for the case of a confining gauge theory. D. Kutasov's lectures dealt with Little String Theories (LST) that are supposed to describe the physics of the NS5-branes. Using the holographic principle, interesting features of LST were deduced by describing the string theory in the background of NS5-branes. E. Verlinde gave a set of lectures on holographic principle in the context of radiation dominated FRW universe. Other topics included lectures by R. Gopakumar on the solitons in non-commutative gauge theories that are relevant in the context of D-branes in the background on anti-symmetric tensor field, and lectures by M. Douglas on D-branes on Calabi-Yau spaces

  9. 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.

  10. 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.

  11. New pseudodimeric aurones as palm pocket inhibitors of Hepatitis C virus RNA-dependent RNA polymerase.

    Science.gov (United States)

    Meguellati, Amel; Ahmed-Belkacem, Abdelhakim; Nurisso, Alessandra; Yi, Wei; Brillet, Rozenn; Berqouch, Nawel; Chavoutier, Laura; Fortuné, Antoine; Pawlotsky, Jean-Michel; Boumendjel, Ahcène; Peuchmaur, Marine

    2016-06-10

    The NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme for Hepatitis C Virus (HCV) replication. In addition to the catalytic site, this enzyme is characterized by the presence of at least four allosteric pockets making it an interesting target for development of inhibitors as potential anti-HCV drugs. Based on a previous study showing the potential of the naturally occurring aurones as inhibitors of NS5B, we pursued our efforts to focus on pseudodimeric aurones that have never been investigated so far. Hence, 14 original compounds characterized by the presence of a spacer between the benzofuranone moieties were synthesized and investigated as HCV RdRp inhibitors by means of an in vitro assay. The most active inhibitor, pseudodimeric aurone 4, induced high inhibition activity (IC50 = 1.3 μM). Mutagenic and molecular modeling studies reveal that the binding site for the most active derivatives probably is the palm pocket I instead of the thumb pocket I as for the monomeric derivatives. PMID:27017550

  12. 2015 Philip S. Portoghese Medicinal Chemistry Lectureship. Curing Hepatitis C Virus Infection with Direct-Acting Antiviral Agents: The Arc of a Medicinal Chemistry Triumph.

    Science.gov (United States)

    Meanwell, Nicholas A

    2016-08-25

    The development of direct-acting antiviral agents that can cure a chronic hepatitis C virus (HCV) infection after 8-12 weeks of daily, well-tolerated therapy has revolutionized the treatment of this insidious disease. In this article, three of Bristol-Myers Squibb's HCV programs are summarized, each of which produced a clinical candidate: the NS3 protease inhibitor asunaprevir (64), marketed as Sunvepra, the NS5A replication complex inhibitor daclatasvir (117), marketed as Daklinza, and the allosteric NS5B polymerase inhibitor beclabuvir (142), which is in late stage clinical studies. A clinical study with 64 and 117 established for the first time that a chronic HCV infection could be cured by treatment with direct-acting antiviral agents alone in the absence of interferon. The development of small molecule HCV therapeutics, designed by medicinal chemists, has been hailed as "the arc of a medical triumph" but may equally well be described as "the arc of a medicinal chemistry triumph". PMID:27501244

  13. 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.

  14. Structure-Based Discovery of Novel Cyclophilin A Inhibitors for the Treatment of Hepatitis C Virus Infections.

    Science.gov (United States)

    Yang, Suhui; K R, Jyothi; Lim, Sangbin; Choi, Tae Gyu; Kim, Jin-Hwan; Akter, Salima; Jang, Miran; Ahn, Hyun-Jong; Kim, Hee-Young; Windisch, Marc P; Khadka, Daulat B; Zhao, Chao; Jin, Yifeng; Kang, Insug; Ha, Joohun; Oh, Byung-Chul; Kim, Meehyein; Kim, Sung Soo; Cho, Won-Jea

    2015-12-24

    Hepatitis C virus (HCV) is a major cause of end-stage liver disease. Direct-acting antivirals (DAAs), including inhibitors of nonstructural proteins (NS3/4A protease, NS5A, and NS5B polymerase), represent key components of anti-HCV treatment, but these are associated with increased drug resistance and toxicity. Thus, the development of host-targeted antiviral agents, such as cyclophilin A inhibitors, is an alternative approach for more effective, selective, and safer treatment. Starting with the discovery of a bis-amide derivative 5 through virtual screening, the lead compound 25 was developed using molecular modeling-based design and systematic exploration of the structure-activity relationship. The lead 25 lacked cytotoxicity, had potent anti-HCV activity, and showed selective and high binding affinity for CypA. Unlike cyclosporin A, 25 lacked immunosuppressive effects, successfully inhibited the HCV replication, restored host immune responses without acute toxicity in vitro and in vivo, and exhibited a high synergistic effect in combination with other drugs. These findings suggest that the bis-amides have significant potential to extend the arsenal of HCV therapeutics. PMID:26613291

  15. Non-geometric Five-branes in Heterotic Supergravity

    CERN Document Server

    Sasaki, Shin

    2016-01-01

    We study T-duality chains of five-branes in heterotic supergravity where the first order $\\alpha'$-corrections are present. By performing the $\\alpha'$-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 $\\alpha'$-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 $\\mathcal{O} (\\alpha')$. On the other hand, the gauge $5^2_2$-brane solution is not a T-fold but show unusual structur...

  16. Identifiction and Detection of Classical Swine Fever Virus in A Pig Farm of Xinxiang%新乡市某猪场猪瘟病毒的鉴别检测

    Institute of Scientific and Technical Information of China (English)

    孔令芸; 陈玲丽; 李冲; 孙相和; 李鹏; 王选年; 银梅

    2015-01-01

    新乡市某猪场发生疑似猪瘟病例,为了鉴别其为疫苗毒还是野毒,采用细胞免疫化学方法和RT-PCR,并对其NS5B基因进行序列测定,序列比对并构建进化树,结果表明,猪只为猪瘟野毒感染,且分离的猪瘟病毒与石门(Shimen)株在基因序列上未发生大的变异。%The suspicious cases of classical swine fever occurred in a pig farm of Xinxiang.The virus was i-dentified by cell immunochemistry method and RT-PCR.The results of the sequence alignment and phylo-genetic tree of NS5B gene showed that the pigs were infected with classical swine fever virus field strain, and the gene sequence variation of this isolate was not obvious.

  17. 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...

  18. 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

    , containing 13 amino acid changes (R114W and V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, and I2340T [NS5A]; A2500S and V2841A [NS5B]), displayed fitness comparable to that of the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD...... 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...... severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high...

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

    Directory of Open Access Journals (Sweden)

    Sadaf Badar

    2012-01-01

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

  20. 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.

  1. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2005-11-01

    Hepatitis C virus infects humans world-wide. The virus genome varies greatly and it has several genotypes. HCV infection is highly prevalent in Central Africa and Cameroon. Initial studies on the genetic variability of HCV showed infection with HCV genotypes 1, 2, and 4. We have now sequenced the NS5b and E2 regions of 156 HCV isolates collected from patients presenting for diagnosis in Yaounde and used the data to describe the distribution of HCV genotypes and subtypes in patients with hepatitis in Cameroon. Genotype 1 was more frequent than Genotypes 4 and 2. Genotypes 1 and 4 were highly heterogeneous, containing many subtypes described previously (1b, 1c, 1e, 1h, 1l, 4f, 4t, 4p, 4k) and unsubtyped groups. There was a systematic phylogenetic concordance between NS5b and E2 sequence clustering. The Genotype 2 sequences did not vary. Neither subject age nor gender influenced HCV distribution. HCV Genotypes 1 and 4 are very heterogeneous in Cameroon, perhaps due to ancient infections. The homogeneity of HCV Genotype 2 indicates its more recent introduction from western Africa. PMID:16173014

  4. 4d N=2 superconformal linear quivers with type IIA duals

    CERN Document Server

    Aharony, Ofer; Berkooz, Micha

    2012-01-01

    We discuss the gravity duals of 4d N=2 superconformal field theories (SCFTs) arising from the low-energy limit of brane configurations of D4-branes stretched between and intersecting NS5-branes and D6-branes. This gives rise to a product of SU(N_i) groups, with bi-fundamental matter between adjacent groups, and extra fundamental hypermultiplets. The most general configuration in 11d (or type IIA) supergravity that is dual to a 4d N=2 SCFT (when the dual of this SCFT is a weakly curved background) was written down by Gaiotto and Maldacena, but finding it explicitly involves solving a complicated Toda equation. This equation simplifies only when the solution can be reduced to type IIA supergravity, so we ask for which SCFTs of this type is there a type IIA dual that is weakly coupled and weakly curved (away from NS5-branes and D6-branes). We find that such solutions (a special case of which was analyzed by Reid-Edwards and Stefanski) exist when there is a large number of gauge groups, with large ranks, and with...

  5. Heterotic/type II triality and instantons on K3

    International Nuclear Information System (INIS)

    A detailed understanding of instanton effects for half-BPS couplings is pursued in theories with 16 supersymmetries. In particular, we investigate the duality between heterotic string on T4 and type IIA on K3 at the T4/Zint2 orbifold point, as well as their higher and lower dimensional versions. We present a remarkably clean quantitative test of the duality at the level of F-circumflex 4 couplings, by completely matching a purely one-loop heterotic amplitude to a purely tree-level type II result. The triality of SO(4,4) and several other miracles are shown to be crucial for the duality to hold. Exact non-perturbative new results for type I', F on K3, M on K3, and IIB on K3 are found, and the general form of D-instanton contributions in type IIA or B on T4/Zint2 is obtained. We also analyze the NS5-brane contributions in type II on K3 X T2, and predict the value mu (N)=sum(d|N) (1/d3) for the bulk contribution to the index of the NS5-brane world-volume theory on K3 X T2. (author)

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

    Science.gov (United States)

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

    2016-02-01

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

  7. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle

    Directory of Open Access Journals (Sweden)

    Changhyun Roh

    2010-04-01

    Full Text Available Changhyun Roh1, Ho-Young Lee2, Sang-Eun Kim2, Sung-Kee Jo11Radiation Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Sinjeong-dong, Jeongeup, Jeonbuk, South Korea; 2Department of Nuclear Medicine, College of Medicine, Seoul National University, South KoreaAbstract: Globally, approximately 170 million people (representing approximately 3% of the population worldwide, are infected with hepatitis C virus (HCV and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinkerTM glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL-1 to 1 ng mL-1 range with a detection limit of 1 ng mL-1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well.Keywords: hepatitis C virus, viral protein, RNA oligonucleotide, quantum dots, biochip

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Transmission of hepatitis C virus among intravenous drug users in the Uppsala region of Sweden

    Directory of Open Access Journals (Sweden)

    Axel Danielsson

    2014-01-01

    Full Text Available Background: Epidemiology and transmission patterns of hepatitis C virus (HCV are important subjects as we enter a new era of treatment with directly acting antivirals (DAAs. The highest prevalence of HCV in developed countries is found among intravenous drug users (IDUs, where unsafe needle sharing practices provide the main route of infection. Efforts to prohibit the continuous spread of HCV among these groups have been initiated by the community services and health care providers. Our goal was to understand how HCV was transmitted among IDUs within a limited population group. We provide a retrospective study (2005–2007 of the HCV transmission patterns in a population of IDUs in the Uppsala region of Sweden. Method: Eighty-two serum samples were collected from IDUs in Uppsala County. Our reverse transcription nested polymerase chain reaction (RT-nested PCR and sequencing method enabled a comprehensive genetic analysis for a broad spectrum of genotypes of two relatively conserved regions, NS5B and NS3, that encodes for the viral polymerase and protease, respectively. HCV RNA in serum samples was amplified and sequenced with in-house primers. Sequence similarities between individuals and subgroups were analyzed with maximum likelihood (ML phylogenetic trees. Published HCV reference sequences from other geographic regions and countries were also included for clarity. Results: Phylogenetic analysis was possible for 59 NS5B (72% and 29 NS3 (35% sequences from Uppsala patients. Additionally, we also included 15 NS3 sequences from Örebro patients, making a total of 44 NS3 sequences for the analysis. By analyzing the NS3 sequences, two transmission sets were found between the IDUs (>98% sequence identity, with one set consisting of two individuals and another set consisting of three individuals. In addition, the phylogenetic analysis done with our serum samples displayed clusters that distinguished them from the reference sequences. Conclusion: Our

  10. Time-resolved investigation of nanosecond discharge in dense gas sustained by short and long high-voltage pulse

    Science.gov (United States)

    Yatom, S.; Gleizer, J. Z.; Levko, D.; Vekselman, V.; Gurovich, V.; Hupf, E.; Hadas, Y.; Krasik, Ya. E.

    2011-12-01

    The results of experimental and numerical studies of the generation of runaway electrons (RAE) in a pressurized air-filled diode under the application of 20 ns, 5 ns and 1 ns duration high-voltage pulses with an amplitude up to 160 kV are presented. It is shown that with a 1 ns pulse, RAE with energy >=20 keV reach the anode prior to the formation of the plasma channel between the cathode and anode. Conversely, with 20 ns or 5 ns pulses, RAE with energy >=20 keV were obtained at the anode only after the formation of the plasma channel. In addition, the high- and low-impedance stages of the development of the discharge were found. Finally, a comparison between experimental and numerical simulation results is presented.

  11. High-fidelity continuous-variable quantum teleportation toward multistep quantum operations

    International Nuclear Information System (INIS)

    The progress in quantum operations of continuous-variable (CV) schemes can be reduced to that in CV quantum teleportation. The fidelity of quantum teleportation of an optical setup is limited by the finite degree of quantum correlation that can be prepared with a pair of finitely squeezed states. Reports of improvement of squeezing level have appeared recently, and we adopted the improved methods in our experimental system of quantum teleportation. As a result, we teleported a coherent state with a fidelity F=0.83±0.01, which is better than any other figures reported to date, to our knowledge. In this paper, we introduce a measure ns, the number of teleportations expected to be carried out sequentially. Our result corresponds to ns=5.0±0.4. It suggests that our improvement would enable us to proceed toward more advanced quantum operations involving multiple steps

  12. Brane polarization is no cure for tachyons

    CERN Document Server

    Bena, Iosif

    2015-01-01

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

  13. 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.

  14. Heterotic-Type IIA Duality and Degenerations of K3 Surfaces

    CERN Document Server

    Braun, Andreas P

    2016-01-01

    We study the duality between four-dimensional N=2 compactifications of Heterotic and Type IIA string theories. Via adiabatic fibration of the duality in six dimensions, Type IIA string theory compactified on a K3-fibred Calabi-Yau threefold has a potential heterotic dual compactification. This adiabatic picture fails whenever the K3 fibre degenerates into multiple components. Guided by monodromy, we identify such degenerate K3 fibres as solitons generalizing the NS5-brane in Heterotic string theory. The theory of degenerations of K3 surfaces can then be used to find which solitons can be present on the heterotic side. Similar to small instanton transitions, these solitons convoy singular transitions between different Calabi-Yau threefolds. Starting from well-known examples of Heterotic-Type IIA duality, such transitions can take us to Type IIA compactifications with unknown heterotic duals.

  15. Probing the Hydrodynamic Limit of (Super)gravity

    CERN Document Server

    Di Dato, Adriana; Pedersen, Andreas Vigand

    2015-01-01

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

  16. Cordes et D-branes dans les espaces-temps courbes

    CERN Document Server

    Ribault, S

    2003-01-01

    This thesis is devoted to the construction and study of D-branes in some curved space-times in string theory. On the one hand, those D-branes are described geometrically as submanifolds subject to Born-Infeld effective dynamics. On the other hand, they can be built microscopically using boundary conformal field theory. We use and compare those two approaches. We also improve them technically : we rewrite Born-Infeld dynamics in a gauge-invariant way, and formulate precise analyticity requirements for the density of open strings on certain D-branes. Our results include the effective description of symmetric D-branes in compact groups, the determination of the complete spectrum of open strings on AdS2 D-branes in AdS3, the exact construction of some D-branes in the cigar SL(2)/U(1), and a geometric description of all D3-branes in NS5-brane backgrounds.

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

    DEFF Research Database (Denmark)

    Pham, Long; Ngo, HT; Lim, YS;

    2012-01-01

    Background & Aims Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc......)-infected cells. In the current study, we have selected and characterized the gene encoding Cyclin A2 (CycA2). Deregulation of CycA2 has been implicated in many types of cancers, including hepatocellular carcinoma. Methods The effects of CycA2 on HCV propagation were investigated by siRNA-mediated knockdown assay......, in vitro and in vivo protein binding assays, luciferase reporter gene assay, and immunoblot assay. Results We showed that siRNA-mediated depletion of CycA2 significantly inhibited HCV replication in both HCV subgenomic replicon cells and HCVcc-infected cells. Furthermore, HCV non-structural 5B (NS5B...

  18. Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Kazi Abdus Salam

    2013-01-01

    Full Text Available Currently, hepatitis C virus (HCV infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin. The new therapy has significantly improved sustained virologic response (SVR; however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.

  19. 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.

  20. A new 6d fixed point from holography

    CERN Document Server

    Apruzzi, Fabio; Tizzano, Luigi

    2016-01-01

    We propose a stringy construction giving rise to a class of interacting and non-supersymmetric CFT's in six dimensions. Such theories may be obtained as an IR conformal fixed point of an RG flow ending up in a $(1, 0)$ theory in the UV. We provide the due holographic evidence in the context of massive type IIA on $\\textrm {AdS}_{7}\\times M_3$, where $M_3$ is topologically an $S^3$. In particular, in this paper we present a 10d flow solution which may be interpreted as a non-BPS bound state of NS5, D6 and $\\overline{\\textrm{D6}}$ branes. Moreover, by adopting its 7d effective desciption, we are able to holographically compute the free energy and the operator spectrum in the novel IR conformal fixed point.

  1. 2d (0,2) Quiver Gauge Theories and D-Branes

    CERN Document Server

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

    2015-01-01

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

  2. Pathogenicity and genetic characterization of a duck Tembusu virus associated with egg-dropping in Muscovy ducks.

    Science.gov (United States)

    Shen, Han-Qin; Lin, Wen-Cheng; Wang, Zhan-Xin; Zhang, Kai; Yan, Zhuan-Qiang; Zhou, Qing-Feng; Qin, Jian-Ping; Xie, Qing-Mei; Bi, Ying-Zuo; Chen, Feng

    2016-09-01

    Duck Tembusu virus (DTMUV) has spread to the major duck-farming region in China, causing acute egg-production drop in Chinese duck population. In this study, we characterized a DTMUV strain (named GD2014) isolated from an egg-production drop duck farm in Guangdong province, South China. The virus was pathogenic to Muscovy duck embryos and caused severe egg production drop for laying Muscovy ducks. The genome sequence of GD2014 shared 97-99% homologies with other waterfowl-origin Tembusu viruses, and shared 89% identities with MM1775 strain isolated from mosquito. Phylogenetic analysis of entire open reading frame (ORF), E gene and NS5 gene indicated that GD2014 belonged to Ntaya group. These results have implications for understanding the orgin, emergence and pathogenicity of DTMUV as well as for the development of vaccines and diagnostics based on epidemiological data. PMID:27354303

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

    CERN Document Server

    Fiorenza, Domenico; Schreiber, Urs

    2016-01-01

    We show that supercocycles on super $L_\\infty$-algebras capture, at the rational level, the twisted cohomological charge structure of the fields of M-theory and of type IIA string theory. We show that rational 4-sphere-valued supercocycles for M-branes in M-theory descend to supercocycles in type IIA string theory to yield the Ramond-Ramond fields predicted by the rational image of twisted K-theory, with the twist given by the B-field. In particular, we derive the M2/M5 $\\leftrightarrow$ F1/Dp/NS5 correspondence via dimensional reduction of sphere-valued $L_\\infty$ supercocycles in rational homotopy theory.

  4. Generation of a synchronized pulse of extraordinary precision using chirped pulse laser

    International Nuclear Information System (INIS)

    We have developed the generation system of synchronized pulses of high precision using chirped pulse laser. The PW laser is synchronized to Gekko XII beams within 10 picosecond by injecting part of the PW laser into the Gekko XII laser system. A part of the 3 ns/6 nm (pulse width/spectral width) output from the front end is stretched to 5.5 ns/5 nm and is then sliced to 1.1 ns/nm width and injected into Gekko XII system. We have obtained 2.5-kJ output energy at a 532-nm wavelength from 12 semi-Gaussian beams. The pulse width is 1.1 ± 0.1 ns (FWHM) and the conversion efficiency from 1 to 0.5 mm was 43%. (author)

  5. Ecdysones from Zoanthus spp. with inhibitory activity against dengue virus 2.

    Science.gov (United States)

    Cheng, Yuan-Bin; Lee, Jin-Ching; Lo, I-Wen; Chen, Shu-Rong; Hu, Hao-Chun; Wu, Yu-Hsuan; Wu, Yang-Chang; Chang, Fang-Rong

    2016-05-01

    Bioassay-guided fractionation of an ethanolic extract of Zoanthus spp. collected in Taiwan has resulted in the isolation of one new ecdysone, zoanthone A (1), along with thirteen known compounds (2-14). The structures of these compounds were determined by spectroscopic methods, especially 2D NMR analyses. The in vitro antiviral activities of all isolated ecdysones (1-14) against dengue virus type 2 (DENV-2) were evaluated using DENV infectious system. New compound (1) exhibited potent antiviral activity (EC50=19.61±2.46μM) with a selectivity index (CC50/EC50) value of 36.7. The structure-activity relationships of isolated ecdysones against DENV-2 were concluded. Molecular docking information of 3 and NS5 polymerase was performed either. PMID:26988299

  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. Heterotic-type IIA duality and degenerations of K3 surfaces

    Science.gov (United States)

    Braun, A. P.; Watari, T.

    2016-08-01

    We study the duality between four-dimensional N = 2 compactifications of heterotic and type IIA string theories. Via adiabatic fibration of the duality in six dimensions, type IIA string theory compactified on a K3-fibred Calabi-Yau threefold has a potential heterotic dual compactification. This adiabatic picture fails whenever the K3 fibre degenerates into multiple components over points in the base of the fibration. Guided by monodromy, we identify such degenerate K3 fibres as solitons generalizing the NS5-brane in heterotic string theory. The theory of degenerations of K3 surfaces can then be used to find which solitons can be present on the heterotic side. Similar to small instanton transitions, these solitons escort singular transitions between different Calabi-Yau threefolds. Starting from well-known examples of heterotic-type IIA duality, such transitions can take us to type IIA compactifications with unknown heterotic duals.

  8. Electrochemical noise evaluation of anodized aluminum. Comparative study against corrosion behaviour in the atmosphere

    International Nuclear Information System (INIS)

    The present work reports the evaluation of aluminum and anodized aluminum by electrochemical noise, as a part of the PATINE/CYTED project of the working group NS5. A visual examination is also made. The samples were exposed at several Ibero-American atmospheres up to 2 years of exposure. Different thickness of anodized aluminum were evaluated. The electrochemical potential noise of the 5 μm unexposed sample (pattern) showed a different behaviour to that showed by the other anodized specimens. This could be due to a slower sealed of the samples of higher thickness. The same behavior was observed on the samples exposed at the rural station. el Pardo. According to the visual examination, the samples of bare aluminum and those of anodized 5 μm thickness were the most affected by pitting corrosion in the highly polluted atmospheres. A good correlation between corrosion behaviour determined by visual examination and EN was obtained. (Author) 4 refs

  9. Three-Charge Black Holes and Quarter BPS States in Little String Theory

    CERN Document Server

    Giveon, Amit; Kutasov, David; Lee, Sungjay

    2015-01-01

    We show that the system of $k$ NS5-branes wrapping $\\mathbb{T}^4\\times S^1$ has non-trivial vacuum structure. Different vacua have different spectra of 1/4 BPS states that carry momentum and winding around the $S^1$. 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.

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

    Directory of Open Access Journals (Sweden)

    Gschwantler M

    2015-01-01

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

  11. 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.

  12. D-branes in non-critical superstrings and duality in N = 1 gauge theories with flavor

    International Nuclear Information System (INIS)

    We study D-branes in the superstring background R3,1 x SL(2, R)k=1/U(1) which are extended in the cigar direction. Some of these branes are new. The branes realize flavor in the four dimensional N = 1 gauge theories on the D-branes localized at the tip of the cigar. We study the analytic properties of the boundary conformal field theories on these branes with respect to their defining parameter and find non- trivial monodromies in this parameter. Through this approach, we gain a better understanding of the brane set-ups in ten dimensions involving wrapped NS5-branes. As one application, using the boundary conformal field theory description of the electric and magnetic D-branes, we can understand electric-magnetic (Seiberg) duality in N = 1 SQCD microscopically in a string theoretic context. (author)

  13. An in vitro Flaviviridae replicase system capable of authentic RNA replication

    International Nuclear Information System (INIS)

    We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication

  14. Subharmonic buncher for the Los Alamos free-electron laser oscillator experiment

    International Nuclear Information System (INIS)

    A high efficiency free-electron laser oscillator experiment is being constructed at Los Alamos National Laboratory. A buncher system has been designed to deliver 30-ps, 5-nC electron bunches to a 20-MeV standing-wave linac at the 60th subharmonic of the 1300-MHz accelerator frequency. The first 108.3-MHz buncher cavity accepts a 5-ns, 5-A peak current pulse from a triode gun. Following a 120-cm drift space, a second 108.3-MHz cavity is used, primarily to enhance the bunching of the trailing half of the bunch. A 1300-MHz cavity with 20-cm drift spaces at the each end completes the beamline components. The bunching process continues into the linac's first three accelerating cells. Two thin iron-shielded lenses and several large-diameter solenoids provide axial magnetic fields for radial focusing

  15. Detection of classical swine fever virus (CSFV) in clinical samples by RT-PCR assay in clinical samples by RT-PCR assay using different pairs of primers

    International Nuclear Information System (INIS)

    The aim was to compare the efficiency of RT-PCT assays using four pairs of primers selected from different regions of the CSFV genome for the detection of CSFV in clinical samples of swine and wild boars. The four RT-PCR assays were able to detect CSFV in all 20 clinical samples which had been collected from dead swine and wild boars during the outbreaks of CSF in Slovakia in 1993 and 1994. The quality of the selected RT-PCR primers was determined as follows: gp55L/gp55U (E2), 324/326 (5'-NC), S1/S2 (NS5B) and gp54L/gp54U (NS2 genomic region). We conclude that gp55L/gp55U primers are the most suitable for direct detection of CSFV by RT-PCR in tissue homogenates of diseased animals

  16. [Research Progress in the Core Proteins of the Classical Swine Fever Virus].

    Science.gov (United States)

    Hou, Yuzhen; Zhao, Dantong; Liu, Guoying; He, Fan; Liu, Bin; Fu, Shaoyin; Hao, Yongqing; Zhang, Wenguang

    2015-09-01

    The core protein (CP) of the classical swine fever virus (CSFV) is one of its structural proteins. Apart from forming the nucleocapsid to protect internal viral genomic RNA, this protein is involved in transcriptional regulation. Also, during viral infection, the CP is involved in interactions with many host proteins. In this review, we combine study of this protein with its disorders, structural/functional characteristics, as well as its interactions with the non-structural proteins NS3, NS5B and host proteins such as SUMO-1, UBC9, OS9 and IQGAP1. We also summarize the important part played by the CP in CSFV pathogenicity, virulence and replication of genomic RNA. We also provide guidelines for further studies in the CP of the CSFV. PMID:26738299

  17. Two-loop partition function in the planar plane-wave matrix model

    Science.gov (United States)

    Spradlin, Marcus; Van Raamsdonk, Mark; Volovich, Anastasia

    2004-12-01

    We perform two independent calculations of the two-loop partition function for the 't Hooft large N limit of the plane-wave matrix model, conjectured to be dual to the decoupled little string theory of a single spherical type IIA NS5-brane. The first is via a direct two-loop path-integral calculation in the matrix model, while the second employs the one-loop dilatation operator of four-dimensional N = 4 Yang-Mills theory truncated to the SU (2 | 4) subsector. We find precise agreement between the results of the two calculations. Various polynomials appearing in the result have rather special properties, possibly related to the large symmetry algebra of the theory or to integrability.

  18. Two-Loop Partition Function in the Planar Plane-Wave Matrix Model

    CERN Document Server

    Spradlin, M; Volovich, A; Spradlin, Marcus; Raamsdonk, Mark Van; Volovich, Anastasia

    2004-01-01

    We perform two independent calculations of the two-loop partition function for the large N 't Hooft limit of the plane-wave matrix model, conjectured to be dual to the decoupled little string theory of a single spherical type IIA NS5-brane. The first is via a direct two-loop path-integral calculation in the matrix model, while the second employs the one-loop dilatation operator of four-dimensional N = 4 Yang-Mills theory truncated to the SU(2|4) subsector. We find precise agreement between the results of the two calculations. Various polynomials appearing in the result have rather special properties, possibly related to the large symmetry algebra of the theory or to integrability.

  19. Perturbative decay of anti-branes in flux backgrounds due to space time instabilities

    CERN Document Server

    Danielsson, Ulf H

    2015-01-01

    In this paper we suggest a new source of perturbative decay of the KPV-state, which might have consequences for the viability of the KKLT-construction. The results do not rely on any direct enhancement of the decay due to flux accumulating on the anti-brane in transverse space. Instead, we note that the system can lower its energy through a sequence of NS5-configurations all the way to the true vacuum, without encountering a barrier, if we allow for clumping of screened charge in space time. The clumping can possibly be a parallel to the Gregory-Laflamme instability of black branes. The results are obtained at large $p$, but for $p/M$ arbitrarily small. It is furthermore argued that the results extend to cases of few or single anti-branes where quantization becomes important. We believe that it is important to investigate this possible effect further to judge whether there are any fatal consequences.

  20. Killing superalgebra deformations of ten-dimensional supergravity backgrounds

    International Nuclear Information System (INIS)

    We explore Lie superalgebra deformations of the Killing superalgebras of some ten-dimensional supergravity backgrounds. We prove the rigidity of the Poincare superalgebras in types I, IIA and IIB, as well as of the Killing superalgebra of the Freund-Rubin vacuum of type IIB supergravity. We also prove rigidity of the Killing superalgebras of the NS5-, D0-, D3-, D4- and D5-branes, whereas we exhibit the possible deformations of the D1-, D2-, D6- and D7-brane Killing superalgebras, as well as of that of the type II fundamental string solutions. We relate the superalgebra deformations of the D2- and D6-branes to those of the (delocalized) M2-brane and the Kaluza-Klein monopole, respectively. The good behaviour under Kaluza-Klein reduction suggests that the deformed superalgebras ought to have a geometric interpretation

  1. On supersymmetry breaking in string theory from gauge theory in a throat

    International Nuclear Information System (INIS)

    We embed the supersymmetry breaking mechanism in N = 1 SQCD of hep-th/0602239 in a smooth superstring theory using D-branes in the background R4 x SL(2)k=1/U(1) which smoothly captures the throat region of an intersecting NS5-brane configuration. A controllable deformation of the supersymmetric branes gives rise to the mass deformation of the magnetic SQCD theory on the branes. The consequent instability on the open string worldsheet can be followed onto a stable non-supersymmetric configuration of D-branes which realize the metastable vacuum configuration in the field theory. The new brane configuration is shown to backreact onto the background such as to produce different boundary conditions for the string fields in the radial direction compared to the supersymmetric configuration. In the string theory, this is interpreted to mean that the supersymmetry breaking is explicit rather than spontaneous. (author)

  2. 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

    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 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 by...

  3. Regulatory mechanisms of viral hepatitis B and C

    Indian Academy of Sciences (India)

    G Waris; A Siddiqui

    2003-04-01

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

  4. 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...

  5. Stable non-supersymmetric vacua in the moduli space of non-critical superstrings

    Energy Technology Data Exchange (ETDEWEB)

    Harmark, Troels [Niels Bohr Institute, Blegdamsvej 17, 2100 Copenhagen O (Denmark)]. E-mail: harmark@nbi.dk; Niarchos, Vasilis [Niels Bohr Institute, Blegdamsvej 17, 2100 Copenhagen O (Denmark)]. E-mail: niarchos@nbi.dk; Obers, Niels A. [Niels Bohr Institute, Blegdamsvej 17, 2100 Copenhagen O (Denmark)]. E-mail: obers@nbi.dk

    2006-12-18

    We study a set of asymmetric deformations of non-critical superstring theories in various dimensions. The deformations arise as Kaehler and complex structure deformations of an orthogonal two-torus comprising of a parallel and a transverse direction in the near-horizon geometry of NS5-branes. The resulting theories have the following intriguing features: Spacetime supersymmetry is broken in a continuous fashion and the masses of the lightest modes are lifted. In particular, no bulk or localized tachyons are generated in the non-supersymmetric vacua. We discuss the effects of these deformations in the context of the holographic duality between non-critical superstrings and little string theories and find solutions of rotating fivebranes in supergravity. We also comment on the generation of a one-loop cosmological constant and determine the effects of the one-loop backreaction to leading order.

  6. Stable non-supersymmetric vacua in the moduli space of non-critical superstrings

    International Nuclear Information System (INIS)

    We study a set of asymmetric deformations of non-critical superstring theories in various dimensions. The deformations arise as Kaehler and complex structure deformations of an orthogonal two-torus comprising of a parallel and a transverse direction in the near-horizon geometry of NS5-branes. The resulting theories have the following intriguing features: Spacetime supersymmetry is broken in a continuous fashion and the masses of the lightest modes are lifted. In particular, no bulk or localized tachyons are generated in the non-supersymmetric vacua. We discuss the effects of these deformations in the context of the holographic duality between non-critical superstrings and little string theories and find solutions of rotating fivebranes in supergravity. We also comment on the generation of a one-loop cosmological constant and determine the effects of the one-loop backreaction to leading order

  7. 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.

  8. Evolution of dengue virus type 2 during two consecutive outbreaks with an increase in severity in southern Taiwan in 2001-2002.

    Science.gov (United States)

    Chen, Hui-Ling; Lin, Su-Ru; Liu, Hsin-Fu; King, Chwan-Chuen; Hsieh, Szu-Chia; Wang, Wei-Kung

    2008-10-01

    To investigate viral determinants and evolution linked to outbreak with increased severity, we examined dengue virus type 2 (DENV-2) sequences from plasma of 31 patients (14 dengue fever, 17 dengue hemorrhagic fever, DHF) continuously during the 2001 and 2002 outbreaks in southern Taiwan, in which both the total cases and proportion of DHF cases increased. Analysis of envelope (E) and full-genome sequences between viruses of the two outbreaks revealed 5 nucleotide changes in E, NS1, NS4A, and NS5 genes. None was identical to those reported in the DENV-2 outbreak in Cuba in 1997, suggesting viral determinants linked to severe outbreak are genotype dependent. Compared with previous reports of lineage turnover years apart, our findings that the 2002 viruses descended from a minor variant of the 2001 viruses in less than 6 months was novel, and may represent a mechanism of evolution of DENV from one outbreak to another. PMID:18840735

  9. Defect (p,q) Five-branes

    CERN Document Server

    Kimura, Tetsuji

    2015-01-01

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

  10. Subharmonic triple buncher for a high-efficiency free electron laser

    International Nuclear Information System (INIS)

    A high-efficiency free electron laser oscillator experiment is being constructed at Los Alamos National Laboratory. A buncher system has been designed to deliver 30-ps, 5-nC electron bunches to a 20-MeV standing-wave linac at the 60th subharmonic of the 1300-MHz accelerator frequency. The first 108.3-MHz buncher cavity accepts a 5-ns, 5-A peak current pulse from a triode gun. Following a 120-cm drift space, a second 108.3-MHz cavity is used, primarily to enhance the bunching of the trailing half of the bunch. A 1300-MHz cavity with 20-cm drift spaces at each end completes the beamline components. The bunching process continues into the linac's first three accelerating cells. Two thin iron-shielded lenses and seven large-diameter solenoids provide axial magnetic fields for radial focusing

  11. Thermodynamic geometry and extremal black holes in string theory

    International Nuclear Information System (INIS)

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

  12. A STUDY ON COMPARISON OF INTRAVENOUS DEXMEDETOMIDINE WITH INTRAVENOUS FENTANYL FOR SUPPRESSION OF HEMODYNAMIC RESPONSES TO LARYNGOSCOPY AND ENDOTRACHEAL INTUBATION DURING GENERAL ANAESTHESIA

    Directory of Open Access Journals (Sweden)

    Nidhi D Patel

    2015-06-01

    Full Text Available Background: Laryngoscopy and intubation is the Gold standard for airway management but this evokes a stress response which is exhibited in the form of changes in heart rate, blood pressure and arrhythmias. This study was prospective, randomized, double blind study to determine whether the fentanyl 2 and micro;g/Kg or dexmedetomidine 1 and micro;g/Kg would decrease the attenuation of hemodynamic response during laryngoscopy and tracheal intubation during general anaesthesia. Methodology: The patients were randomly allocated into two groups. In Group D cases (n=30 received injection Dexmedetomidine 1 and micro;g/kg diluted to 10ml NS IV over 10min using syringe pump prior to intubation and 5ml of NS 5 min. prior to intubation. In Group F cases (n=30 received 2 and micro;g/kg diluted to 5ml NS 5min. prior to intubation and 20ml NS in infusion pump over 10 min., prior to intubation. Results: The age and weight of the cases in both the groups are comparable. It was observed that mean HR increased in both groups D and F immediately after endotracheal intubation. The systolic blood pressure was highly significant in group F as compared to group D during laryngoscopy and intubation, 1, 3, 5 and 10 min after intubation (p<0.000. Ramsay sedation score was and #8805; 4 in all patients in group D and was and #8804; 3 in group F. Dexmedetomidine has higher sedation score but no respiratory depression. Conclusion: We concluded that dexmedetomidine in dose 1 and micro;gm/kg i.v. is more effective in attenuating the hemodynamic pressor responses to laryngoscopy and intubation than Inj. Fentanyl 2 and micro;gm/kg i.v. when given as pre-medicant without significant side effects. [Natl J Med Res 2015; 5(2.000: 127-131

  13. Prevalence of hepatitis G virus infection and homology of different viral strains in Southern China

    Institute of Scientific and Technical Information of China (English)

    Gang Li; Hui-Hui Ma; Geroge KK Lau; Yin-Kit Leung; Chun-Lan Yao; Yu-Tian Chong; Wen-Hui Tang; Ji-Lu Yao

    2002-01-01

    AIM: To investigate the prevalence of hepatitis G virus (HGV) infection and to analyse the homology of different HGV strains in Southern China.METHODS: A total of 1993 sera from different groups in Guangdong, Hong Kong, and Yunnan were detected by reverse transcription polymerase chain reaction (RT-PCR). The nucleotide sequences of 5'untranslated region (5'UTR) derived from 20 strains and NS5 region from 3 strains were determined.RESULTS: The positive rate of HGV RNA was 0.89 % in community population, 2.57 % in blood donors, 17.86 % in intravenous drug abusers, 14.13 % in patients with hemodialysis, 13.66 % in those with hepatocellular carcinoma, 25.30 % in non A-E hepatitis, 7.22 % in hepatitis bone marrow transplantation, respectively. The homology was 90.40-100 % in 5'UTR among different strains, while that of NS5 region was 93.3-94 % in nucleotide sequence, and 97-99.2 % in amino acid sequence.CONCLUSION: These results showed that there was a high incidence of HGV infection in patients from Southern China, being treated for bone marrow transplantation, hepatocellular carcinoma and those on haemodialysis. Furthermore, there was also a high frequency of co-infection of HGV with HBV,HCV, non A-E viral hepatitis and that among intravenous drug abusers. The study also showed that sequence variation in different strains was associated with geographical factors but there was no significant difference in 5'UTR in circulating viruses between different patient groups. Finally, by sequential analysis of viral species present in individual patients over a three months period there was no evidence of sequence variation in the 5' UTR.

  14. Profile of paritaprevir/ritonavir/ombitasvir plus dasabuvir in the treatment of chronic hepatitis C virus genotype 1 infection

    Directory of Open Access Journals (Sweden)

    Smith MA

    2015-11-01

    Full Text Available Michael A Smith, Alice LimDepartment of Pharmacy Practice and Pharmacy Administration, Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA, USAAbstract: Over the last several years, many advances have been made in the treatment of chronic hepatitis C virus (HCV infection with the development of direct-acting antivirals. Paritaprevir/ritonavir/ombitasvir with dasabuvir (PrOD is a novel combination of a nonstructural (NS 3/4A protein inhibitor boosted by ritonavir, an NS5A protein inhibitor, and an NS5B nonnucleoside polymerase inhibitor. This review aims to discuss the pharmacology, efficacy, safety, drug interactions, and viral drug resistance of PrOD in the treatment of HCV genotype 1 infections. Phase I, II, and III human and animal studies that describe the pharmacology, pharmacokinetics, efficacy, and safety of PrOD for HCV were identified and included. Studies that evaluated patients without cirrhosis (n=2,249 and with cirrhosis (n=422 demonstrated that PrOD for 12 or 24 weeks was effective at achieving sustained virologic response rates (>90% in patients with genotype 1a or 1b HCV infection. Although indicated for the treatment of HCV genotype 1 infection, PrOD is also recommended for the treatment of HCV in patients coinfected with HIV. Additionally, promising data exist for the use of PrOD in liver-transplant recipients. The most common adverse drug events associated with PrOD included nausea, pruritus, insomnia, diarrhea, asthenia, dry skin, vomiting, and anemia. The high efficacy rates seen coupled with a favorable side effect profile seen with PrOD with or without ribavirin have led to its addition as a recommended treatment regimen for HCV genotype 1 infection.Keywords: direct-acting antiviral, interferon-free, ribavirin-free

  15. Profile of paritaprevir/ritonavir/ombitasvir plus dasabuvir in the treatment of chronic hepatitis C virus genotype 1 infection.

    Science.gov (United States)

    Smith, Michael A; Lim, Alice

    2015-01-01

    Over the last several years, many advances have been made in the treatment of chronic hepatitis C virus (HCV) infection with the development of direct-acting antivirals. Paritaprevir/ritonavir/ombitasvir with dasabuvir (PrOD) is a novel combination of a nonstructural (NS) 3/4A protein inhibitor boosted by ritonavir, an NS5A protein inhibitor, and an NS5B nonnucleoside polymerase inhibitor. This review aims to discuss the pharmacology, efficacy, safety, drug interactions, and viral drug resistance of PrOD in the treatment of HCV genotype 1 infections. Phase I, II, and III human and animal studies that describe the pharmacology, pharmacokinetics, efficacy, and safety of PrOD for HCV were identified and included. Studies that evaluated patients without cirrhosis (n=2,249) and with cirrhosis (n=422) demonstrated that PrOD for 12 or 24 weeks was effective at achieving sustained virologic response rates (>90%) in patients with genotype 1a or 1b HCV infection. Although indicated for the treatment of HCV genotype 1 infection, PrOD is also recommended for the treatment of HCV in patients coinfected with HIV. Additionally, promising data exist for the use of PrOD in liver-transplant recipients. The most common adverse drug events associated with PrOD included nausea, pruritus, insomnia, diarrhea, asthenia, dry skin, vomiting, and anemia. The high efficacy rates seen coupled with a favorable side effect profile seen with PrOD with or without ribavirin have led to its addition as a recommended treatment regimen for HCV genotype 1 infection. PMID:26622169

  16. Biomedical Mutation Analysis (BMA): A software tool for analyzing mutations associated with antiviral resistance

    Science.gov (United States)

    Salvatierra, Karina; Florez, Hector

    2016-01-01

    Introduction: Hepatitis C virus (HCV) is considered a major public health problem, with 200 million people infected worldwide. The treatment for HCV chronic infection with pegylated interferon alpha plus ribavirin inhibitors is unspecific; consequently, the treatment is effective in only 50% of patients infected. This has prompted the development of direct-acting antivirals (DAA) that target virus proteins. These DAA have demonstrated a potent effect in vitro and in vivo; however, virus mutations associated with the development of resistance have been described. Objective: To design and develop an online information system for detecting mutations in amino acids known to be implicated in resistance to DAA. Materials and methods:    We have used computer applications, technological tools, standard languages, infrastructure systems and algorithms, to analyze positions associated with resistance to DAA for the NS3, NS5A, and NS5B genes of HCV. Results: We have designed and developed an online information system named Biomedical Mutation Analysis (BMA), which allows users to calculate changes in nucleotide and amino acid sequences for each selected sequence from conventional Sanger and cloning sequencing using a graphical interface. Conclusion: BMA quickly, easily and effectively analyzes mutations, including complete documentation and examples. Furthermore, the development of different visualization techniques allows proper interpretation and understanding of the results. The data obtained using BMA will be useful for the assessment and surveillance of HCV resistance to new antivirals, and for the treatment regimens by selecting those DAA to which the virus is not resistant, avoiding unnecessary treatment failures. The software is available at: http://bma.itiud.org.

  17. A Human Vaccine Strategy Based On Chimpanzee Adenoviral and MVA Vectors That Primes, Boosts and Sustains Functional HCV Specific T-Cell Memory*

    Science.gov (United States)

    Swadling, Leo; Capone, Stefania; Antrobus, Richard D.; Brown, Anthony; Richardson, Rachel; Newell, Evan W.; Halliday, John; Kelly, Christabel; Bowen, Dan; Fergusson, Joannah; Kurioka, Ayako; Ammendola, Virginia; Sorbo, Mariarosaria Del; Grazioli, Fabiana; Esposito, Maria Luisa; Siani, Loredana; Traboni, Cinzia; Hill, Adrian; Colloca, Stefano; Davis, Mark; Nicosia, Alfredo; Cortese, Riccardo; Folgori, Antonella; Klenerman, Paul; Barnes, Eleanor

    2015-01-01

    A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies and assessment of host immunity during acute infection highlight the critical role that effective T-cell immunity plays in viral control. In this first-in-man study we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A and NS5B proteins of HCV genotype-1b. Analysis employed single cell mass cytometry (CyTOF), and HLA class-I peptide tetramer technology in healthy human volunteers. We show that HCV specific T-cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8+ and CD4+ HCV specific T-cells targeting multiple HCV antigens. Sustained memory and effector T-cell populations are generated and T-cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) following heterologous MVA boost. We have developed a HCV vaccine strategy, with durable, broad, sustained and balanced T-cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine. PMID:25378645

  18. Large-density field theory, viscosity and ‘2kF’ singularities from string duals

    International Nuclear Information System (INIS)

    We analyze systems where an effective large-N expansion arises naturally in gauge theories without a large number of colors: a sufficiently large charge density alone can produce a perturbative string ('tHooft) expansion. One example is simply the well-known NS5/F1 system dual to AdS3 × T4 × S3, here viewed as a 5+1 dimensional theory at finite density. This model is completely stable, and we find that the existing string-theoretic solution of this model yields two interesting results. First, it indicates that the shear viscosity is not corrected by α′ effects in this system. For flow perpendicular to the F1 strings the viscosity to entropy ratio take the usual value 1/4π, but for flow parallel to the F1s it vanishes as T2 at low temperature. Secondly, it encodes singularities in correlation functions coming from low-frequency modes at a finite value of the momentum along the T4 directions. This may provide a strong coupling analogue of finite density condensed matter systems for which fermionic constituents of larger operators contribute so-called ‘2kF’ singularities. In the NS5/F1 example, stretched strings on the gravity side play the role of these composite operators. We explore the analogue for our system of the Luttinger relation between charge density and the volume bounded by these singular surfaces. This model provides a clean example where the string-theoretic UV completion of the gravity dual to a finite density field theory plays a significant and calculable role. (paper)

  19. Interaction of Hepatitis C virus proteins with pattern recognition receptors

    Directory of Open Access Journals (Sweden)

    Imran Muhammad

    2012-06-01

    Full Text Available Abstract Hepatitis C virus (HCV is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I as the receptors for intracellular viral double stranded RNA (dsRNA, and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C, non structural 3/4 A (NS3/4A and non structural 5A (NS5A have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

  20. A rapid immunization strategy with a live attenuated tetravalent dengue vaccine elicits protective neutralizing antibody responses in non-human primates

    Directory of Open Access Journals (Sweden)

    Yuping eAmbuel

    2014-06-01

    Full Text Available Dengue viruses (DENVs cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine (TDV that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2 and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3 and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0 at two different anatomical locations with a needle-free disposable syringe jet injection (DSJI delivery devices (PharmaJet in non-human primates (NHP. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (two months later vaccination schedule. In addition, the vaccine induced CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3 and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post- challenge. RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas.

  1. Inhibition of hepatitis C virus infection by DNA aptamer against NS2 protein.

    Science.gov (United States)

    Gao, Yimin; Yu, Xiaoyan; Xue, Binbin; Zhou, Fei; Wang, Xiaohong; Yang, Darong; Liu, Nianli; Xu, Li; Fang, Xiaohong; Zhu, Haizhen

    2014-01-01

    NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies. PMID:24587329

  2. The role of HCV proteins on treatment outcomes.

    Science.gov (United States)

    Kumthip, Kattareeya; Maneekarn, Niwat

    2015-01-01

    For many years, the standard of treatment for hepatitis C virus (HCV) infection was a combination of pegylated interferon alpha (Peg-IFN-α) and ribavirin for 24-48 weeks. This treatment regimen results in a sustained virologic response (SVR) rate in about 50% of cases. The failure of IFN-α-based therapy to eliminate HCV is a result of multiple factors including a suboptimal treatment regimen, severity of HCV-related diseases, host factors and viral factors. In recent years, advances in HCV cell culture have contributed to a better understanding of the viral life cycle, which has led to the development of a number of direct-acting antiviral agents (DAAs) that target specific key components of viral replication, such as HCV NS3/4A, HCV NS5A, and HCV NS5B proteins. To date, several new drugs have been approved for the treatment of HCV infection. Application of DAAs with IFN-based or IFN-free regimens has increased the SVR rate up to >90% and has allowed treatment duration to be shortened to 12-24 weeks. The impact of HCV proteins in response to IFN-based and IFN-free therapies has been described in many reports. This review summarizes and updates knowledge on molecular mechanisms of HCV proteins involved in anti-IFN activity as well as examining amino acid variations and mutations in several regions of HCV proteins associated with the response to IFN-based therapy and pattern of resistance associated amino acid variants (RAV) to antiviral agents. PMID:26666318

  3. Inhibition of hepatitis C virus production by aptamers against the core protein.

    Science.gov (United States)

    Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong; Zhu, Haizhen

    2014-02-01

    Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C. PMID:24307579

  4. 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.

  5. Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

    Directory of Open Access Journals (Sweden)

    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.

  6. Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue.

    Science.gov (United States)

    Amraiz, Deeba; Zaidi, Najam-Us-Sahar Sadaf; Fatima, Munazza

    2016-10-01

    NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4mM. The induced cultures were then grown for 20h at 18°C and cells were harvested by centrifugation at 6000xg for 15min at 4°C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems. PMID:27542741

  7. [Novel treatments for hepatitis C virus infection in chronic kidney disease].

    Science.gov (United States)

    Fabrizi, Fabrizio; Messa, Piergiorgio

    2016-01-01

    Recent evidence has been accumulated showing a negative impact of chronic hepatitis C virus infection on survival in patients with chronic kidney disease. Moreover, it appears that anti-HCV positive status has been associated with an increased risk of developing chronic kidney disease in the adult general population. These reports have emphasized the need for safe and effective therapies for hepatitis C virus infection in the chronic kidney disease population. Treatment of HCV has made considerable progress with the approval of interferon-free, direct-acting antiviral drug-based combination therapies among patients with intact kidneys; but a paucity of information exists regarding chronic kidney disease patients. The first published report on the antiviral treatment of hepatitis C among patients with chronic kidney disease (stage 4-5) and HCV genotype 1 concerns the combination of grazoprevir (NS3/4A protease inhibitor) and elbasvir (NS5A inhibitor); excellent safety and efficacy (sustained viral response, 94.3% 115/122) have been reached. In another study, the 3-D regimen (ombitasvir/ paritaprevir/ ritonavir/ dasabuvir with or without ribavirin) has been administered to CKD (stage 4-5) patients with genotype 1 (n=20); the rate of sustained viral response was excellent (90%, 18/20) and no patients discontinued treatment due to adverse events. Preliminary data on the combined treatment of sofosbuvir (NS5B inhibitor) and simeprevir (NS3/4A inhibitor) has given a viral response of 89% (34/38), but the size of the study group (n=38 patients with end-stage renal disease) was small. Thus, the evidence in the medical literature concerning use of DAAs in CKD population is encouraging even if it has a preliminary nature. Also, several points need to be addressed regarding the use of DAAs in CKD population including their impact on survival, costs, and drug-drug interactions. PMID:27545640

  8. First Description of Hepacivirus and Pegivirus Infection in Domestic Horses in China: A Study in Guangdong Province, Heilongjiang Province and Hong Kong District

    Science.gov (United States)

    Xu, Tao; He, Dong; Wang, Zengchao; Ou, Shudan; Jia, Kun; Yuan, Liguo; Li, Shoujun

    2016-01-01

    Since 2012, three viruses, known as equine hepacivirus (EqHV), equine pegivirus (EPgV) and Theiler’s disease-associated virus (TDAV), have been discovered in equines. Given that these viruses are the newest members of the Flaviviridae family, genomic information concerning circulating EqHV, EPgV and TDAV strains around the world is limited. To date, no genetic surveillance studies have been performed on these three viruses in the equine population of China. Here, a total of 177 serum samples were collected from equines across China between 2014 and 2015. Using PCR, we detected viral RNA in the serum samples, six of which were EqHV positive and two of which were EPgV positive. Co-infection with the two viruses was not observed among the Chinese equines studied, and TDAV RNA was not detected in the equine serum samples collected for this study. Phylogenetic analysis of partial NS5B open reading frame (ORF), NS3 ORF, and 5’ untranslated region nucleotide sequences from EqHV as well as partial NS3 ORF sequence from EPgV indicated that EqHV and EPgV have evolved into two main clades by themselves, both of which are circulating in China. Based on the partial NS5B and NS3 ORF sequences of EqHV, the sequences of one clade were also split into two subclades. This study enriches our knowledge of the geographic distribution of these three equine viruses. PMID:27182887

  9. Acute hepatitis C in a chronically HIV-infected patient: Evolution of different viral genomic regions

    Institute of Scientific and Technical Information of China (English)

    Diego Flichman; Veronica Kott; Silvia Sookoian; Rodolfo Campos

    2003-01-01

    AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV infected patient that seroconverted to anti HCV antibodies were sequenced, from the event of superinfection through a period of 17 months and in a late sample (42nd month). Hypervariable genomic regions of HIV (V3 loop of the gp120) and HCV (HVR-1 on the E2 glycoprotein gene) were studied. In order to analyze genomic regions involved in different biological functions and with the cellular immune response, HCV core and NS5A were also chosen to be sequenced. Amplification of the different regions was done by RT-PCR and directly sequenced. Confirmation of sequences was done on reamplified material. Nucleotide sequences of the different time points were aligned with CLUSTAL W 1.5, and the corresponding amino acid ones were deduced.RESULTS: Hypervariable genomic regions of both viruses (HVR1 and gp120 V3 loop) presented several nonsynonymous changes but, while in the gp120 V3 loop mutations were detected in the sample obtained right after HCV superinfection and maintained throughout, they occurred following a sequential and cumulative pattern in the HVR1. In the NS5A region of HCV, two amino acid changes were detected during the follow-up period, whereas the core region presented several amino acid replacements, once the HCV chronic infection had been established.CONCLUSION: During the HIV-HCV superinfection, each genomic region analyzed shows a different evolutionary pattem.Most of the nucleotide substitutions observed are nonsynonymous and clustered in previously described epitopes,thus suggesting an immune-driven evolutionary process.

  10. 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

  11. 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. PMID:25962112

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

    Directory of Open Access Journals (Sweden)

    Rujipat Wasitthankasem

    Full Text Available The majority of hepatitis C virus (HCV infection results in chronic infection, which can lead to liver cirrhosis and hepatocellular carcinoma. Global burden of hepatitis C virus (HCV is estimated at 150 million individuals, or 3% of the world's population. The distribution of the seven major genotypes of HCV varies with geographical regions. Since Asia has a high incidence of HCV, we assessed the distribution of HCV genotypes in Thailand and Southeast Asia. From 588 HCV-positive samples obtained throughout Thailand, we characterized the HCV 5' untranslated region, Core, and NS5B regions by nested PCR. Nucleotide sequences obtained from both the Core and NS5B of these isolates were subjected to phylogenetic analysis, and genotypes were assigned using published reference genotypes. Results were compared to the epidemiological data of HCV genotypes identified within Southeast Asian. Among the HCV subtypes characterized in the Thai samples, subtype 3a was the most predominant (36.4%, followed by 1a (19.9%, 1b (12.6%, 3b (9.7% and 2a (0.5%. While genotype 1 was prevalent throughout Thailand (27-36%, genotype 3 was more common in the south. Genotype 6 (20.9% constituted subtype 6f (7.8%, 6n (7.7%, 6i (3.4%, 6j and 6m (0.7% each, 6c (0.3%, 6v and 6xa (0.2% each and its prevalence was significantly lower in southern Thailand compared to the north and northeast (p = 0.027 and p = 0.030, respectively. Within Southeast Asia, high prevalence of genotype 6 occurred in northern countries such as Myanmar, Laos, and Vietnam, while genotype 3 was prevalent in Thailand and Malaysia. Island nations of Singapore, Indonesia and Philippines demonstrated prevalence of genotype 1. This study further provides regional HCV genotype information that may be useful in fostering sound public health policy and tracking future patterns of HCV spread.

  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. PMID:25962112

  14. New treatment for hepatitis C in chronic kidney disease, dialysis, and transplant.

    Science.gov (United States)

    Fabrizi, Fabrizio; Martin, Paul; Messa, Piergiorgio

    2016-05-01

    The evidence that chronic hepatitis C plays a detrimental role in survival among patients on maintenance dialysis or renal transplant recipients promotes the antiviral treatment of hepatitis C virus (HCV) among chronic kidney disease patients. Also, it seems that HCV infection is associated with an increased risk of developing chronic kidney disease in the adult general population. Interferon-based regimens have provided limited efficacy and safety among chronic kidney disease patients, whereas the advent of the new direct-acting antivirals for the treatment of hepatitis C (launched over the past 5 years) has given the opportunity to reach sustained virologic response rates of 90% for many patient groups. Unfortunately, poor information exists regarding the antiviral treatment of hepatitis C in the chronic kidney disease population. The first published data on the treatment of hepatitis C among patients with chronic kidney disease (stage 4-5) and HCV genotype 1 regard the grazoprevir (NS3/4A protease inhibitor) and elbasvir (NS5A inhibitor) combination; excellent efficacy (sustained viral response, 94.3%; 115/122) and safety have been achieved. Preliminary evidence on the combined treatment of sofosbuvir (NS5B inhibitor) and simeprevir (NS3/4A inhibitor) has given a viral response of 89%, but the size of the study group (n = 38 patients with end-stage renal disease) was small. Some phase 2 and 3 clinical trials based on other antiviral combinations (3D regimen, sofosbuvir/ledipasvir, or other sofosbuvir-containing approaches) are ongoing. Thus, the antiviral regimens based on direct-acting antivirals promise to play a pivotal role in the eradication of hepatitis C among kidney disease patients. Direct-acting antivirals are very expensive; in an era of cost containment this is a crucial point either in developed and developing countries. Adverse drug reactions resulting from concomitantly administered medications are another ongoing concern for patients

  15. Therapie der chronischen Hepatitis C: Der Beginn einer neuen Ära

    Directory of Open Access Journals (Sweden)

    Stättermayer AF

    2014-01-01

    Full Text Available Im letzten Jahrzehnt stellte die Kombination aus pegyliertem Interferon-α (PegINF-α und Ribavirin (RBV die Standardtherapie bei Patienten mit chronischer Hepatitis C dar. Mit der Zulassung der Protease-Inhibitoren Telaprevir und Boceprevir stehen seit 2011 erstmals direkt antiviral wirksame Substanzen („direct-acting antiviral agents“ [DAA] für die Therapie des HCV-Genotyps 1 zur Verfügung. Zwar konnte hiermit der Therapieerfolg deutlich erhöht werden, gleichzeitig führte die neue Triple-Therapie allerdings auch zu vermehrten Nebenwirkungen und höheren Therapiekosten. Zusätzliche Probleme wie Virusresistenz, Medikamenteninteraktionen und eine hohe Anzahl an täglich einzunehmenden Tabletten erschweren die antivirale Therapie mit Protease-Inhibitoren der ersten Generation. Insbesondere Patienten mit fortgeschrittener Lebererkrankung, welche den dringendsten Bedarf für eine antivirale Therapie aufweisen, haben ein hohes Risiko, schwere Nebenwirkungen unter der Triple-Therapie zu entwickeln. Die Entwicklungen in der Therapie der Hepatitis C sind rasant. So stehen gegenwärtig mit der Zulassung von Sofosbuvir (Sovaldi®, NS5BInhibitor, Simeprevir (Olysio®, Protease-Inhibitor und Daclatasvir (Daklinza®, NS5A-Inhibitor in Österreich 3 weitere direkt antiviral wirkende Substanzen zur Verfügung, welche in der Kombination bei Genotyp 1 (GT1 und Genotyp 4 (GT4 somit erstmalig eine Interferon-freie Therapie erlauben. Für die HCV-Genotypen 2 und 3 (GT2, GT3 ist die Interferon-freie Therapie in Form von Sofosbuvir/Ribavirin ebenfalls bereits Realität. Eine Reihe zusätzlicher Substanzen wird in Kürze folgen. Eine Vielzahl von klinischen Studien erbrachte diesbezüglich vielversprechende Ergebnisse. Diese zusätzlichen wirksamen, nebenwirkungsarmen, hocheffektiven und kürzeren Therapien für alle Patienten mit chronischer HCV-Infektion werden in absehbarer Zeit zur Verfügung stehen.

  16. Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection

    Directory of Open Access Journals (Sweden)

    Bala Shashi

    2012-07-01

    Full Text Available Abstract Background Hepatitis C Virus (HCV, a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA regulate inflammation (miR-155, -146a and -125b as well as hepatocyte function (miR-122. Methods Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection. Results We found that monocytes from chronic HCV infected treatment-naïve (cHCV but not treatment responder patients showed increased expression of miR-155, a positive regulator of TNFα, and had increased TNFα production compared to monocytes of normal controls. After LPS stimulation, miR-155 levels were higher in monocytes from cHCV patients compared to controls. MiR-125b, which has negative regulatory effects on inflammation, was decreased in cHCV monocytes compared to controls. Stimulation of normal monocytes with TLR4 and TLR8 ligands or HCV core, NS3 and NS5 recombinant proteins induced a robust increase in both miR-155 expression and TNFα production identifying potential mechanisms for in vivo induction of miR-155. Furthermore, we found increased serum miR-155 levels in HCV patients compared to controls. Serum miR-125b and miR-146a levels were also increased in HCV patients. Serum levels of miR-122 were elevated in cHCV patients and correlated with increased ALT and AST levels and serum miR-155 levels. Conclusion In conclusion, our novel data demonstrate that miR-155, a positive regulator of inflammation, is upregulated both in monocytes and in the serum of patients with chronic HCV infection. Our study suggests that HCV core, NS3, and NS5 proteins or TLR4 and TLR8 ligands can mediate

  17. Proteome-wide screening reveals immunodominance in the CD8 T cell response against classical swine fever virus with antigen-specificity dependent on MHC class I haplotype expression.

    Directory of Open Access Journals (Sweden)

    Giulia Franzoni

    Full Text Available Vaccination with live attenuated classical swine fever virus (CSFV vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core₂₄₁₋₂₅₅ PESRKKLEKALLAWA and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY, or minimal length antigenic peptides: E2₉₉₆₋₁₀₀₃ YEPRDSYF, NS2₁₂₂₃₋₁₂₃₀ STVTGIFL and NS5A₃₀₇₀₋₃₀₇₈ RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS₁₂₂₃₋₁₂₃₀ STVTGIFL and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed

  18. Changing the face of hepatitis C management – the design and development of sofosbuvir

    Directory of Open Access Journals (Sweden)

    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

  19. 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.)

  20. 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)

    Jeffers, Lennox J.; Ravendhran, Natarajan; Shiffman, Mitchell L.; Poulos, John; Sulkowski, Mark S.; Gitlin, Norman; Workowski, Kimberly; Zhu, Yanni; Yang, Jenny C.; Pang, Phillip S.; McHutchison, John G.; Muir, Andrew J.; Howell, Charles; Kowdley, Kris; Afdhal, Nezam; Reddy, K. Rajender

    2015-01-01

    Black patients chronically infected with genotype 1 hepatitis C virus (HCV) have historically had lower rates of response to interferon‐based treatment than patients of other races. In the phase 3 ION program, the single‐tablet regimen of the NS5A inhibitor ledipasvir and NS5B nucleotide polymerase inhibitor sofosbuvir was shown to be safe and highly effective in the general population. The aim of this study was to evaluate the safety and efficacy of ledipasvir/sofosbuvir in black patients using data from the three open‐label ION clinical trials, which evaluated the safety and efficacy of 8, 12, and 24 weeks of ledipasvir/sofosbuvir with or without ribavirin for the treatment of treatment‐naïve and treatment‐experienced patients with genotype 1 HCV, including those with compensated cirrhosis. The primary endpoint was sustained virologic response at 12 weeks after the end of therapy (SVR12). For our analysis, rates of SVR12, treatment‐emergent adverse events, and graded laboratory abnormalities were analyzed in black versus non‐black patients. Of the 1949 patients evaluated, 308 (16%) were black. On average, black patients were older, had higher body mass index, were more likely to be IL28B non‐CC, and had a lower serum alanine aminotransferase at baseline than non‐black patients. Overall, 95% of black and 97% of non‐black patients achieved SVR12. The rate of relapse was 3% in black patients as compared with 2% in non‐black patients. The most common adverse events included fatigue, headache, nausea, and insomnia. The majority of adverse events occurred more frequently in the ribavirin‐containing arms of the studies. No differences were observed in overall safety by race. Conclusion: A once‐daily dosage of ledipasvir/sofosbuvir was similarly effective in black and non‐black patients with genotype 1 HCV infection. The addition of ribavirin did not appear to increase SVR12 but was associated with higher rates of adverse events. (Hepatology

  1. Hepatitis C Virus Genotypes

    Directory of Open Access Journals (Sweden)

    Kayhan Azadmanesh

    2005-09-01

    throughout the viral genome(9.Genomic Organization of HCVThis virus has a positive-sense single-stranded RNA genome of about 10 kb containing one long open reading frame (ORF. All its proteins are encoded in a single open reading frame that encodes a polyprotein of approximately 3010 amino acids(10 (Fig. 1, with genes coding for structural proteins situated towards the N-terminus of the genome and non-structural genes located near the C-terminus. The structural genes code for the capsid protein (C, or Core and the envelope glycoproteins (E1, E2. The first 27 amino acids of the E2 gene constitute the hypervariable region 1 (HVR1, which is the most variable region of the genome and appears to be involved in virus evasion of the immune system and disease progression. The non-structural genes code for a protease (NS2, NS3 and its cofactor (NS4A, a helicase (NS3, a protein of unknown function (NS4B, a phosphoprotein (NS5A, and an RNA-dependent RNA polymerase (NS5B. In addition, the HCV genome has 5'-UTR and 3'- UTR untranslated regions (UTRs that are involved in control of viral translation. Correspondence:Malek H. Ahmadipour, Department of Hepatitis and AIDS,Pasteur Institute of Iran, Tehran, IranTel: +98 21 6696 92 91Fax: +98 21 6646 51 32E-mail: m_ahmadipour@yahoo.com It has a complicated secondary structure comprised of at least seven stem-loops important for ribosome entry and presumably, for viral RNA replication(11, 12. The translation of HCV RNA begins at the internal ribosome entry site (IRES and at the 40S ribosomal subunit in the absence of external factors, which makes the HCV translation efficient(13. Thus, the secondary structure of IRES plays an important role in HCV replication(14, and single nucleotide changes within this region could alter viral replication characteristics. HCV replication via RNA-dependent RNApolymerase is very error-prone and generates mutations at an estimated rate of 10-5 mutations per nucleotide per replication. This high mutation rate is

  2. Stability, Tunneling and Flux Changing de Sitter Transitions in the Large Volume String Scenario

    CERN Document Server

    de Alwis, S; Hatefi, E; Quevedo, F

    2013-01-01

    We study the non-perturbative stability of the Large Volume Scenario (LVS) of IIB string compactifications, by analysing transitions mediated by the Brown-Teitelboim (BT) brane nucleations and by Coleman De Luccia tunneling (CDL). We find that, as long as the effective field theory description holds, the LVS AdS minima are stable despite being non-supersymmetric. This opens the possibility of having a CFT dual. Metastable de Sitter vacua behave differently depending on the uplifting mechanism. We find explicit expressions for the different decay rates in terms of exponentials of the volume. Among the transitions of dS to dS those with increasing volume and decreasing vacuum energy are preferred, though dS decays to AdS (big-crunch sinks) have higher probability. Transitions via the CDL mechanism to decompactification are exponentially suppressed compared to these. The BT decays correspond to flux/D3 brane transitions mediated by the nucleation of D5/NS5 branes. We compare our results with previous analysis fo...

  3. Rotating Shaft Tilt Angle Measurement Using an Inclinometer

    Science.gov (United States)

    Luo, Jun; Wang, Zhiqian; Shen, Chengwu; Wen, Zhuoman; Liu, Shaojin; Cai, Sheng; Li, Jianrong

    2015-10-01

    This paper describes a novel measurement method to accurately measure the rotating shaft tilt angle of rotating machine for alignment or compensation using a dual-axis inclinometer. A model of the rotating shaft tilt angle measurement is established using a dual-axis inclinometer based on the designed mechanical structure, and the calculation equation between the rotating shaft tilt angle and the inclinometer axes outputs is derived under the condition that the inclinometer axes are perpendicular to the rotating shaft. The reversal measurement method is applied to decrease the effect of inclinometer drifts caused by temperature, to eliminate inclinometer and rotating shaft mechanical error and inclinometer systematic error to attain high measurement accuracy. The uncertainty estimation shows that the accuracy of rotating shaft tilt angle measurement depends mainly on the inclinometer uncertainty and its uncertainty is almost the same as the inclinometer uncertainty in the simulation. The experimental results indicate that measurement time is 4 seconds; the range of rotating shaft tilt angle is 0.002° and its standard deviation is 0.0006° using NS-5/P2 inclinometer, whose precision and resolution are ±0.01° and 0.0005°, respectively.

  4. Quantum billiards in multidimensional models with branes

    International Nuclear Information System (INIS)

    gravitational D-dimensional model with l scalar fields and several forms is considered. When a cosmological-type diagonal metric is chosen, an electromagnetic composite brane ansatz is adopted and certain restrictions on the branes are imposed; the conformally covariant Wheeler-DeWitt (WDW) equation for the model is studied. Under certain restrictions asymptotic solutions to WDW equation are found in the limit of the formation of the billiard walls which reduce the problem to the so-called quantum billiard on the (D+l-2)-dimensional Lobachevsky space. Two examples of quantum billiards are considered. The first one deals with 9-dimensional quantum billiard for D = 11 model with 330 four-forms which mimic space-like M2- and M5-branes of D = 11 supergravity. The second one deals with the 9-dimensional quantum billiard for D = 10 gravitational model with one scalar field, 210 four-forms and 120 three-forms which mimic space-like D2-, D4-, FS1- and NS5-branes in D = 10 IIA supergravity. It is shown that in both examples wave functions vanish in the limit of the formation of the billiard walls (i.e. we get a quantum resolution of the singularity for 11D model) but magnetic branes could not be neglected in calculations of quantum asymptotic solutions while they are irrelevant for classical oscillating behavior when all 120 electric branes are present. (orig.)

  5. Phylogenetic Analysis and Epidemic History of Hepatitis C Virus Genotype 2 in Tunisia, North Africa.

    Science.gov (United States)

    Rajhi, Mouna; Ghedira, Kais; Chouikha, Anissa; Djebbi, Ahlem; Cheikh, Imed; Ben Yahia, Ahlem; Sadraoui, Amel; Hammami, Walid; Azouz, Msaddek; Ben Mami, Nabil; Triki, Henda

    2016-01-01

    HCV genotype 2 (HCV-2) has a worldwide distribution with prevalence rates that vary from country to country. High genetic diversity and long-term endemicity were suggested in West African countries. A global dispersal of HCV-2 would have occurred during the 20th century, especially in European countries. In Tunisia, genotype 2 was the second prevalent genotype after genotype 1 and most isolates belong to subtypes 2c and 2k. In this study, phylogenetic analyses based on the NS5B genomic sequences of 113 Tunisian HCV isolates from subtypes 2c and 2k were carried out. A Bayesian coalescent-based framework was used to estimate the origin and the spread of these subtypes circulating in Tunisia. Phylogenetic analyses of HCV-2c sequences suggest the absence of country-specific or time-specific variants. In contrast, the phylogenetic grouping of HCV-2k sequences shows the existence of two major genetic clusters that may represent two distinct circulating variants. Coalescent analysis indicated a most recent common ancestor (tMRCA) of Tunisian HCV-2c around 1886 (1869-1902) before the introduction of HCV-2k in 1901 (1867-1931). Our findings suggest that the introduction of HCV-2c in Tunisia is possibly a result of population movements between Tunisia and European population following the French colonization. PMID:27100294

  6. Recent Immersed Bz X-ray Diode Experiments

    Science.gov (United States)

    Cooper, G. M.; McLean, J.; Davitt, R.; Goldsack, T. J.

    2002-12-01

    The immersed Bz diode is being fielded on one of the AWE Superswarf machines which provides a 55ns, 5.5MV, 35kA electron beam. The external magnetic field, up to 25Tesla, is produced by a solenoid which is driven by a 624μF, 22kV capacitor bank. The magnetic field constrains the electron beam to a small diameter at the target which results in a small x-ray source size. Recent experiments to try and reduce the source size include investigation of shaped field solenoids and the effects of reducing the cathode diameter. The inclusion of a time resolved source size diagnostic has provided more information on the behaviour of the diode. One of the better Bz shots has produced 72R@1m with a 4.0mm spot. This compares to a standard paraxial diode 80R@1m with a 5.3mm spot and the enhanced vacuum cell paraxial diode 65R@1m with a 4.0mm spot. Future investigations aimed at reducing the spot size will include providing a better vacuum in the diode and a possible reduction in the pre-pulse on the diode.

  7. Hydrogen/Deuterium Exchange Kinetics Demonstrate Long Range Allosteric Effects of Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase.

    Science.gov (United States)

    Deredge, Daniel; Li, Jiawen; Johnson, Kenneth A; Wintrode, Patrick L

    2016-05-01

    New nonnucleoside analogs are being developed as part of a multi-drug regimen to treat hepatitis C viral infections. Particularly promising are inhibitors that bind to the surface of the thumb domain of the viral RNA-dependent RNA polymerase (NS5B). Numerous crystal structures have been solved showing small molecule non-nucleoside inhibitors bound to the hepatitis C viral polymerase, but these structures alone do not define the mechanism of inhibition. Our prior kinetic analysis showed that nonnucleoside inhibitors binding to thumb site-2 (NNI2) do not block initiation or elongation of RNA synthesis; rather, they block the transition from the initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex. Here we have mapped the effect of three NNI2 inhibitors on the conformational dynamics of the enzyme using hydrogen/deuterium exchange kinetics. All three inhibitors rigidify an extensive allosteric network extending >40 Å from the binding site, thus providing a structural rationale for the observed disruption of the transition from distributive initiation to processive elongation. The two more potent inhibitors also suppress slow cooperative unfolding in the fingers extension-thumb interface and primer grip, which may contribute their stronger inhibition. These results establish that NNI2 inhibitors act through long range allosteric effects, reveal important conformational changes underlying normal polymerase function, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:27006396

  8. 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.

  9. Interpretation of the spectrum of Sn II: experimental and theoretical transition probabilities

    International Nuclear Information System (INIS)

    The optical emission a from laser produced plasma generated by 1064 nm irradiation of Sn/Pb alloys targets at a flux of 2.1010 W cm-2 was recorded and analyzed between 200 and 700 nm. The local thermodynamic equilibrium (LTE) conditions and plasma homogeneity have been checked. The analysis of Sn II was checked, with the support of parametric studies of the mixed configurations in both parities, by means of a self-consistent-field method to generate one-electron orbitals. The parametric description of the 5s5p2 configuration is improved by taking into account 5s25d, 5s26d and 5s27d mixing effects. Eigenfunctions were used to derive theoretical values of the transition probabilities. Experimental transition probabilities for 36 lines of Sn II arising from the 5s2ns, 5s2np, 5s2nd, 5s2nf, and 5s5p2 configurations of Sn II have been determined. Some values have been compared with the available data in the literature and are in a good agreement. The coincidence between several experimental and theoretical transition probabilities obtained in this work is remarked. (orig.)

  10. PP-Wave / CFT_2 Duality

    CERN Document Server

    Gomis, J P; Strominger, A; Gomis, Jaume; Motl, Lubos; Strominger, Andrew

    2002-01-01

    We investigate the pp-wave limit of the AdS_3\\times S^3\\times K3 compactification of Type IIB string theory from the point of view of the dual Sym_N(K3) CFT. It is proposed that a fundamental string in this pp-wave geometry is dual to the c=6 effective string of the Sym_N(K3) CFT, with the string bits of the latter being composed of twist operators. The massive fundamental string oscillators correspond to certain twisted Virasoro generators in the effective string. It is shown that both the ground states and the genus expansion parameter (at least in the orbifold limit of the CFT) coincide. Surprisingly the latter scales like J^2/N rather than the J^4/N^2 which might have been expected. We demonstrate a leading-order agreement between the pp-wave and CFT particle spectra. For a degenerate special case (one NS 5-brane) an intriguing complete agreement is found.

  11. Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

    Science.gov (United States)

    Chem, Y K; Chua, K B; Malik, Y; Voon, K

    2015-06-01

    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection. PMID:26691263

  12. Fate of ZN domain wall in hot holographic QCD

    International Nuclear Information System (INIS)

    We first study ZN-domain walls in a deconfined phase of Witten's D4-brane background of pure SU(N) Yang-Mills theory, motivated by a recent work in the case of N = 4 SYM. Similarly to it, we propose that for a large domain wall charge k ∼ N, it is described by k D2-branes blown up into a NS5-brane wrapping S3 inside S4 via Myers effect, and we calculate the tension by suitable U-duality. We find a precise Casimir scaling for the tension formula. We then study the fate of ZN-vacua in a presence of fundamental flavors in quenched approximation via gauge/gravity correspondence. In the case of D3/D7 system where one can vary the mass mq of flavors, we show that there is a phase transition at Tc ∼ mq, below which the ZN-vacua survive while they are lifted above the critical temperature. We analytically calculate the energy lift of k'th vacua in the massless case, both in the D3/D7 system and in the Sakai-Sugimoto model. (author)

  13. The many faces of brane-flux annihilation

    CERN Document Server

    Gautason, Fridrik Freyr; Van Riet, Thomas

    2015-01-01

    Fluxes can decay via the nucleation of Brown-Teitelboim bubbles, but when the decaying fluxes induce D-brane charges this process must be accompanied with an annihilation of D-branes. This occurs via dynamics inside the bubble wall as was well described for (anti-)D3 branes branes annihilating against 3-form fluxes. In this paper we extend this to the other Dp branes with p smaller than seven. Generically there are two decay channels: one for the RR flux and one for the NSNS flux. The RR channel is accompanied by brane annihilation that can be understood from the Dp branes polarising into D(p+2) branes, whereas the NSNS channel corresponds to Dp branes polarising into NS5 branes or KK5 branes. We illustrate this with the decay of antibranes probing local toroidal throat geometries obtained from T-duality of the D6 solution in massive type IIA. We show that anti-Dp branes are metastable against annihilation in these backgrounds, at least at the probe level.

  14. Hepatitis C Virus Protein Interaction Network Analysis Based on Hepatocellular Carcinoma.

    Science.gov (United States)

    Han, Yuewen; Niu, Jun; Wang, Dong; Li, Yuanyuan

    2016-01-01

    Epidemiological studies have validated the association between hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC). An increasing number of studies show that protein-protein interactions (PPIs) between HCV proteins and host proteins play a vital role in infection and mediate HCC progression. In this work, we collected all published interaction between HCV and human proteins, which include 455 unique human proteins participating in 524 HCV-human interactions. Then, we construct the HCV-human and HCV-HCC protein interaction networks, which display the biological knowledge regarding the mechanism of HCV pathogenesis, particularly with respect to pathogenesis of HCC. Through in-depth analysis of the HCV-HCC interaction network, we found that interactors are enriched in the JAK/STAT, p53, MAPK, TNF, Wnt, and cell cycle pathways. Using a random walk with restart algorithm, we predicted the importance of each protein in the HCV-HCC network and found that AKT1 may play a key role in the HCC progression. Moreover, we found that NS5A promotes HCC cells proliferation and metastasis by activating AKT/GSK3β/β-catenin pathway. This work provides a basis for a detailed map tracking new cellular interactions of HCV and identifying potential targets for HCV-related hepatocellular carcinoma treatment. PMID:27115606

  15. Genotypes and viral load of hepatitis C virus among persons attending a voluntary counseling and testing center in Ethiopia.

    Science.gov (United States)

    Abreha, Tesfay; Woldeamanuel, Yimtubezinash; Pietsch, Corinna; Maier, Melanie; Asrat, Daniel; Abebe, Almaz; Hailegiorgis, Bereket; Aseffa, Abraham; Liebert, Uwe Gerd

    2011-05-01

    The prevalence of different genotypes of hepatitis C virus (HCV) in Ethiopia is not known. HCV genotypes influence the response to therapy with alpha-interferon alone or in combination with ribavirin. A cross sectional study was conducted on attendees of voluntary counseling and testing center. Serum samples from 1,954 (734 HIV positive and 1,220 HIV negative) individuals were screened for HCV antibody. Active HCV infection was confirmed by quantitative PCR in 18 of the 71 samples with anti-HCV antibodies. The HCV viral load ranged from 39,650 to 9,878,341 IU/ml (median 1,589,631 IU/ml) with no significant difference [χ(2)(17) = 18.00, P = 0.389] between persons positive or negative for HIV. The viral load of HCV was, however, higher in older study subjects (r = 0.80, P = 0.000). HCV genotypes were determined using the VERSANT HCV Genotype Assay (LiPA) and sequence analysis of the NS5b region of the HCV genome. Diverse HCV genotypes were found including genotypes 1, 2, 4, and 5. There was no difference in the distribution regarding the HIV status. As in other parts of the world, genotyping of HCV must be considered whenever HCV is incriminated as a cause of hepatitis. PMID:21351106

  16. N=(4,4) Gauged Linear Sigma Models for Defect Five-branes

    CERN Document Server

    Kimura, Tetsuji

    2015-01-01

    We study two-dimensional ${\\cal N}=(4,4)$ gauged linear sigma model (GLSM). Its low energy effective theory is a nonlinear sigma model whose target space gives rise to a configuration of five-branes in string theory. In this article we focus on sigma models for NS5-branes, KK5-branes and an exotic $5^2_2$-brane. In particular, we carefully analyze the GLSM for an exotic $5^2_2$-brane whose background configuration is multi-valued. The exotic $5^2_2$-brane is a concrete example of nongeometric configuration in string theory. We find that the exotic feature originates from the string winding coordinate in a very clear way. In order to complete this analysis, we propose a duality transformation formula which converts an ${\\cal N}=(2,2)$ chiral superfield in F-term to a twisted chiral superfield coupled to an unconstrained complex superfield. This article is a short review based on arXiv:1304.4061 in collaboration with Shin Sasaki.

  17. Epidemiological history and phylogeography of West Nile virus lineage 2.

    Science.gov (United States)

    Ciccozzi, Massimo; Peletto, Simone; Cella, Eleonora; Giovanetti, Marta; Lai, Alessia; Gabanelli, Elena; Acutis, Pier Luigi; Modesto, Paola; Rezza, Giovanni; Platonov, Alexander E; Lo Presti, Alessandra; Zehender, Gianguglielmo

    2013-07-01

    West Nile virus (WNV) was first isolated in Uganda. In Europe WNV was sporadically detected until 1996, since then the virus has been regularly isolated from birds and mosquitoes and caused several outbreaks in horses and humans. Phylogenetic analysis showed two main different WNV lineages. The lineage 1 is widespread and segregates into different subclades (1a-c). WNV-1a includes numerous strains from Africa, America, and Eurasia. The spatio-temporal history of WNV-1a in Europe was recently described, identifying two main routes of dispersion, one in Eastern and the second in Western Europe. The West Nile lineage 2 (WNV-2) is mainly present in sub-Saharan Africa but has been recently emerged in Eastern and Western European countries. In this study we reconstruct the phylogeny of WNV-2 on a spatio-temporal scale in order to estimate the time of origin and patterns of geographical dispersal of the different isolates, particularly in Europe. Phylogeography findings obtained from E and NS5 gene analyses suggest that there were at least two separate introductions of WNV-2 from the African continent dated back approximately to the year 1999 (Central Europe) and 2000 (Russia), respectively. The epidemiological implications and clinical consequences of lineage 1 and 2 cocirculation deserve further investigations. PMID:23542457

  18. Little string theory from a double-scaled matrix model

    International Nuclear Information System (INIS)

    Following Lin and Maldacena, we find exact supergravity solutions dual to a class of vacua of the plane wave matrix model by solving an electrostatics problem. These are asymptotically near-horizon D0-brane solutions with a throat associated with NS5-brane degrees of freedom. We determine the precise limit required to decouple the asymptotic geometry and leave an infinite throat solution found earlier by Lin and Maldacena, dual to Little String Theory on S5. By matching parameters with the gauge theory, we find that this corresponds to a double scaling limit of the plane wave matrix model in which N→∞ and the 't Hooft coupling λ scales as ln4(N), which we speculate allows all terms in the genus expansion to contribute even at infinite N. Thus, the double-scaled matrix quantum mechanics gives a Lagrangian description of Little String Theory on S5, or equivalently a ten-dimensional string theory with linear dilaton background

  19. Stable non-supersymmetric supergravity solutions from deformations of the Maldacena-Nunez background

    International Nuclear Information System (INIS)

    We study a deformation of the type IIB Maldacena-Nunez background which arises as the near-horizon limit of NS5 branes wrapped on a two-cycle. This background is dual to a 'little string theory compactified on a two-sphere, a theory which at low energies includes four-dimensional N=1 super Yang-Mills theory. The deformation we study corresponds to a mass term for some of the scalar fields in this theory, and it breaks supersymmetry completely. In the language of seven-dimensional SO(4) gauged supergravity the deformation involves (at leading order) giving a VEV, depending only on the radial coordinate, to a particular scalar field. We explicitly construct the corresponding solution at leading order in the deformation, both in seven-dimensional and in ten-dimensional supergravity, and we verify that it completely breaks supersymmetry. Since the original background had a mass gap and we are performing a small deformation, the deformed background is guaranteed to be stable even though it is not supersymmetric. (author)

  20. Design, synthesis and biological evaluation of pyrido[2,3-d]pyrimidin-7-(8H)-ones as HCV inhibitors.

    Science.gov (United States)

    Camarasa, Marta; Puig de la Bellacasa, Raimon; González, Àlex L; Ondoño, Raül; Estrada, Roger; Franco, Sandra; Badia, Roger; Esté, José; Martínez, Miguel Ángel; Teixidó, Jordi; Clotet, Bonaventura; Borrell, José I

    2016-06-10

    The design and selection of a combinatorial library of pyrido[2,3-d]pyrimidin-7(8H)-ones (4) has allowed the synthesis of 121 compounds, using known and new synthetic methodologies, and the evaluation of the inhibitory activity against hepatitis C virus (HCV) genotype 1b replicon. Among these compounds, 21{4,10} and 24{2,10} presented very high activities [EC50 = 0.027 μM (CC50 = 5.3 μM) and EC50 = 0.034 μM (CC50 = 13.5 μM), respectively] and high selectivity indexes, 196 and 397. These values are similar to the EC50 reported for sofosbuvir (2) (0.048 μM) using a similar methodological approach and the same virus subtype. 21{4,10} and 24{2,10} are obtained through shorter synthetic itineraries than sofosbuvir and 24{2,10} is achiral contrary to sofosbuvir which presents 4 stereogenic centers. In silico studies suggest that 21{4,10} and 24{2,10} inhibits NS5B polymerase through allosteric site binding. PMID:27054294

  1. Oscillating supertubes and neutral rotating black hole microstates

    International Nuclear Information System (INIS)

    The construction of neutral black hole microstates is an important problem, with implications for the information paradox. In this paper we conjecture a construction of non-supersymmetric supergravity solutions describing D-brane configurations which carry mass and angular momentum, but no other conserved charges. We first study a classical string solution which locally carries dipole winding and momentum charges in two compact directions, but globally carries no net winding or momentum charge. We investigate its backreaction in the D1-D5 duality frame, where this object becomes a supertube which locally carries oscillating dipole D1-D5 and NS1-NS5 charges, and again carries no net charge. In the limit of an infinite straight supertube, we find an exact supergravity solution describing this object. We conjecture that a similar construction may be carried out based on a class of two-charge non-supersymmetric D1-D5 solutions. These results are a step towards demonstrating how neutral black hole microstates may be constructed in string theory

  2. Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    Science.gov (United States)

    Dixit, Updesh; Pandey, Ashutosh K.; Mishra, Priya; Sengupta, Amitabha; Pandey, Virendra N.

    2016-01-01

    Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3′ NTR and domain IIId of the HCV-IRES in the 5′ NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication. PMID:27106056

  3. Viral genome imaging of hepatitis C virus to probe heterogeneous viral infection and responses to antiviral therapies.

    Science.gov (United States)

    Ramanan, Vyas; Trehan, Kartik; Ong, Mei-Lyn; Luna, Joseph M; Hoffmann, Hans-Heinrich; Espiritu, Christine; Sheahan, Timothy P; Chandrasekar, Hamsika; Schwartz, Robert E; Christine, Kathleen S; Rice, Charles M; van Oudenaarden, Alexander; Bhatia, Sangeeta N

    2016-07-01

    Hepatitis C virus (HCV) is a positive single-stranded RNA virus of enormous global health importance, with direct-acting antiviral therapies replacing an immunostimulatory interferon-based regimen. The dynamics of HCV positive and negative-strand viral RNAs (vRNAs) under antiviral perturbations have not been studied at the single-cell level, leaving a gap in our understanding of antiviral kinetics and host-virus interactions. Here, we demonstrate quantitative imaging of HCV genomes in multiple infection models, and multiplexing of positive and negative strand vRNAs and host antiviral RNAs. We capture the varying kinetics with which antiviral drugs with different mechanisms of action clear HCV infection, finding the NS5A inhibitor daclatasvir to induce a rapid decline in negative-strand viral RNAs. We also find that the induction of host antiviral genes upon interferon treatment is positively correlated with viral load in single cells. This study adds smFISH to the toolbox available for analyzing the treatment of RNA virus infections. PMID:27128351

  4. Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

    International Nuclear Information System (INIS)

    We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC5) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78 ± 21 μM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 μM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin

  5. 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.

  6. Radiative lifetimes in MoI using a novel atomic beam source

    International Nuclear Information System (INIS)

    Radiative lifetimes of the 4d5(a6S)5pz7P0 levels and the 4d5(a6S)5pz5P0 levels in MoI are reported as follows: tau(z7P04) = 15.9 ns, tau(z7P03) = 17.0 ns, tau(z7P02) = 17.1 ns, tau(z5P03) = 22.3 ns, tau(z5P02) = 22.1 ns, and tau(z5P01) = 21.7 ns (+-5%). The lifetimes are measured using time resolved laser induced fluorescence and a novel atomic beam source. This novel hollow cathode effusive beam source produces an intense beam of ground state Mo atoms and metastable Mo atoms. Our measurements on the z7P0 levels are in agreement with earlier lifetime measurements. Our measurements on the z5P0 levels are the first direct radiative lifetime measurements of these levels; the z5P0 measurements are of particular interest because the a5S → z5P0 multiplet is used to determine the solar abundance of Mo. (orig.)

  7. The Laser Vegetation Imaging Sensor: a medium-altitude, digitisation-only, airborne laser altimeter for mapping vegetation and topography

    Science.gov (United States)

    Blair, J. Bryan; Rabine, David L.; Hofton, Michelle A.

    The Laser Vegetation Imaging Sensor (LVIS) is an airborne, scanning laser altimeter, designed and developed at NASA's Goddard Space Flight Center (GSFC). LVIS operates at altitudes up to 10 km above ground, and is capable of producing a data swath up to 1000 m wide nominally with 25-m wide footprints. The entire time history of the outgoing and return pulses is digitised, allowing unambiguous determination of range and return pulse structure. Combined with aircraft position and attitude knowledge, this instrument produces topographic maps with dm accuracy and vertical height and structure measurements of vegetation. The laser transmitter is a diode-pumped Nd:YAG oscillator producing 1064 nm, 10 ns, 5 mJ pulses at repetition rates up to 500 Hz. LVIS has recently demonstrated its ability to determine topography (including sub-canopy) and vegetation height and structure on flight missions to various forested regions in the US and Central America. The LVIS system is the airborne simulator for the Vegetation Canopy Lidar (VCL) mission (a NASA Earth remote sensing satellite due for launch in year 2000), providing simulated data sets and a platform for instrument proof-of-concept studies. The topography maps and return waveforms produced by LVIS provide Earth scientists with a unique data set allowing studies of topography, hydrology, and vegetation with unmatched accuracy and coverage.

  8. Non-Gaussian signatures arising from warm inflation driven by geometric tachyon

    International Nuclear Information System (INIS)

    In a warm inflationary scenario, the initial seeds of density perturbation arise from thermal fluctuations of the inflaton field. These fluctuations in principle have Gaussian distribution. In a Gaussian distribution the density perturbation can be expressed as the two point correlation function. Thus if in an inflationary model the density perturbation is expressed as correlation function of order higher than two, these fluctuations are non-Gaussian in nature. A simple inflationary model containing single scalar field, slow roll, canonical kinetic term and vacuum initial state can produce a tiny amount of non-Gaussianity which are very small to be detected by any experiment. Non-Gaussianity can also arise in inflationary models containing multiple scalar fields. For an inflationary scenario with single scalar field, non-Gaussianity can be expressed in terms of bi-spectrum however for multi field Inflation, it is expressed in terms of trispectrum etc. In this piece of work, the warm inflationary scenario, driven by a D3 brane due to the presence of a stack of k coincident NS 5 branes is considered and the non-Gaussian effects in such an inflationary scenario has been analysed by measuring the bispectrum of the gravitational field fluctuations generated during the warm inflation in strong dissipative regime. The bi-spectrum of the Inflation is expressed in terms of the parameter fNL and it is seen that the value of fNL parameter lies well within the limit observed by WMAP7

  9. Hepatitis B and C prevalences among blood donors in the south region of Brazil

    Directory of Open Access Journals (Sweden)

    H. C. F. F. Vasconcelos

    1994-12-01

    Full Text Available The prevalence of hepatitis B and C infection has been determined in a seroepidemiological survey among blood donors from the south of Brazil (Florianópolis, State of Santa Catarina. These markers has also been correlated with the levels of alanine aminotransferase (ALT, a surrogate marker to prevent post-transfusion hepatitis. Sera from 5000 donors were randomly collected in the period of April to November 1991. The prevalences of HBsAg, anti-HBs and anti-HBc were respectively 0.78, 7.02 and 13.98. The anti-HCV prevalence after confirmation testing with line immunoassay (LIA, was 1.14. Normal values of ALT ( = 70 U/ml in 2.48. The positivity of anti-HCV antibodies increased with the elevation of ALT levels. This correlation was not observed in relation to HBsAg. There exists a diversity in the recognition of HCV epitopes among HCV positive donors. Via the confirmation test used, we could observe that 94.7 of donors recognize the structural core antigen. Besides that, we observed that 5.26 of the HCV reactive sera recognized only epitopes located in the NS4 and/or NS5 region, indicating the importance of these epitopes for the improvement of assays.

  10. Antibody recognition of the dengue virus proteome and implications for development of vaccines.

    Science.gov (United States)

    Fernandez, Stefan; Cisney, Emily D; Tikhonov, Alexander P; Schweitzer, Barry; Putnak, Robert J; Simmons, Monika; Ulrich, Robert G

    2011-04-01

    Dengue is a mosquito-borne infection caused by four distinct serotypes of dengue virus, each appearing cyclically in the tropics and subtropics along the equator. Although vaccines are currently under development, none are available to the general population. One of the main impediments to the successful advancement of these vaccines is the lack of well-defined immune correlates of protection. Here, we describe a protein microarray approach for measuring antibody responses to the complete viral proteome comprised of the structural (capsid, membrane, and envelope) and nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) components of all four dengue virus serotypes (1 to 4). We examined rhesus macaques vaccinated with tetravalent vaccines consisting of live-attenuated virus (LAV) or purified inactivated virus (PIV), followed by boosting with LAV and challenging with wild-type dengue virus. We detected temporal increases in antibodies against envelope proteins in response to either vaccine, while only the PIV/LAV vaccination strategy resulted in anticapsid antibodies. In contrast to results from vaccination, naïve macaques challenged with wild-type viruses of each serotype demonstrated a balanced response to nonstructural and structural components, including responses against the membrane protein. Our results demonstrate discriminating details concerning the nature of antibody responses to dengue virus at the proteomic level and suggest the usefulness of this information for vaccine development. PMID:21270280

  11. Hyperbolic Spaces in String and M-Theory

    International Nuclear Information System (INIS)

    We describe string-theory and d=11 supergravity solutions involving symmetric spaces of constant negative curvature. Many examples of non-supersymmetric string compactifications on hyperbolic spaces Hr of finite volume are given in terms of suitable cosets of the form Hr and Γ, where Γ is a discrete group. We describe in some detail the cases of the non-compact hyperbolic spaces F2 and F3, representing the fundamental regions of H2 and H3 under SL(2,Z) and the Picard group, respectively. By writing AdS as a U(1) fibration, we obtain new solutions where AdS2p+1 gets untwisted by T-duality to /bf RxSU(p,1)/(SU(p)xU(1)). Solutions with time-dependent dilaton field are also constructed by starting with a solution with NS5-brane flux over H3. A new class of non-supersymmetric conformal field theories can be defined via holography. (author)

  12. Inflation from Supergravity with Gauged R-symmetry in de Sitter Vacuum

    CERN Document Server

    Antoniadis, I; Isono, H; Knoops, R

    2016-01-01

    We study the cosmology of a recent model of supersymmetry breaking, in the presence of a tuneable positive cosmological constant, based on a gauged shift symmetry of a string modulus that can be identified with the string dilaton. The minimal spectrum of the `hidden' supersymmetry breaking sector consists then of a vector multiplet that gauges the shift symmetry of the dilaton multiplet and when coupled to the MSSM leads to a distinct low energy phenomenology depending on one parameter. Here we study the question if this model can also lead to inflation by identifying the dilaton with the inflaton. We find that this is possible if the K\\"ahler potential is modified by a term that has the form of NS5-brane instantons, leading to an appropriate inflationary plateau around the maximum of the scalar potential, depending on two extra parameters. This model is consistent with present cosmological observations without modifying the low energy particle phenomenology associated to the minimum of the scalar potential.

  13. Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells.

    Science.gov (United States)

    Dixit, Updesh; Pandey, Ashutosh K; Mishra, Priya; Sengupta, Amitabha; Pandey, Virendra N

    2016-06-20

    Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3' NTR and domain IIId of the HCV-IRES in the 5' NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication. PMID:27106056

  14. Short communication. Further evidence of lineage 2 West Nile Virus in Culex pipiens of North-Eastern Italy

    Directory of Open Access Journals (Sweden)

    Gioia Capelli

    2013-09-01

    Full Text Available West Nile Virus lineage 1 (WNV lin1 emerged in North-Eastern Italy in 2008 and, since then, it has been detected in animals, humans and mosquitoes. Three years later, in the same area, a lineage 2 (lin2 strain of WNV was found in birds and vectors. On August the 21st, during the 2012 WNV entomological surveillance plan, a WNV lin2 strain was detected by RT-PCR in a pool of Culex pipiens mosquitoes captured in Veneto region. According to the alignment of the partial sequences of the NS5 and NS3 genes, no differences between this Italian lineage 2 strain and the Nea Santa-Greece-2010 WNV isolate (Gr-10 were observed. Similarly to the Gr-10 strain, the putative NS3 aminoacid sequences of the Italian strain showed proline in position 249 instead of histidine (H249P. Although proline in position 249 has been suggested to increase the virulence of WNV strains, neither human nor veterinary cases associated to this strain have been reported in the region. A prompt mosquito disinfestation was organized to avoid the spread of this potential threatening virus. The simultaneous circulation of both WNV lineage 1 and 2 confirms North-Eastern Italy as a high risk area for WNV emergence and highlights the need for a continuous surveillance.

  15. 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. PMID:27101072

  16. Isolation and genetic characterization of a tembusu virus strain isolated from mosquitoes in Shandong, China.

    Science.gov (United States)

    Tang, Y; Diao, Y; Chen, H; Ou, Q; Liu, X; Gao, X; Yu, C; Wang, L

    2015-04-01

    Tembusu virus (TMUV) is a flavivirus, presumed to be a mosquito-borne flavivirus of the Ntaya virus subgroup. To date, however, there have been no reports indicating that mosquitoes are involved in the spread of TMUV. In this study, we report the first isolation of TMUV from Culex mosquitoes. We describe the isolation and characterization of a field strain of TMUV from mosquitoes collected in Shandong Province, China. The virus isolate, named TMUV-SDMS, grows well in mosquito cell line C6/36, in Vero and duck embryo fibroblast (DEF) cell lines, and causes significant cytopathic effects in these cell cultures. The TMUV-SDMS genome is a single-stranded RNA, 10 989 nt in length, consisting of a single open reading frame encoding a polyprotein of 3410 amino acids, with 5' and 3' untranslated regions of 142 and 617 nt, respectively. Phylogenetic analysis of the E and NS5 genes revealed that the TMUV-SDMS is closely related to the TMUV YY5 and BYD strains which cause severe egg-drop in ducks. The 3'NTR of TMUV-SDMS contains two pairs of tandem repeat CS and one non-duplicate CS, which have sequence similarities to the same repeats in the YY5 and BYD strains. Our findings indicate that mosquitoes carrying the TMUV may play an important role in the spread of this virus and in disease outbreak. PMID:23711093

  17. Hepatitis C Virus Protein Interaction Network Analysis Based on Hepatocellular Carcinoma.

    Directory of Open Access Journals (Sweden)

    Yuewen Han

    Full Text Available Epidemiological studies have validated the association between hepatitis C virus (HCV infection and hepatocellular carcinoma (HCC. An increasing number of studies show that protein-protein interactions (PPIs between HCV proteins and host proteins play a vital role in infection and mediate HCC progression. In this work, we collected all published interaction between HCV and human proteins, which include 455 unique human proteins participating in 524 HCV-human interactions. Then, we construct the HCV-human and HCV-HCC protein interaction networks, which display the biological knowledge regarding the mechanism of HCV pathogenesis, particularly with respect to pathogenesis of HCC. Through in-depth analysis of the HCV-HCC interaction network, we found that interactors are enriched in the JAK/STAT, p53, MAPK, TNF, Wnt, and cell cycle pathways. Using a random walk with restart algorithm, we predicted the importance of each protein in the HCV-HCC network and found that AKT1 may play a key role in the HCC progression. Moreover, we found that NS5A promotes HCC cells proliferation and metastasis by activating AKT/GSK3β/β-catenin pathway. This work provides a basis for a detailed map tracking new cellular interactions of HCV and identifying potential targets for HCV-related hepatocellular carcinoma treatment.

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

    Directory of Open Access Journals (Sweden)

    Margo A. Brinton

    2013-12-01

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

  19. Treatment of early-stage erythematotelangiectatic rosacea with a Q-switched 595-nm Nd:YAG laser.

    Science.gov (United States)

    Goo, Boncheol Leo; Kang, Jin-Soo; Cho, Sung Bin

    2015-06-01

    Erythematotelangiectatic rosacea presents as persistent erythema and telangiectasia with frequent flushing and blushing on the facial and extrafacial skin. Additionally, papulopustular rosacea shows acneiform papules, pustules, and nodules with persistent plaque-form edema. Despite garnering only grade-C or -D level recommendations, a 585-nm or 595-nm flashlamp-pumped pulsed-dye laser can be considered as an effective therapeutic modality for the treatment of rosacea in patients who are refractory to topical and/or systemic treatments. In this report, treatment with a Q-switched 595-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) laser with low non-purpuragenic fluence proved to be safe and effective in treating early-stage erythematotelangiectatic rosacea in two female Korean patients. Laser treatment for rosacea was delivered with the settings of pulse energy of 0.4-0.5 J/cm(2), pulse duration of 5-10 ns, 5-mm spot size, 5 Hz, and 500 shots. Additionally, we found that remarkable therapeutic effects were achieved for both rosacea and melasma by combining Q-switched quick pulse-to-pulse 1,064-nm Nd:YAG and Q-switched 595-nm Nd:YAG laser treatments, which required only the changing of handpieces equipped with solid dye. In conclusion, we suggest that treatment with a Q-switched 595-nm Nd:YAG laser with low fluence may provide an additional therapeutic option for treating early-stage erythematotelangiectatic rosacea. PMID:25549817

  20. Genetic Variability of Bovine Viral Diarrhea Virus and Evidence for a Possible Genetic Bottleneck during Vertical Transmission in Persistently Infected Cattle.

    Directory of Open Access Journals (Sweden)

    Natalie Dow

    Full Text Available Bovine viral diarrhea virus (BVDV, a Pestivirus in the family Flaviviridae, is an economically important pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI cattle, which shed large quantities of virus throughout life. Despite the absence of an acquired immunity against BVDV in these PI cattle there are strong indications of viral variability that are of clinical and epidemiological importance. In this study the variability of E2 and NS5B sequences in multiple body compartments of PI cattle were characterized using clonal sequencing. Phylogenetic analyses revealed that BVDV exists as a quasispecies within PI cattle. Viral variants were clustered by tissue compartment significantly more often than expected by chance alone with the central nervous system appearing to be a particularly important viral reservoir. We also found strong indications for a genetic bottleneck during vertical transmission from PI animals to their offspring. These quasispecies analyses within PI cattle exemplify the role of the PI host in viral propagation and highlight the complex dynamics of BVDV pathogenesis, transmission and evolution.

  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. Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints.

    Directory of Open Access Journals (Sweden)

    October M Sessions

    Full Text Available Dengue viruses (DENV cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients' sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.

  3. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  4. Biological and molecular characterization of classical swine fever challenge virus from India

    Directory of Open Access Journals (Sweden)

    Parveen Kumar

    2015-03-01

    Full Text Available Aim: The aim of this study was biological and molecular characterization of classical swine fever (CSF challenge virus from India. Materials and Methods: CSF challenge virus maintained at Division of Biological standardization was experimentally infected to two seronegative piglets. The biological characterization was done by clinical sign and symptoms along with postmortem findings. For molecular characterization 5’-nontranslated region, E2 and NS5B regions were amplified by reverse transcription polymerase chain reaction and sequenced. The sequences were compared with that of reference strains and the local field isolates to establish a phylogenetic relation. Results: The virus produced symptoms of acute disease in the piglets with typical post-mortem lesions. Phylogenetic analysis of the three regions showed that the current Indian CSF Challenge virus is having maximum similarity with the BresciaX strain (USA and Madhya Pradesh isolate (India and is belonging to subgroup 1.2 under Group 1. Conclusion: Based on biological and molecular characterization of CSF challenge virus from India is described as a highly virulent virus belonging to subgroup 1.2 under Group 1 along with some field isolates from India and Brescia strain.

  5. The IR obstruction to UV completion for Dante's Inferno model with higher-dimensional gauge theory origin

    Science.gov (United States)

    Furuuchi, Kazuyuki; Koyama, Yoji

    2016-06-01

    We continue our investigation of large field inflation models obtained from higher-dimensional gauge theories, initiated in our previous study [1]. We focus on Dante's Inferno model which was the most preferred model in our previous analysis. We point out the relevance of the IR obstruction to UV completion, which 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. Current Drug Discovery for Anti-hepatitis C Virus Targeting NS4B.

    Science.gov (United States)

    Wang, Zhenya; Chen, Xinli; Wu, Chunli; Xu, Haiwei; Liu, Hongmin

    2016-01-01

    Hepatitis C virus (HCV) infection is a major worldwide epidemic disease. It is estimated that more than 170 million individuals are infected with HCV and with three to four million new cases each year. Many new direct-acting antiviral (DAA) agents that specifically target HCV NS3 protease or NS5B polymerase inhibitors are therefore in development, with a significant effect for the patient and for the market recently. The non-structural 4B (NS4B) protein, is among the least characterized of the HCV proteins. A variety of functions have been recognized for NS4B, such as the ability to induce the membranous web replication platform, RNA binding and NTPase activity. In order to maximize antiviral efficacy and prevent the emergence of resistance, novel NS4B inhibitors have been subjected to pharmacological studies. In this review, we discussed current understanding of the structure and function of NS4B, and novel drug discoveries targeting NS4B as anti-hepatitis C virus such as sulfonamide, piperidine, carboxamide, piperazinone and quinoline derivatives within the last three years. PMID:26585935

  7. Z p charged branes in flux compactifications

    Science.gov (United States)

    Berasaluce-González, M.; Cámara, P. G.; Marchesano, F.; Uranga, A. M.

    2013-04-01

    We consider 4d string compactifications in the presence of fluxes, and classify particles, strings and domain walls arising from wrapped branes which have charges conserved modulo an integer p, and whose annihilation is catalized by fluxes, through the Freed-Witten anomaly or its dual versions. The Z p -valued strings and particles are associated to Z p discrete gauge symmetries, which we show are realized as discrete subgroups of 4d U(1) symmetries broken by their Chern-Simons couplings to the background fluxes. We also describe examples where the discrete gauge symmetry group is actually non-Abelian. The Z p -valued domain walls separate vacua which have different flux quanta, yet are actually equivalent by an integer shift of axion fields (or further string duality symmetries). We argue that certain examples are related by T-duality to the realization of discrete gauge symmetries and Z p charges from torsion (co)homology. At a formal level, the groups classifying these discrete charges should correspond to a generalization of K-theory in the presence of general fluxes (and including fundamental strings and NS5-branes).

  8. In-silico analysis of putative HCV epitopes against Pakistani human leukocyte antigen background: An approach towards development of future vaccines for Pakistani population.

    Science.gov (United States)

    Ashraf, Naeem Mahmood; Bilal, Muhammad; Mahmood, Malik Siddique; Hussain, Aadil; Mehboob, Muhammad Zubair

    2016-09-01

    Mounting burden of HCV-infected individuals and soaring cost of treatment is a serious source of unease for developing countries. Numbers of various approaches have been anticipated to develop a vaccine against HCV but the majority of them proved ineffective. Development of vaccine by considering geographical distribution of HCV genotypes and host genetics shows potential. In this research article, we have tried to predict most putative HCV epitopes which are efficiently restricted by most common HLA alleles in Pakistani population through different computational algorithms. Thirteen selected, experimentally identified epitopes sequences were used to derived consensus sequences in all genotypes of HCV. Obtained consensus sequences were used to predict their binding affinities with most prevalent HLA alleles in Pakistani population. Two Class-I epitopes from NS4B region, one from Class-I epitope from NS5A and one Class-II epitope from NS3 region showed effective binding and proved to be highly putative to boost immune response. A cocktail of these four have been checked for population coverage and they gave 75.53% for Pakistani Asian and 70.77% for Pakistani Mixed populations with no allergenic response. Computational algorithms are robust way to shortlist potential candidate epitopes for vaccine development but further, in vivo and in-vitro studies are required to confirm their immunogenic properties. PMID:27166094

  9. Role of different regions of the hepatitis C virus genome in the therapeutic response to interferon-based treatment.

    Science.gov (United States)

    Khaliq, Saba; Latief, Noreen; Jahan, Shah

    2014-01-01

    Hepatitis C virus (HCV) is considered a significant risk factor in HCV-induced liver diseases and development of hepatocellular carcinoma (HCC). Nucleotide substitutions in the viral genome result in its diversification into quasispecies, subtypes and distinct genotypes. Different genotypes vary in their infectivity and immune response due to these nucleotide/amino acid variations. The current combination treatment for HCV infection is pegylated interferon α (PEG-IFN-α) with ribavirin, with a highly variable response rate mainly depending upon the HCV genotype. Genotypes 2 and 3 are found to respond better than genotypes 1 and 4, which are more resistant to IFN-based therapies. Different studies have been conducted worldwide to explore the basis of this difference in therapy response, which identified some putative regions in the HCV genome, especially in Core and NS5a, and to some extent in the E2 region, containing specific sequences in different genotypes that act differently with respect to the IFN response. In the review, we try to summarize the role of HCV proteins and their nucleotide sequences in association with treatment outcome in IFN-based therapy. PMID:23851652

  10. Ribavirin: Past, present and future

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    Before the advent of direct acting antiviral agents (DAAs)ribavirin, associated to pegylated-interferon playeda crucial role in the treatment of chronic hepatitis C,preventing relapses and breakthroughs. In the presentera of new potent DAAs, a place is still devoted tothe drug. Ribavirin associated with sofosbuvir alone isefficient in the treatment of most cases of G2 infectedpatients. All options currently available for the lastdifficult-to-treat cirrhotic G3 patients contain ribavirin.Reducing treatment duration to 12 wk in G1 or G4cirrhotic compensated patients is feasible thanks toribavirin. Retreating patients with acquired anti NS5Aresistance-associated variants using ribavirin-basedstrategies could be useful. The addition of ribavirin withDAAs combinations however, leads to more frequentbut mild adverse events especially in cirrhotic patients.Preliminary data with interferon-free second generationDAAs combinations without ribavirin suggest thatfuture of the drug is jeopardized even in difficult-totreatpatients The optimization of ribavirin dosageaccording to an early monitoring of blood levels hasbeen suggested to be relevant in double therapy withpeginterferon or sofosbuvir but not with very potentcombinations of more than two DAAs.

  11. CuO nanoparticles: Synthesis, characterization, optical properties and interaction with amino acids

    International Nuclear Information System (INIS)

    Cupric oxide (CuO) nanoparticles with an average size of 6 nm have been successfully prepared by an alcothermal method. The prepared CuO nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier-transform infrared (FT-IR) and UV-visible absorption spectroscopy. A strong sharp emission under UV excitation is reported from the prepared CuO nanoparticles. The results show that the CuO nanoparticles have high dispersion and narrow size distribution. The fluorescence emission spectra display an intense sharp emission at 365 nm and weak broad intensity emission at 470 nm. Picosecond fluorescence measurements of the nanoparticles suggest bi-exponential function giving time constants of τ1 (330 ps, 94.21%) and τ2 (4.69 ns, 5.79%). In neutral and alkaline solutions, Zeta potential values of CuO nanoparticles are negative, due to the adsorption of COO- group via the coordination of bidentate. At low pH the zeta potential value is positive due to the increased potential of H+ ions in solution. Comparative UV-visible absorption experiments with the model amino acid compounds of positive and negative charges as arginine and aspartic acid, respectively confirmed the negative surface of CuO nanoparticles. The results should be extremely useful for understanding the mode of the interaction with biological systems. This binding process also affects the particle's behavior inside the body.

  12. Detection of novel insect flavivirus sequences integrated in Aedes albopictus (Diptera: Culicidae in Northern Italy

    Directory of Open Access Journals (Sweden)

    Tenorio Antonio

    2009-07-01

    Full Text Available Abstract The presence of DNA sequences integrated from a new flavivirus related to Cell Fusing Agent and Kamiti River Virus was identified in wild Aedes albopictus mosquito populations from the provinces of Trentino and Padova, Northern Italy. Field work was developed during August–October 2007 with BG-traps, and mosquitoes were screened for flavivirus and alphavirus. No alphavirus was detected, indicating that Chikungunya virus is not present in these mosquitoes in Trentino and Padova area. However, 21% of the pools were positive for flavivirus, further recognised with BLAST as similar to Kamiti River Virus. Phylogenetical analysis with 708 nucleotides from the NS5 gene identified this virus as a new member of the insect flavivirus clade, together with others like Kamiti River Virus, Cell Fusing Agent or Culex flavivirus, and in the group of those transmitted by Aedes. Furthermore, the treatment with RNAse, indicated that this flavivirus should be integrated in the genome of Ae. albopictus. These results propose that these sequences are transmitted by both sexes, and with different prevalence in the studied populations, and support the idea of a widespread distribution of integrated genomes in several mosquitoes from different areas, as first demonstrated with Cell Silent Agent. Evolutionary implications of this discovery and application in flavivirus phylogeny are discussed.

  13. Indian National Association for Study of the Liver (INASL) Guidance for Antiviral Therapy Against HCV Infection: Update 2016.

    Science.gov (United States)

    Puri, Pankaj; Saraswat, Vivek A; Dhiman, Radha K; Anand, Anil C; Acharya, Subrat K; Singh, Shivaram P; Chawla, Yogesh K; Amarapurkar, Deepak N; Kumar, Ajay; Arora, Anil; Dixit, Vinod K; Koshy, Abraham; Sood, Ajit; Duseja, Ajay; Kapoor, Dharmesh; Madan, Kaushal; Srivastava, Anshu; Kumar, Ashish; Wadhawan, Manav; Goel, Amit; Verma, Abhai; Shalimar; Pandey, Gaurav; Malik, Rohan; Agrawal, Swastik

    2016-06-01

    India contributes significantly to the global burden of HCV. While the nucleoside NS5B inhibitor sofosbuvir became available in the Indian market in March 2015, the other directly acting agents (DAAs), Ledipasvir and Daclatasvir, have only recently become available in the India. The introduction of these DAA in India at a relatively affordable price has led to great optimism about prospects of cure for these patients as not only will they provide higher efficacy, but combination DAAs as all-oral regimen will result in lower side effects than were seen with pegylated interferon alfa and ribavirin therapy. Availability of these newer DAAs has necessitated revision of INASL guidelines for the treatment of HCV published in 2015. Current considerations for the treatment of HCV in India include the poorer response of genotype 3, nonavailability of many of the DAAs recommended by other guidelines and the cost of therapy. The availability of combination DAA therapy has simplified therapy of HCV with decreased reliance of evaluation for monitoring viral kinetics or drug related side effects. PMID:27493460

  14. HIV/HCV共感染合并血友病患者利巴韦林联合干扰素治疗前后特异性细胞免疫反应的研究%Study of cellular immune responses following treatment with interferon and ribavirin in patients of HIV/hepatitis C virus co-infection in combination with hemophilia

    Institute of Scientific and Technical Information of China (English)

    吕盈盈; 王江蓉; 廖晶; 谷俊朝

    2011-01-01

    Objective To study the hepatitis C virus(HCV)-specific immune responses following treatment with interferon and ribavirin in subjects with HIV-1/HCV co-infection in combination with hemophilia. Methods Immune responses were studied in a treatment trial with pegylated interferon alfa (PEG-IFN) and ribavirin(R). By using HCV core antigens,NS3 and NS5,enzyme-linked immunosorbent spots on peripheral blood mononuclear cells,products of IFN-y,IL-2,IL-4,IL-10 were measured. Immunologic,virologic and clinical variables were modeled as identifying factors associated with HCV virological response before and after 72 weeks ( SVR) treatment in 36 patients. Results There were no significant differences in baseline IFN-y and IL-2 immune responses and higher IL-10 to NS3 in subjects with VR versus non-respnnders. Subjects who had significant decreases in IL-10 responses at week 72 compared with baseline for NS3,NS5,were more likely to be VR. Using baseline immunological responses and clinical data in CART models, patients who were randomized to PEC1FN/R and had high IL-10 responses to summed HCV proteins were more likely to be VR. In SVR,IL-2 production decreased moderately, 1L-4 secreted little. The main correlation of SVR for genotype-1 subjects maintained HCV-specific IFN-7 responses from baseline to week 72. Conclusions In subjects of HIV and HCV co-infection combined with hemophilia, decrease in HCV-specific IL-10 and IL-2 responses during treatment and maintenance of IFN-y responses with IFN and ribavirin were associated with 24 or 72 week virological response.%目的 研究HIV/HCV共感染合并血友病患者在利巴韦林联合干扰素治疗过程中的细胞免疫反应,了解此类患者在抗HCV治疗过程中HCV的损伤机制和免疫重建过程.方法 收集36例HIV/HCV共感染合并血友病患者,分别在干扰素和利巴韦林治疗前、治疗结束24周后采集外周血单个核细胞( PBMC),Elispot方法检测在HCV核心抗原、NS3和NS5

  15. Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain

    Science.gov (United States)

    Wu, Xiaogang; Shi, Ying; Yan, Dawei; Li, Xuesong; Yan, Pixi; Gao, Xuyuan; Zhang, Yuee; Yu, Lei; Ren, Chaochao; Li, Guoxin; Yan, Liping; Teng, Qiaoyang; Li, Zejun

    2016-01-01

    The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV. PMID:27248497

  16. Effect of Retinoic Acid on Lung Injury in Hyperoxia-Exposed Newborn Rats

    Institute of Scientific and Technical Information of China (English)

    常立文; 容志惠; 张谦慎; 钱莉玲

    2003-01-01

    To investigate whether treatment with retinoic acid (RA) could improve level of lung alveolarization and influence lung collagen in newborn rats exposed to hyperoxia, newborn SpragueDawley rats aged 2 days were randomly assigned to 8 groups: (1) air, (2) O2, (3) air+NS, (4)O2 +NS, (5) air+dex, (6) O2+dex, (7) air+RA and (8) O2+RA. Group 2, 4 6 and 8 were kept in chambers containing 85 % oxygen, the values were checked 3 times a day. The other 4 groups were exposed to room air. Level of alveolarization and lung collagen were analyzed at age of 14 or 21 days through radial alveolar counts, alveolar airspace measurements, type Ⅰ , Ⅲ collagen immunohistochemical methods (SP method) and image processing system. Transforming growth factor-β receptors and procollagen mRNA accumulation were examined at age of 14 days through immunohistochemical methods and in situ hybridization. Our results showed that radial alveolar counts were increased and distal airspace was enlarged in group 8. Type I collagen was markedly increased, and transforming growth factor-β receptors and procollagen mRNA were decreased by retinoic acid in bronchial epithelial cells, alveolar epithelial cells and alveolar intersitium. It is concluded that retinoic acid can partially reverse lung development arrest during exposure to hyperoxia by increasing lung collagen.

  17. Elliptic Genus of E-strings

    CERN Document Server

    Kim, Joonho; Lee, Kimyeong; Park, Jaemo; Vafa, Cumrun

    2014-01-01

    We study a family of 2d N=(0,4) gauge theories which describes at low energy the dynamics of E-strings, the M2-branes suspended between a pair of M5 and M9 branes. The gauge theory is engineered using a duality with type IIA theory, leading to the D2-branes suspended between an NS5-brane and 8 D8-branes on an O8-plane. We compute the elliptic genus of this family of theories, and find agreement with the known results for single and two E-strings. The partition function can in principle be computed for arbitrary number of E-strings, and we compute them explicitly for low numbers. We test our predictions against the partially known results from topological strings, as well as from the instanton calculus of 5d Sp(1) gauge theory. Given the relation to topological strings, our computation provides the all genus partition function of the refined topological strings on the canonical bundle over 1/2 K3.

  18. Oscillating supertubes and neutral rotating black hole microstates

    CERN Document Server

    Mathur, Samir D

    2014-01-01

    The construction of neutral black hole microstates is an important problem, with implications for the information paradox. In this paper we conjecture a construction of non-supersymmetric supergravity solutions describing D-brane configurations which carry mass and angular momentum, but no other conserved charges. We first study a classical string solution which locally carries dipole winding and momentum charges in two compact directions, but globally carries no net winding or momentum charge. We investigate its backreaction in the D1-D5 duality frame, where this object becomes a supertube which locally carries oscillating dipole D1-D5 and NS1-NS5 charges, and again carries no net charge. In the limit of an infinite straight supertube, we find an exact supergravity solution describing this object. We conjecture that a similar construction may be carried out based on a class of two-charge non-supersymmetric D1-D5 solutions. These results are a step towards demonstrating how neutral black hole microstates may ...

  19. Massive S-matrix of AdS{sub 3}×S{sup 3}×T{sup 4} superstring theory with mixed 3-form flux

    Energy Technology Data Exchange (ETDEWEB)

    Hoare, B., E-mail: ben.hoare@physik.hu-berlin.de [Institut für Physik, Humboldt-Universität zu Berlin, Newtonstraße 15, D-12489 Berlin (Germany); Tseytlin, A.A., E-mail: tseytlin@imperial.ac.uk [The Blackett Laboratory, Imperial College, London SW7 2AZ (United Kingdom)

    2013-08-11

    The type IIB supergravity AdS{sub 3}×S{sup 3}×T{sup 4} background with mixed RR and NSNS 3-form fluxes is a near-horizon limit of a non-threshold bound state of D5–D1 and NS5–NS1 branes. The corresponding superstring world-sheet theory is expected to be integrable, opening the possibility of computing its exact spectrum for any values of the coefficient q of the NSNS flux and the string tension. In (arXiv:1303.1447) we have found the tree-level S-matrix for the massive BMN excitations in this theory, which turned out to have a simple dependence on q. Here, by analyzing the constraints of symmetry and integrability, we propose an exact massive-sector dispersion relation and the exact S-matrix for this world-sheet theory. The S-matrix generalizes its recent construction in the q=0 case in (arXiv:1303.5995)

  20. Massive S-matrix of AdS3×S3×T4 superstring theory with mixed 3-form flux

    Science.gov (United States)

    Hoare, B.; Tseytlin, A. A.

    2013-08-01

    The type IIB supergravity AdS3×S3×T4 background with mixed RR and NSNS 3-form fluxes is a near-horizon limit of a non-threshold bound state of D5-D1 and NS5-NS1 branes. The corresponding superstring world-sheet theory is expected to be integrable, opening the possibility of computing its exact spectrum for any values of the coefficient q of the NSNS flux and the string tension. In arXiv:1303.1447 we have found the tree-level S-matrix for the massive BMN excitations in this theory, which turned out to have a simple dependence on q. Here, by analyzing the constraints of symmetry and integrability, we propose an exact massive-sector dispersion relation and the exact S-matrix for this world-sheet theory. The S-matrix generalizes its recent construction in the q=0 case in arXiv:1303.5995. This is a consequence of the fact that parity symmetry is broken with the introduction of the NSNS flux. However, charge conjugation composed with parity is still a symmetry.

  1. Oscillating supertubes and neutral rotating black hole microstates

    Science.gov (United States)

    Mathur, Samir D.; Turton, David

    2014-04-01

    The construction of neutral black hole microstates is an important problem, with implications for the information paradox. In this paper we conjecture a construction of non-supersymmetric supergravity solutions describing D-brane configurations which carry mass and angular momentum, but no other conserved charges. We first study a classical string solution which locally carries dipole winding and momentum charges in two compact directions, but globally carries no net winding or momentum charge. We investigate its backreaction in the D1-D5 duality frame, where this object becomes a supertube which locally carries oscillating dipole D1-D5 and NS1-NS5 charges, and again carries no net charge. In the limit of an infinite straight supertube, we find an exact supergravity solution describing this object. We conjecture that a similar construction may be carried out based on a class of two-charge non-supersymmetric D1-D5 solutions. These results are a step towards demonstrating how neutral black hole microstates may be constructed in string theory.

  2. Massive S-matrix of AdS_3 x S^3 x T^4 superstring theory with mixed 3-form flux

    CERN Document Server

    Hoare, B

    2013-01-01

    The type IIB supergravity AdS_3 x S^3 x T^4 background with mixed RR and NSNS 3-form fluxes is a near-horizon limit of a non-threshold bound state of D5-D1 and NS5-NS1 branes. The corresponding superstring world-sheet theory is expected to be integrable, opening the possibility of computing its exact spectrum for any values of the coefficient q of the NSNS flux and the string tension. In arXiv:1303.1447 we have found the tree-level S-matrix for the massive BMN excitations in this theory, which turned out to have a simple dependence on q. Here, by analyzing the constraints of symmetry and integrability, we propose an exact massive-sector dispersion relation and the exact S-matrix for this world-sheet theory. The S-matrix generalizes its recent construction in the q=0 case in arXiv:1303.5995.

  3. Ribavirin: Past, present and future

    Science.gov (United States)

    Loustaud-Ratti, Véronique; Debette-Gratien, Marilyne; Jacques, Jérémie; Alain, Sophie; Marquet, Pierre; Sautereau, Denis; Rousseau, Annick; Carrier, Paul

    2016-01-01

    Before the advent of direct acting antiviral agents (DAAs) ribavirin, associated to pegylated-interferon played a crucial role in the treatment of chronic hepatitis C, preventing relapses and breakthroughs. In the present era of new potent DAAs, a place is still devoted to the drug. Ribavirin associated with sofosbuvir alone is efficient in the treatment of most cases of G2 infected patients. All options currently available for the last difficult-to-treat cirrhotic G3 patients contain ribavirin. Reducing treatment duration to 12 wk in G1 or G4 cirrhotic compensated patients is feasible thanks to ribavirin. Retreating patients with acquired anti NS5A resistance-associated variants using ribavirin-based strategies could be useful. The addition of ribavirin with DAAs combinations however, leads to more frequent but mild adverse events especially in cirrhotic patients. Preliminary data with interferon-free second generation DAAs combinations without ribavirin suggest that future of the drug is jeopardized even in difficult-to-treat patients: The optimization of ribavirin dosage according to an early monitoring of blood levels has been suggested to be relevant in double therapy with peginterferon or sofosbuvir but not with very potent combinations of more than two DAAs. PMID:26807208

  4. Hyper-K\\"{a}hler with Torsion, T-duality, and Defect (p,q) Five-branes

    CERN Document Server

    Kimura, Tetsuji; Yata, Masaya

    2014-01-01

    We investigate the five-branes interpretation of a hyper-K\\"{a}hler geometry with torsion (HKT). This geometry is obtained by a conformal transformation of the Taub-NUT space which represents a single Kaluza-Klein five-brane. This HKT would represent an NS5-brane on the Taub-NUT space. In order to explore the HKT further, we compactify one transverse direction, and study the $O(2,2;{\\mathbb Z}) = SL(2,{\\mathbb Z}) \\times SL(2,{\\mathbb Z})$ monodromy structure associated with two-torus. Performing the conjugate transformation, we obtain a new solution whose physical interpretation is the defect $(p,q)$ five-branes on the ALG space. Throughout this analysis, we understand that the HKT represents the coexistent state of two kinds of five-branes. This situation is different from composite states such as $(p,q)$ five-branes or $(p,q)$ seven-branes in type IIB theory. We also study the T-dualized system of the HKT. We again find a new solution which also indicates the defect $(p,q)$ five-branes on another ALG space...

  5. Semi-doubled Sigma Models for Five-branes

    CERN Document Server

    Kimura, Tetsuji

    2015-01-01

    We study two-dimensional ${\\cal N}=(2,2)$ gauge theory and its dualized system in terms of complex (linear) superfields and their alternatives. Although this technique itself is not new, we can obtain a new model, the so-called "semi-doubled" GLSM. Similar to doubled sigma model, this involves both the original and dual degrees of freedom simultaneously, whilst the latter only contribute to the system via topological interactions. Applying this to the ${\\cal N}=(4,4)$ GLSM for H-monopoles, i.e., smeared NS5-branes, we obtain its T-dualized systems in quite an easy way. As a bonus, we also obtain the semi-doubled GLSM for an exotic $5^3_2$-brane whose background is locally nongeometric. In the low energy limit, we construct the semi-doubled NLSM which also generates the conventional string worldsheet sigma models. In the case of the NLSM for $5^3_2$-brane, however, we find that the Dirac monopole equation does not make sense any more because the physical information is absorbed into the divergent part via the ...

  6. 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.

  7. Holographic compactifications of (1,0) theories from massive IIA supergravity

    CERN Document Server

    Apruzzi, Fabio; Passias, Achilleas; Rota, Andrea; Tomasiello, Alessandro

    2015-01-01

    We describe three analytic classes of infinitely many AdS_d BPS solutions of massive IIA supergravity, for d = 7, 5, 4. The three classes are related by simple universal maps. For example, the AdS_7 x M_3 solutions (where M_3 is topologically S^3) are mapped to AdS_5 x Sigma_2 x M'_3, where Sigma_2 is a Riemann surface of genus g >= 2 and the metric on M'_3 is obtained by distorting M_3 in a certain way. The solutions can have localized D6 or O6 sources, as well as an arbitrary number of D8-branes. The AdS_7 case (previously known only numerically) is conjecturally dual to an NS5-D6-D8 system. The field theories in three and four dimensions are not known, but their number of degrees of freedom can be computed at leading order. The AdS_4 solutions have numerical "attractor" generalizations that might be useful for flux compactification purposes.

  8. Six-Dimensional Superconformal Theories and their Compactifications from Type IIA Supergravity

    Science.gov (United States)

    Apruzzi, Fabio; Fazzi, Marco; Passias, Achilleas; Rota, Andrea; Tomasiello, Alessandro

    2015-08-01

    We describe three analytic classes of infinitely many AdSd supersymmetric solutions of massive IIA supergravity, for d =7 ,5 ,4 . The three classes are related by simple universal maps. For example, the AdS7×M3 solutions (where M3 is topologically S3 ) are mapped to AdS5×Σ2×M3' , where Σ2 is a Riemann surface of genus g ≥2 and the metric on M3' is obtained by distorting M3 in a certain way. The solutions can have localized D6 or O6 sources, as well as an arbitrary number of D8-branes. The AdS7 case (previously known only numerically) is conjecturally dual to an NS5-D6-D8 system. The field theories in three and four dimensions are not known, but their number of degrees of freedom can be computed in the supergravity approximation. The AdS4 solutions have numerical "attractor" generalizations that might be useful for flux compactification purposes.

  9. Anti-hepatitis C virus activity of 3-hydroxy caruilignan C from Swietenia macrophylla stems.

    Science.gov (United States)

    Wu, S-F; Lin, C-K; Chuang, Y-S; Chang, F-R; Tseng, C-K; Wu, Y-C; Lee, J-C

    2012-05-01

    Chronic hepatitis C virus (HCV) infection ultimately leads to chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma (HCC). As the standard treatment is not completely efficacious, a safer and more effective agent against HCV infection needs to be developed. In this report, we demonstrated that 3-hydroxy caruilignan C (3-HCL-C) isolated from Swietenia macrophylla stems exhibited high anti-HCV activity at both protein and RNA levels at nontoxic concentrations, with an EC(50) value of 10.5 ± 1.2 μm. Combinations of 3-HCL-C and interferon-α (IFN-α), an HCV NS5B polymerase inhibitor (2'-C-methylcytidine; NM-107) or an HCV NS3/4A protease inhibitor (Telaprevir; VX-950) increased the suppression of HCV RNA replication. The results suggested that 3-HCL-C may be a potential anti-viral agent. We then demonstrated that 3-HCL-C interfered with HCV replication by inducing IFN-stimulated response element transcription and IFN-dependent anti-viral gene expression. PMID:22497816

  10. Prevalence of hepatitis C virus (HCV coinfection in HIV infected individuals in south India and characterization of HCV genotypes

    Directory of Open Access Journals (Sweden)

    Ponamgi SPD

    2009-01-01

    Full Text Available Purpose: To determine anti-HCV antibodies and genomic subtype of HCV in 1487 confirmed human immunodeficiency virus (HIV positive samples. Methods: A total of 1487 confirmed HIV-positive samples were tested for anti-HCV antibodies by using a third generation ELISA kit (Ortho 3.0 and by RT PCR for HCV. HIV and HCV coinfected samples were selected for HCV genotyping by RFLP and subtyping with NS5-type specific primers. Results: A total of 1487 HIV-infected serum samples were screened for HCV infection, of which, a 1443 (97.04% were negative and 45 (3.02% were coinfected. HIV-HCV coinfection was predominant in the age group 41-50 years (51.1%. HCV genotyping and subtyping was done for the 45 HCV RNA-positive specimens of which genotype 1 was observed in 31 (68.8% and genotype 3 was observed in 14 (31.1% subjects. Further subtyping analysis showed the genotype 1b in 23 (51.1%, 1a in eight (17.7%, 3a in 10 (22.2% and 3b in four (8.8% subjects. Conclusion: HIV and HCV seroprevalence is higher in South India, and the most prevalent genotype in coinfection was genotype 1b.

  11. Heterotic supergravity on manifolds with Killing spinors

    International Nuclear Information System (INIS)

    The present work deals with the construction of heterotic string backgrounds on manifolds with real Killing spinors. The latter have played an important role in string theory for a long time, mainly due to Baer's correspondence between Killing spinors on a manifold M and parallel spinors on the cone over M. Given the fact that parallel spinors always lead to exact supergravity BPS backgrounds, it implies that the cone admits a solution of the BPS equations. Furthermore, in type II string theory and in M-theory it is possible to place a brane at the tip of the cone, in appropriate dimensions, and the resulting supergravity solutions are exactly known. In the limit far away from the brane they converge to the empty space solution, whereas in the near horizon limit one obtains a so-called Freund-Rubin solutions, consisting of an anti-de Sitter space times our base manifold M. In heterotic supergravity on the other hand two types of brane-like solutions are known; the NS5-brane, consisting of an R4-factor with fluxes and a transverse 6-dimensional Minkowski space, and what is sometimes called the gauge solitonic branes. These come equipped with an instanton gauge field on some Euclidean space Rp, which carries further non-vanishing fluxes, and again a transverse (10-p)-dimensional Minkowski space. The possible values for p that appeared in the literature so far are p=4, 7 and 8, and the corresponding instantons are the famous BPST and octonionic instantons. Manifolds with real Killing spinors have been classified: besides the round spheres they are either 6-dimensional nearly Kaehler, 7-dimensional nearly parallel G2, Sasaki-Einstein, or 3-Sasakian. I present a generalization of the gauge solitonic branes to the cone over any real Killing spinor manifold, based upon this classification. In particular, this involves the construction of instantons on the cone. Additionally, I show that for homogeneous manifolds with real Killing spinors there is a solution similar to the

  12. First isolation and identification of Zika virus in China%我国首例寨卡病毒的分离与鉴定

    Institute of Scientific and Technical Information of China (English)

    吴德; 谈琦琪; 孙九峰; 周惠琼; 管大伟; 张欢; 宁丹; 柯昌文

    2016-01-01

    目的:建立寨卡病毒分离方法,为寨卡病毒的分离积累经验。方法将寨卡病毒血清阳性标本通过颅内注射途径接种1~3日龄BALB/c乳鼠,接种6日后处死,提取乳鼠脑、心、肝、脾、肺、肾、肌肉、皮肤和肠组织,均浆取上清提取病毒核酸,real-time RT-PCR进行核酸检测鉴定。阳性脑组织上清进行乳鼠传代接种。分离的病毒株用巢式PCR进行扩增,所获得的序列用MEGA6.0软件进行系统进化分析。结果从接种寨卡病毒的乳鼠脑、心和肝等组织中均可检测出寨卡病毒核酸,其中心和脑组织寨卡病毒Ct值最低,分别为24.4和25.3个循环。传代乳鼠病毒接种后2日可在乳鼠脑组织中检测出病毒核酸。巢式PCR成功扩增出1034个碱基大小的片段,进化分析结果显示本次分离的毒株属于亚洲族系。结论利用乳鼠颅内接种法,成功分离1株寨卡病毒,该病毒属于亚洲族系。%Objective To establish a method for the isolation of Zika virus and to gather experi-ences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples posi-tive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nu-cleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The se-quences of NS5 gene were analyzed by using MEGA6. 0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle thresh-old (Ct) values of 24. 4 and 25. 3, respectively. The second generation of Zika virus was identified in suck-ling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of

  13. Perspectivas futuras en el tratamiento de la hepatitis crónica C Future perspective in the treatment of chronic hepatitis C

    Directory of Open Access Journals (Sweden)

    J.V. Fernández-Montero

    2011-06-01

    Full Text Available Se exponen las principales líneas de investigación en nuevos fármacos y estrategias de tratamiento contra la hepatitis vírica tipo C. Esta es una enfermedad que supone un importante problema de salud pública, con más de 700.000 afectados en España y con una importante prevalencia entre las personas privadas de libertad. Las limitaciones del actual tratamiento contra la hepatitis vírica C han forzado la investigación de nuevos fármacos que se van sustanciando en dos líneas principales de productos, algunos de ellos pronto disponibles en el mercado: los inhibidores de la serín-proteasa NS3/4ª (telaprevir, boceprevir, danoprevir o vaniprevir y los inhibidores de la RNA-polimerasa NS5B (RG-7128, RG-7227, Filibuvir, ANA-598, estos últimos en una fase de desarrollo algo más temprana. Previsiblemente habrá que utilizar estos nuevos fármacos añadidos al tratamiento estándar actual de interferón pegilado más ribavirina y en esas condiciones de uso todos estos fármacos han demostrado ya una mayor efectividad que el tratamiento estándar actual. A pesar de este esperanzador panorama, estos fármacos tienen limitaciones como el desarrollo de resistencias, la toxicidad o el poco conocimiento que tenemos de su efectividad en genotipos virales distintos del 1. No obstante su aparición abre muchas nuevas posibilidades en el tratamiento de esta enfermedad.The main lines of research into new drugs and treatment strategies against type C viral hepatitis are described. This disease is a major public health problem, with more than 700,000 people affected by the illness in Spain and with a high degree of prevalence amongst prison inmates. Limitations on current treatment for viral hepatitis C have led to research into new drugs in the form of two main product lines, some of which are soon to be available on the market: NS3/4ª serine-protease inhibitors (telaprevir, boceprevir, danoprevir and vaniprevir and the NS5B RNA polymerase inhibitors

  14. 猪源牛病毒性腹泻病毒SD0803株细胞传代研究%BIOLOGICAL CHARACTERISTICS OF PASSAGED ON MDBK CELLS BOVINE VIRAL DIARRHEA VIRUS OF PIG ORIGIN

    Institute of Scientific and Technical Information of China (English)

    孙春清; 邓宇; 张宏彪; 龙进学; 韦祖樟; 童光志; 袁世山

    2012-01-01

    To develop a Bovine viral diarrhea virus(BVDV) vaccine,the BVDV strain SD0803 of pig origin was passaged in MDBK cells for 40 times.Viral RNA was extracted from the 40th passage virus and 5 segments were amplified in RT-PCR.The complete genomic sequence was megaligned and compared with its parental virus using DNASTAR software.The results showed that the homology between the 40th passage and parental virus was 99.8% in nucleotides and 99.6 % in amino acids.Twenty three nucleotide mutations were identified,of which 15 were sense mutations.Amino acid mutations were mainly located on E2 and NS5B.To compare the growth characteristics between the parent virus and passaged viruses,supernatants were collected from infected MDCK cells at passages 1,10,20,30 and 40,and measured the amounts of released viral RNAs in RT-PCR.The multi-step growth curves showed that the parent virus and passaged viruses had high replication efficiency in MDBK cells,and shared similar growth properties.Some mutations that occurred during virus passages had no effect on the virus titers as determined by titration.%为研究猪源牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)生物学特性,将本实验室分离得到的SD0803毒株在马-达氏牛肾细胞(mardin-darby bovine kidney,MDBK)上连续传40代,提取第40代病毒基因组RNA,设计扩增及测序引物,用RT-PCR方法分5段扩增第40代病毒基因片段,并进行全长测序,利用DNASTAR软件进行序列拼接及分析,与亲代病毒SD0803序列比对分析;用Real-time PCR方法测定第1、10、20、30和40代细胞上清中的病毒复制效率,并绘制多步生长曲线。测序结果表明,第40代病毒与亲代病毒核苷酸序列的同源性为99.8%,氨基酸序列的同源性为99.6%,其中有23处发生核苷酸突变,14处为有义突变,氨基酸变化主要集中在E2和NS5B区域。多步生长曲线显示,传代病毒和亲本病毒均能在MDBK细胞上获得较高的复制效率,并

  15. Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

    Science.gov (United States)

    Wose Kinge, Constance N.; Espiritu, Christine; Prabdial-Sing, Nishi; Sithebe, Nomathamsaqa Patricia

    2014-01-01

    Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds. PMID:24982066

  16. Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation.

    Science.gov (United States)

    Li, Jiawen; Johnson, Kenneth A

    2016-05-01

    Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:26851276

  17. DENGUE OUTBREAK IN MATO GROSSO STATE, MIDWESTERN BRAZIL.

    Science.gov (United States)

    Heinen, Letícia Borges da Silva; Zuchi, Nayara; Cardoso, Belgath Fernandes; Santos, Marcelo Adriano Mendes Dos; Nogueira, Mauricio Lacerda; Dezengrini-Slhessarenko, Renata

    2015-12-01

    Dengue virus (DENV) is the most frequent arbovirus worldwide. In this study, we report a large outbreak in Mato Grosso State (MT). Serum samples from 604 patients with acute febrile illness for less than five days were inoculated in C6/36 cells, then infected cells were subjected to an indirect immunofluorescence test for DENV serotypes and yellow fever virus. Serum samples were submitted to a multiplex-semi-nested-RT-PCR for 11 flaviviruses. DENV-4 was isolated in 150/604 (24.8%) and DENV-1 in 19/604 (3.1%) specimens. By RT-PCR, 331 (54.8%) samples tested positive for DENV; 321 had single infections (DENV-4 n = 305; DENV-1 n = 15; DENV-3 n = 1), nine had co-infections of DENV-1/DENV-4, and one of DENV-2/DENV-4. DENV-4 was detected in 315/331 (95.2%) positive patients from 17 municipalities, and DENV-1 in 24/331 (7.2%) patients from five cities in north-central MT and the city of Cuiaba. The incidence of infection was higher in patients aged 20-39 (142/331; 42.9%). The NS5 partial nucleotide sequence of DENV-1 was most similar to that of genotype V, DENV-2 to Southeast Asian/American, DENV-3 to genotype III, and DENV-4 to genotype II strains, considered the most frequent strains in Brazil. This outbreak coincided with the introduction of DENV-4 in the state. Cuiaba was hyperendemic for the four DENV serotypes, highlighting the necessity for arbovirus surveillance in MT. PMID:27049702

  18. Neoadjuvant chemotherapy for radioinduced osteosarcoma of the extremity: The Rizzoli experience in 20 cases

    International Nuclear Information System (INIS)

    Purpose: Evaluate treatment and outcome of 20 patients with radioinduced osteosarcoma (RIO). Because of previous primary tumor treatment, RIO protocols were different from others we used for non-RIO. Patients and Methods: Between 1983 and 1998, we treated 20 RIO patients, ages 4-36 years (mean 16 years), with chemotherapy (two cycles before surgery, three postoperatively). The first preoperative cycle consisted of high-dose Methotrexate (HDMTX)/Cisplatinum (CDP)/Adriamycin (ADM) and the second of HDMTX/CDP/Ifosfamide (IFO). The three postoperative treatments were performed with cycles of MTX/CDP; IFO was used as single agent per cycle repeated three times. Results: Two patients received palliative treatment because their osteosarcoma remained unresectable after preoperative chemotherapy. The remaining 18 patients had surgery (7 amputations, 11 resections); histologic response to preoperative chemotherapy was good in 8 patients, poor in 10. At a mean follow-up of 11 years (range, 7-22 years), 9 patients remained continuously disease-free, 10 died from osteosarcoma and 1 died from a third neoplasm (myeloid acute leukemia). These results are not significantly different from those achieved in 754 patients with conventional osteosarcoma treated in the same period with protocols used for conventional treatment. However, this later group had an 18% 3-year event-free survival after treatment of relapse vs. 0% in the RIO group. Conclusion: Treated with neoadjuvant chemotherapy RIO seem to have an outcome that is not significantly different from that of comparable patients with conventional primary high grade osteosarcoma (5-year event-free survival: 40% vs. 60%, p = NS; 5-year overall survival 40% vs. 67%, p < 0.01)

  19. Dengue virus type 3 adaptive changes during epidemics in São Jose de Rio Preto, Brazil, 2006-2007.

    Science.gov (United States)

    Villabona-Arenas, Christian Julian; Mondini, Adriano; Bosch, Irene; Schimdt, Diane J; Schimitt, Diane; Calzavara-Silva, Carlos E; Zanotto, Paolo M de A; Nogueira, Maurício L

    2013-01-01

    Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak. PMID:23667626

  20. Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses.

    Science.gov (United States)

    Sironi, Manuela; Forni, Diego; Clerici, Mario; Cagliani, Rachele

    2016-09-01

    The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian

  1. dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

    Directory of Open Access Journals (Sweden)

    Eliane F. Meurs

    2012-10-01

    Full Text Available The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2a. As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2a kinase, its participation in the regulation of the NF-kB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV. PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2a-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

  2. Hepatitis C virus (HCV genotype 1 subtype identification in new HCV drug development and future clinical practice.

    Directory of Open Access Journals (Sweden)

    Stéphane Chevaliez

    Full Text Available BACKGROUND: With the development of new specific inhibitors of hepatitis C virus (HCV enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s for accurate HCV genotype 1 subtype determination. METHODOLOGY/PRINCIPAL FINDINGS: A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5' non-coding region (5'NCR by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%-29.5% of cases, and HCV subtype 1b in 9.5%-8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5'NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases. CONCLUSIONS/SIGNIFICANCE: In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5'NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-01-07

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

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

    International Nuclear Information System (INIS)

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

  5. Molecular characterization, distribution, and dynamics of hepatitis C virus genotypes in blood donors in Colombia.

    Science.gov (United States)

    Mora, Mónica Viviana Alvarado; Romano, Camila Malta; Gomes-Gouvêa, Michele Soares; Gutiérrez, Maria Fernanda; Carrilho, Flair José; Pinho, João Renato Rebello

    2010-11-01

    Hepatitis C virus (HCV) is a frequent cause of acute and chronic hepatitis and a leading cause for cirrhosis of the liver and hepatocellular carcinoma. HCV is classified in six major genotypes and more than 70 subtypes. In Colombian blood banks, serum samples were tested for anti-HCV antibodies using a third-generation ELISA. The aim of this study was to characterize the viral sequences in plasma of 184 volunteer blood donors who attended the "Banco Nacional de Sangre de la Cruz Roja Colombiana," Bogotá, Colombia. Three different HCV genomic regions were amplified by nested PCR. The first of these was a segment of 180 bp of the 5'UTR region to confirm the previous diagnosis by ELISA. From those that were positive to the 5'UTR region, two further segments were amplified for genotyping and subtyping by phylogenetic analysis: a segment of 380 bp from the NS5B region; and a segment of 391 bp from the E1 region. The distribution of HCV subtypes was: 1b (82.8%), 1a (5.7%), 2a (5.7%), 2b (2.8%), and 3a (2.8%). By applying Bayesian Markov chain Monte Carlo simulation, it was estimated that HCV-1b was introduced into Bogotá around 1950. Also, this subtype spread at an exponential rate between about 1970 to about 1990, after which transmission of HCV was reduced by anti-HCV testing of this population. Among Colombian blood donors, HCV genotype 1b is the most frequent genotype, especially in large urban conglomerates such as Bogotá, as is the case in other South American countries. PMID:20872715

  6. 2'-O methylation of the viral mRNA cap by West Nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo.

    Directory of Open Access Journals (Sweden)

    Kristy J Szretter

    Full Text Available Prior studies have shown that 2'-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2'-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT and a mutant in the NS5 gene (WNV-E218A lacking 2'-O methylation of the 5' viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1⁻/⁻ mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS and a greater than 16,000-fold decrease in LD(50 values in Ifit1⁻/⁻ compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1⁻/⁻ brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8⁺ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2'-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.

  7. Scintillation properties of lead sulfate

    International Nuclear Information System (INIS)

    We report on the scintillation properties of lead sulfate (PbSO4), a scintillator that show promise as a high energy photon detector. It physical properties are well suited for gamma detection, as its has a density of 6.4 gm/cm3, a 1/e attenuation length for 511 keV photons of 1.2 cm, is not affected by air or moisture, and is cut and polished easily. In 99.998% pure PbSO4 crystals at room temperature excited by 511 keV annihilation photons, the fluorescence decay lifetime contains significant fast components having 1.8 ns (5%) and 19 ns (36%) decay times, but with longer components having 95 ns (36%) and 425 ns (23%) decays times. The peak emission wavelength is 335 nm, which is transmitted by borosilicate glass windowed photomultiplier tubes. The total scintillation light output increases with decreasing temperature fro 3,200 photons/MeV at +45 degrees C to 4, 900 photons/MeV at room temperature (+25 degrees C) and 68,500 photons/MeV at -145 degrees C. In an imperfect, 3 mm cube of a naturally occurring mineral form of PbSO4 (anglesite) at room temperature, a 511 keV photopeak is seen with a total light output of 60% that BGO. There are significant sample to sample variations of the light output among anglesite samples, so the light output of lead sulfate may improve when large synthetic crystals become available. 10 refs

  8. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    International Nuclear Information System (INIS)

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV

  9. [Novel methods of hepatitis C treatment and prevention].

    Science.gov (United States)

    Chmielewska, Alicja M; Rychłowska, Małgorzata; Król, Ewelina; Solarz, Karolina; Bieńkowska-Szewczyk, Krystyna

    2015-01-01

    Despite available treatment, Hepatitis C remains one of most serious burdens to public health. Current therapy based on pegylated interferon-alpha and ribavirin has significant side effects and its effectiveness varies for different genotypes of the virus. Four novel drugs - viral protease inhibitors (telaprevir, boceprevir, simeprevir) and polymerase inhibitor - sofosbuvir have been introduced in last years for use in combination with standard-of-care treatment. For the first time interferon free therapies were approved with the use of combination of sofosbuvir+ribavirin. New therapies improve virological response rates but also increase the cost, side effects and raise the issue of drug resistance. Numerous novel anti-HCV compounds have been evaluated in advanced clinical trials including inhibitors of viral proteins (protease, polymerase and NS5A) and inhibitors of host factors involved in HCV replication (cyclophilin A, microRNA - miR-122). New interferon-free therapies reducing severe side effects are expected to enter the market within few months. At the same time efforts are undertaken to determine the host and viral factors with predictive value for HCV treatment response, enabling personalized therapy approach. The main success in this field was the discovery of interleukin IL28B polymorphism, which correlates with positive standard-of-care treatment response. An effective vaccination may be an alternative for antiviral drugs, but no anti-HCV vaccine is available currently. It is well proved that successful vaccination should induce antibody and T-cell responses specific against a range of HCV genotypes. With this aim, new subunit and genetic candidate vaccines have been evaluated in I and II phase clinical trials. This review summarizes the recent developments in the field of new drug development and vaccine studies against hepatitis C virus. PMID:26400881

  10. Logical design of anti-prion agents using NAGARA.

    Science.gov (United States)

    Ma, Biao; Yamaguchi, Keiichi; Fukuoka, Mayuko; Kuwata, Kazuo

    2016-01-22

    To accelerate the logical drug design procedure, we created the program "NAGARA," a plugin for PyMOL, and applied it to the discovery of small compounds called medical chaperones (MCs) that stabilize the cellular form of a prion protein (PrP(C)). In NAGARA, we constructed a single platform to unify the docking simulation (DS), free energy calculation by molecular dynamics (MD) simulation, and interfragment interaction energy (IFIE) calculation by quantum chemistry (QC) calculation. NAGARA also enables large-scale parallel computing via a convenient graphical user interface. Here, we demonstrated its performance and its broad applicability from drug discovery to lead optimization with full compatibility with various experimental methods including Western blotting (WB) analysis, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) measurements. Combining DS and WB, we discovered anti-prion activities for two compounds and tegobuvir (TGV), a non-nucleoside non-structural protein NS5B polymerase inhibitor showing activity against hepatitis C virus genotype 1. Binding profiles predicted by MD and QC are consistent with those obtained by SPR and NMR. Free energy analyses showed that these compounds stabilize the PrP(C) conformation by decreasing the conformational fluctuation of the PrP(C). Because TGV has been already approved as a medicine, its extension to prion diseases is straightforward. Finally, we evaluated the affinities of the fragmented regions of TGV using QC and found a clue for its further optimization. By repeating WB, MD, and QC recursively, we were able to obtain the optimum lead structure. PMID:26723253

  11. Molecular characterization of new emerging GII.17 norovirus strains from South China.

    Science.gov (United States)

    Xue, Liang; Wu, Qingping; Cai, Weicheng; Zhang, Jumei; Guo, Weipeng

    2016-06-01

    Noroviruses are still the primary cause of non-bacterial acute gastroenteritis worldwide. Recently, a novel GII.17 norovirus variant emerged and caused an infection peak in the cold season of 2014/2015 in some Asian countries, including China. In this study, in order to understand the evolutionary advantage of the novel variant, complete genomic sequences of GII.17 NoV strains from South China were comprehensively analyzed. Pairwise alignments of new GII.17 genomes with representative sequences of each GII genotype were performed. Inconsistent homology was observed between different protein-encoding regions, of which VPg (NS5) and P2 were found to be the most conserved and variable ones, respectively. The differences between new sequences and other reported GII.17 genomes were also compared, and 84 mismatched nucleotides were found, resulting in 15 amino acid changes. Then, all capsid sequences of different GII.17 NoV variants were collected for multiple alignments, and a total of 87 spots were identified during their evolution process. Homology modeling of capsid proteins of four GII.17 variants was carried out based on comparison with GII.4 VA387 strain, and structural differences were mainly embodied in five extended loops. Furthermore, positions of potential conformational epitope regions of new GII.17 variants were found more similar or adjacent to those of GII.4 rather than those of the former GII.17 variants. In summary, nine GII.17 strains from South China were extensively characterized based on their complete genomes, and a different distribution pattern of epitope residues was predicted on the new GII.17 variant capsid from that of the former ones. PMID:26923075

  12. Oversizing and Restenosis with Self-Expanding Stents in Iliofemoral Arteries

    Energy Technology Data Exchange (ETDEWEB)

    Saguner, Ardan M., E-mail: ardan.saguner@usz.ch; Traupe, Tobias; Raeber, Lorenz; Hess, Nina [University Hospital, Swiss Cardiovascular Center (Switzerland); Banz, Yara [University of Bern, Institute of Pathology (Switzerland); Saguner, Arhan R.; Diehm, Nicolas; Hess, Otto M. [University Hospital, Swiss Cardiovascular Center (Switzerland)

    2012-08-15

    Purpose: Uncoated self-expanding nitinol stents (NS) are commonly oversized in peripheral arteries. In current practice, 1-mm oversizing is recommended. Yet, oversizing of NS may be associated with increased restenosis. To provide further evidence, NS were implanted in porcine iliofemoral arteries with a stent-to-artery-ratio between 1.0 and 2.3. Besides conventional uncoated NS, a novel self-expanding NS with an antiproliferative titanium-nitride-oxide (TiNOX) coating was tested for safety and efficacy. Methods: Ten uncoated NS and six TiNOX-coated NS (5-6 mm) were implanted randomly in the iliofemoral artery of six mini-pigs. After implantation, quantitative angiography (QA) was performed for calculation of artery and minimal luminal diameter. Follow-up was performed by QA and histomorphometry after 5 months. Results: Stent migration, stent fracture, or thrombus formation were not observed. All stents were patent at follow-up. Based on the location of the stent (iliac/femoral) and the stent-to-artery-ratio, stent segments were divided into 'normal-sized' (stent-to-artery-ratio < 1.4, n = 12) and 'oversized' (stent-to-artery-ratio {>=} 1.4, n = 9). All stent segments expanded to their near nominal diameter during follow-up. Normal-sized stent segments increased their diameter by 6% and oversized segments by 29%. A significant correlation between oversizing and restenosis by both angiography and histomorphometry was observed. Restenosis rates were similar for uncoated NS and TiNOX-coated NS. Conclusions: TiNOX-coated NS are as safe and effective as uncoated NS in the porcine iliofemoral artery. All stents further expand to near their nominal diameter during follow-up. Oversizing is linearly and positively correlated with neointimal proliferation and restenosis, which may not be reduced by TiNOX-coating.

  13. Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil

    Science.gov (United States)

    Drumond, Betania Paiva; da Silva Fagundes, Luiz Gustavo; Rocha, Raissa Prado; Fumagalli, Marcilio Jorge; Araki, Carlos Shigueru; Colombo, Tatiana Elisa; Nogueira, Mauricio Lacerda; Castilho, Thiago Elias; da Silveira, Nelson José Freitas; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-01-01

    Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1–4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population. PMID:26887252

  14. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

    2016-01-01

    The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. PMID:26574011

  15. Hepatitis C Virus Subtype and Evolution Characteristic Among Drug Users, Men Who Have Sex With Men, and the General Population in Beijing, China.

    Science.gov (United States)

    Jiao, Yang; Zhang, Xiaoxi; Wang, Chen; Li, Li; Liu, Jie; Bar, Katharine J; Wei, Huamian; Hu, Yao; Huang, Ping; Zeng, Zhaoli; Jiang, Shulin; Du, Jialiang; Shao, Yiming; Metzger, David; Li, Shuming; Ma, Liying

    2016-02-01

    The aim of this study was to characterize the current molecular epidemiology of hepatitis C virus (HCV) infection and evaluate the evolutionary patterns of HCV subtypes in Beijing, China, among different subpopulations.The whole blood samples and behavioral data were collected from a total of 10,354 subjects, including drug users (DUs), men who have sex with men (MSM), and the general population, in Beijing from 2010 to 2011. Samples were tested for HCV infection using both enzyme-linked immunosorbent assay (ELISA) and real-time PCR. All viremic subjects were then sequenced by nested PCR over core/E1 and NS5B regions. Phylogenetic and phylogeographic analysis was performed by BEAST software.In total, 217 subjects (2.1%) were tested positive for HCV by antibody or vRNA-based testing. HCV prevalence rates for DUs, MSM, and the general population were 26.2%, 0.54%, and 0.37%, respectively. The 156 HCV RNA-positive samples were sequenced. Nine HCV genotypes, including 1a, 1b, 2a, 3a, 3b, 6a, 6n, 6u and 6v, were detected. The most prevalent subtypes were 3b (36.09%), 1b (32.54%), and 3a (16.57%). Bayesian evolutionary analysis estimated that the time of introduction of subtype 1b into Beijing was 2004 (95% CI: 1997.7, 2007.7), with subtypes 3a and 3b being introduced later in 2006. Evolutionary analyses further suggested that subtype 1b from Beijing and Shanghai were closely related, whereas subtype 3a sequences were more similar with sequences from Yunnan, Guangzhou, Hong Kong, and Jiangsu. Subtype 3b sequences were closely related to those from Yunnan, Guangdong, and Hong Kong.Thus, the current HCV epidemic in Beijing is complex, heavily affecting DUs, and involving multiple genotypes that likely spread from different regions in China with its large migrant population. PMID:26871798

  16. A novel small molecule inhibitor of hepatitis C virus entry.

    Directory of Open Access Journals (Sweden)

    Carl J Baldick

    Full Text Available Small molecule inhibitors of hepatitis C virus (HCV are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc, blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

  17. Comparative genomic analysis of pre-epidemic and epidemic Zika virus strains for virological factors potentially associated with the rapidly expanding epidemic

    Science.gov (United States)

    Zhu, Zheng; Chan, Jasper Fuk-Woo; Tee, Kah-Meng; Choi, Garnet Kwan-Yue; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Tse, Herman; Yuen, Kwok-Yung

    2016-01-01

    Less than 20 sporadic cases of human Zika virus (ZIKV) infection were reported in Africa and Asia before 2007, but large outbreaks involving up to 73% of the populations on the Pacific islands have started since 2007, and spread to the Americas in 2014. Moreover, the clinical manifestation of ZIKV infection has apparently changed, as evident by increasing reports of neurological complications, such as Guillain–Barré syndrome in adults and congenital anomalies in neonates. We comprehensively compared the genome sequences of pre-epidemic and epidemic ZIKV strains with complete genome or complete polyprotein sequences available in GenBank. Besides the reported phylogenetic clustering of the epidemic strains with the Asian lineage, we found that the topology of phylogenetic tree of all coding regions is the same except that of the non-structural 2B (NS2B) coding region. This finding was confirmed by bootscan analysis and multiple sequence alignment, which suggested the presence of a fragment of genetic recombination at NS2B with that of Spondweni virus. Moreover, the representative epidemic strain possesses one large bulge of nine bases instead of an external loop on the first stem-loop structure at the 3′-untranslated region just distal to the stop codon of the NS5 in the 1947 pre-epidemic prototype strain. Fifteen amino acid substitutions are found in the epidemic strains when compared with the pre-epidemic strains. As mutations in other flaviviruses can be associated with changes in virulence, replication efficiency, antigenic epitopes and host tropism, further studies would be important to ascertain the biological significance of these genomic changes. PMID:26980239

  18. Circulation of a Meaban-like virus in yellow-legged gulls and seabird ticks in the western Mediterranean basin.

    Directory of Open Access Journals (Sweden)

    Audrey Arnal

    Full Text Available In recent years, a number of zoonotic flaviviruses have emerged worldwide, and wild birds serve as their major reservoirs. Epidemiological surveys of bird populations at various geographical scales can clarify key aspects of the eco-epidemiology of these viruses. In this study, we aimed at exploring the presence of flaviviruses in the western Mediterranean by sampling breeding populations of the yellow-legged gull (Larus michahellis, a widely distributed, anthropophilic, and abundant seabird species. For 3 years, we sampled eggs from 19 breeding colonies in Spain, France, Algeria, and Tunisia. First, ELISAs were used to determine if the eggs contained antibodies against flaviviruses. Second, neutralization assays were used to identify the specific flaviviruses present. Finally, for colonies in which ELISA-positive eggs had been found, chick serum samples and potential vectors, culicid mosquitoes and soft ticks (Ornithodoros maritimus, were collected and analyzed using serology and PCR, respectively. The prevalence of flavivirus-specific antibodies in eggs was highly spatially heterogeneous. In northeastern Spain, on the Medes Islands and in the nearby village of L'Escala, 56% of eggs had antibodies against the flavivirus envelope protein, but were negative for neutralizing antibodies against three common flaviviruses: West Nile, Usutu, and tick-borne encephalitis viruses. Furthermore, little evidence of past flavivirus exposure was obtained for the other colonies. A subset of the Ornithodoros ticks from Medes screened for flaviviral RNA tested positive for a virus whose NS5 gene was 95% similar to that of Meaban virus, a flavivirus previously isolated from ticks of Larus argentatus in western France. All ELISA-positive samples subsequently tested positive for Meaban virus neutralizing antibodies. This study shows that gulls in the western Mediterranean Basin are exposed to a tick-borne Meaban-like virus, which underscores the need of exploring

  19. Epidemic History of Hepatitis C Virus among Patients with Inherited Bleeding Disorders in Iran.

    Science.gov (United States)

    Samimi-Rad, Katayoun; Rahimnia, Ramin; Sadeghi, Mahdi; Malekpour, Seyed Amir; Marzban, Mona; Keshvari, Maryam; Kiani, Seyed Jalal; Alavian, Seyed-Moayed

    2016-01-01

    The high rate of hepatitis C virus (HCV) infection among transfusion related risk groups such as patients with inherited bleeding disorders highlighting the investigation on prevalent subtypes and their epidemic history among this group. In this study, 166 new HCV NS5B sequences isolated from patients with inherited bleeding disorders together with 29 sequences related to hemophiliacs obtained from a previous study on diversity of HCV in Iran were analyzed. The most prevalent subtype was 1a (65%), followed by 3a (18.7%),1b (14.5%),4(1.2%) and 2k (0.6%). Subtypes 1a and 3a showed exponential expansion during the 20th century. Whereas expansion of 3a started around 20 years earlier than 1a among the study patients, the epidemic growth of 1a revealed a delay of about 10 years compared with that found for this subtype in developed countries. Our results supported the view that the spread of 3a reached the plateau 10 years prior to the screening of blood donors for HCV. Rather, 1a reached the plateau when screening program was implemented. The differences observed in the epidemic behavior of HCV-1a and 3a may be associated with different transmission routes of two subtypes. Indeed, expansion of 1a was more commonly linked to blood transfusion, while 3a was more strongly associated to drug use and specially IDU after 1960. Our findings also showed HCV transmission through blood products has effectively been controlled from late 1990s. In conclusion, the implementation of strategies such as standard surveillance programs and subsiding antiviral treatments seems to be essential to both prevent new HCV infections and to decline the current and future HCV disease among Iranian patients with inherited bleeding disorders. PMID:27611688

  20. Genotype Distribution and Molecular Epidemiology of Hepatitis C Virus in Hubei, Central China.

    Directory of Open Access Journals (Sweden)

    Jing Peng

    Full Text Available Little is known about the molecular epidemiology of hepatitis C virus (HCV infection in Central China.A total of 570 patients from Hubei Province in central China were enrolled. These patients were tested positive for HCV antibody prior to blood transfusion. Among them, 177 were characterized by partial NS5B and/or Core-E1 sequences and classified into five subtypes: 1b, 83.0% (147/177; 2a, 13.0% (23/177; 3b, 2.3% (4/177; 6a, 1.1% (2/177; 3a, 0.6% (1/177. Analysis of genotype-associated risk factors revealed that paid blood donation and transfusion before 1997 were strongly associated with subtypes 1b and 2a, while some subtype 2a cases were also found in individuals with high risk sexual behaviors; subtypes 3b, 6a, and 3a were detected only in intravenous drug users. Phylogeographic analyses based on the coalescent datasets demonstrated that 1b, 2a, 3b, and 6a were locally epidemic in Hubei Province. Among them, subtype 1b Hubei strains may have served as the origins of this subtype in China, and 2a and 3b Hubei strains may have descended from the northwest and southwest of China, respectively, while 6a Hubei strains may have been imported from the central south and southwest.The results suggest that the migration patterns of HCV in Hubei are complex and variable among different subtypes. Implementation of mandatory HCV screening before donation has significantly decreased the incidence of transfusion-associated HCV infection since 1997. More attention should be paid to intravenous drug use and unsafe sexual contact, which may have become new risk factors for HCV infection in Hubei Province.

  1. A novel hepatitis C virus genotyping method based on liquid microarray.

    Directory of Open Access Journals (Sweden)

    Cesar A B Duarte

    Full Text Available The strategy used to treat HCV infection depends on the genotype involved. An accurate and reliable genotyping method is therefore of paramount importance. We describe here, for the first time, the use of a liquid microarray for HCV genotyping. This liquid microarray is based on the 5'UTR - the most highly conserved region of HCV - and the variable region NS5B sequence. The simultaneous genotyping of two regions can be used to confirm findings and should detect inter-genotypic recombination. Plasma samples from 78 patients infected with viruses with genotypes and subtypes determined in the Versant™ HCV Genotype Assay LiPA (version I; Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany were tested with our new liquid microarray method. This method successfully determined the genotypes of 74 of the 78 samples previously genotyped in the Versant™ HCV Genotype Assay LiPA (74/78, 95%. The concordance between the two methods was 100% for genotype determination (74/74. At the subtype level, all 3a and 2b samples gave identical results with both methods (17/17 and 7/7, respectively. Two 2c samples were correctly identified by microarray, but could only be determined to the genotype level with the Versant™ HCV assay. Genotype "1" subtypes (1a and 1b were correctly identified by the Versant™ HCV assay and the microarray in 68% and 40% of cases, respectively. No genotype discordance was found for any sample. HCV was successfully genotyped with both methods, and this is of prime importance for treatment planning. Liquid microarray assays may therefore be added to the list of methods suitable for HCV genotyping. It provides comparable results and may readily be adapted for the detection of other viruses frequently co-infecting HCV patients. Liquid array technology is thus a reliable and promising platform for HCV genotyping.

  2. CARACTERIZACIÓN BIOLÓGICA DE VARIANTES DE PLACA DE LA CEPA VACUNAL 17D CONTRA LA FIEBRE AMARILLA

    Directory of Open Access Journals (Sweden)

    María Rojas

    2009-09-01

    Full Text Available La vacuna colombiana 17D, contiene por lo menos cuatro fenotipos, denominados pequeño (0.3 – 1.2 mm, mediano (1.3 – 2.1 mm, grande (2.2 – 3.0 mm y extragrande (>3.1 mm. La composición y distribución porcentual de esos fenotipos, varió entre lotes y entre ampolletas de un mismo lote. Cada variante fue clonada por dilución de la vacuna y su efecto virulento fue analizado en ratones; el fenotipo de placa pequeño estuvo ligeramente sub representado en los lotes analizados y mostró una virulencia similar a la de la cepa silvestre neurotrópica Francesa (LD50 > 10-6, mientras que el fenotipo predominante y mas atenuado fue el mediano (LD50: 10-4. Los fenotipos grande y extragrande mostraron una virulencia intermedia (LD 50: 10 – 5  con relación a los anteriores. Los análisis de secuencia de las variantes sobre una región comprendida entre el extremo 3´NS5 y el inicio de 3´NCR, mostró la cercanía entre aquellas variantes con algún grado de virulencia, y entre la variante atenuada y la vacuna colombiana. La heterogeneidad de la vacuna 17D, constituye una evidencia de la estructura de quasiespecies propia de los virus RNA y señala cómo los casos de reacciones post vacunales  adversas pueden estar asociados con la aplicación de vacunas fabricadas a partir de cepas virales atenuadas.  

  3. Novel dengue virus inhibitor 4-HPR activates ATF4 independent of protein kinase R-like Endoplasmic Reticulum Kinase and elevates levels of eIF2α phosphorylation in virus infected cells.

    Science.gov (United States)

    Fraser, J E; Wang, C; Chan, K W K; Vasudevan, S G; Jans, D A

    2016-06-01

    Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic. PMID:26965420

  4. Innate immunity to dengue virus infection and subversion of antiviral responses.

    Science.gov (United States)

    Green, Angela M; Beatty, P Robert; Hadjilaou, Alexandros; Harris, Eva

    2014-03-20

    Dengue is a major public health issue in tropical and subtropical regions worldwide. The four serotypes of dengue virus (DENV1-DENV4) are spread primarily by Aedes aegypti and Aedes albopictus mosquitoes, whose geographic range continues to expand. Humans are the only host for epidemic strains of DENV, and the virus has developed sophisticated mechanisms to evade human innate immune responses. The host cell's first line of defense begins with an intracellular signaling cascade resulting in production of interferon α/β (IFN-α/β), which promotes intracellular antiviral responses and helps initiates the adaptive response during the course of DENV infection. In response, DENV has developed numerous ways to subvert these intracellular antiviral responses and directly inhibit cellular signaling cascades. Specifically, DENV manipulates the unfolded protein response and autophagy to counter cellular stress and delay apoptosis. The DENV non-structural protein NS4B and subgenomic flavivirus RNA interfere with the RNA interference pathway by inhibiting the RNase Dicer. During heterotypic secondary DENV infection, subneutralizing antibodies can enable viral uptake through Fcγ receptors and down-regulate signaling cascades initiated via the pattern recognition receptors TLR-3 and MDA5/RIG-I, thus reducing the antiviral state of the cell. The DENV NS2B/3 protein cleaves human STING/MITA, interfering with induction of IFN-α/β. Finally, DENV NS2A, NS4A, and NS4B complex together to block STAT1 phosphorylation, while NS5 binds and promotes degradation of human STAT2, thus preventing formation of the STAT1/STAT2 heterodimer and its transcriptional induction of interferon stimulating genes. Here, we discuss the host innate immune response to DENV and the mechanisms of immune evasion that DENV has developed to manipulate cellular antiviral responses. PMID:24316047

  5. HIV, HBV, and HCV molecular epidemiology among trans (transvestites, transsexuals, and transgender) sex workers in Argentina.

    Science.gov (United States)

    Carobene, Mauricio; Bolcic, Federico; Farías, María Sol Dos Ramos; Quarleri, Jorge; Avila, María Mercedes

    2014-01-01

    Commercial sex work is frequent among male-to-female transvestites, transsexuals and transgenders in Argentina, leading to high susceptibility to HIV, HBV, and HCV among other sexually transmitted infections. In a global context of scarce data on the trans sex workers population, this study was aimed to study the genomic characterization of these viruses. Plasma presence of HIV, HBV, and HCV genomic material was evaluated in samples from 273 trans sex workers. Genomic sequences of HIV-gag, pol, and vif-vpu genes, HBV-S gene, and HCV-5'UT and NS5B genes were obtained. Molecular characterization involved phylogenetic analysis and several in silico tools. Resistance-associated mutations in HIV and HBV pol genes were also analyzed. The HIV genomic characterization in 62 trans sex workers samples showed that 54.8% of the isolates corresponded to BF intersubtype recombinants, and 38.7% to subtype B. The remaining were classified as subtypes C (4.8%) and A (1.6%). HBV and HCV co-infection prevalence among HIV positive trans sex workers yielded rates of 3.2% and 6.5% respectively. Drug resistance-associated mutations were found in 12/62 (19%) HIV pol sequences, but none among HBV. Based on phylogenetic relationships, HIV isolates characterized as subtypes BF and B appeared intermingled with those from other high-risk groups. Despite trans sex workers declared not to have received antiviral treatment, complex drug resistance-associated mutation patterns were found in several HIV isolates. Planned prevention, screening, and treatment are needed to reduce further transmission and morbidity. PMID:24123155

  6. First isolation of West Nile virus from a dromedary camel.

    Science.gov (United States)

    Joseph, Sunitha; Wernery, Ulrich; Teng, Jade Ll; Wernery, Renate; Huang, Yi; Patteril, Nissy Ag; Chan, Kwok-Hung; Elizabeth, Shyna K; Fan, Rachel Yy; Lau, Susanna Kp; Kinne, Jörg; Woo, Patrick Cy

    2016-01-01

    Although antibodies against West Nile virus (WNV) have been detected in the sera of dromedaries in the Middle East, North Africa and Spain, no WNV has been isolated or amplified from dromedary or Bactrian camels. In this study, WNV was isolated from Vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in Dubai. Complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. There was no clustering of the present WNV with other WNVs isolated in other parts of the Middle East. Within lineage 1a, the dromedary WNV occupied a unique position, although it was most closely related to other WNVs of cluster 2. Comparative analysis revealed that the putative E protein encoded by the genome possessed the original WNV E protein glycosylation motif NYS at E154-156, which contained the N-linked glycosylation site at N-154 associated with increased WNV pathogenicity and neuroinvasiveness. In the putative NS1 protein, the A70S substitution observed in other cluster 2 WNVs and P250, which has been implicated in neuroinvasiveness, were present. In addition, the foo motif in the putative NS2A protein, which has been implicated in neuroinvasiveness, was detected. Notably, the amino-acid residues at 14 positions in the present dromedary WNV genome differed from those in most of the closely related WNV strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative E and NS5 proteins. The present study is the first to demonstrate the isolation of WNV from dromedaries. This finding expands the possible reservoirs of WNV and sources of WNV infection. PMID:27273223

  7. Dengue virus type 3 adaptive changes during epidemics in Sao Jose de Rio Preto, Brazil, 2006-2007.

    Directory of Open Access Journals (Sweden)

    Christian Julian Villabona-Arenas

    Full Text Available Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS and nonsynonimous (dN substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1 and 8 under significant purifying selection (dN/dS < 1. We found several polymorphic amino acid coding sites in the envelope (15, NS1 (17, NS2A (11, and NS5 (24 genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

  8. Regulation of Hepatitis C Virus Replication and Gene Expression by the MAPK-ERK Pathway

    Institute of Scientific and Technical Information of China (English)

    Rongjuan Pei; Xiaoyong Zhang; Song Xu; Zhongji Meng; Michael Roggendorf; Mengji Lu; Xinwen Chen

    2012-01-01

    The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle.In the present study using a Huh7 cell line Con1 with an HCV replicon,we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-α signalling.Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells.It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site.Consistently,a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays.Thus,the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication.In addition,cyclin-dependent kinases (CDKs) downstream of ERK may also be involved in the modulation of HCV replication since roscovitine,an inhibitor of CDKs had a similar effect to that of U0126.Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels.Further,the replication of HCV replicon in Conl cells was inhibited by IFN-α.The inhibitory effect of IFN-α could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs.It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

  9. Tembusu-like flavivirus (Perak virus) as the cause of neurological disease outbreaks in young Pekin ducks.

    Science.gov (United States)

    Homonnay, Zalán Gábor; Kovács, Edit Walkóné; Bányai, Krisztián; Albert, Mihály; Fehér, Enikő; Mató, Tamás; Tatár-Kis, Tímea; Palya, Vilmos

    2014-01-01

    A neurological disease of young Pekin ducks characterized by ataxia, lameness, and paralysis was observed at several duck farms in Malaysia in 2012. Gross pathological lesions were absent or inconsistent in most of the cases, but severe and consistent microscopic lesions were found in the brain and spinal cord, characterized by non-purulent panencephalomyelitis. Several virus isolates were obtained in embryonated duck eggs and in cell cultures (Vero and DF-1) inoculated with the brain homogenates of affected ducks. After exclusion of other viruses, the isolates were identified as a flavivirus by flavivirus-specific reverse transcription-polymerase chain reaction (RT-PCR) assays. Inoculation of 2-week-old Pekin ducks with a flavivirus isolate by the subcutaneous or intramuscular route resulted in typical clinical signs and histological lesions in the brain and spinal cord. The inoculated virus was detected by RT-PCR from organ samples of ducks with clinical signs and histological lesions. With a few days delay, the disease was also observed among co-mingled contact control birds. Phylogenetic analysis of NS5 and E gene sequences proved that the isolates were representatives of a novel phylogenetic group within clade XI (Ntaya virus group) of the Flavivirus genus. This Malaysian Duck Tembusu Virus (DTMUV), named Perak virus, has moderate genomic RNA sequence similarity to a related DTMUV identified in China. In our experiment the Malaysian strain of DTMUV could be transmitted in the absence of mosquito vectors. These findings may have implications for the control and prevention of this emerging group of flaviviruses. PMID:25299764

  10. Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    Betania Paiva Drumond

    2016-03-01

    Full Text Available Abstract Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1–4 are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population.

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

    Directory of Open Access Journals (Sweden)

    Po-Yuan Ke

    Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  12. Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

    Directory of Open Access Journals (Sweden)

    Wan Zhixiang

    2005-07-01

    Full Text Available Abstract Background Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR-based assays and enzyme-linked immunosorbent assays (ELISA are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Methods Chemically modified glass slides were used as solid supports (named chip, on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. Results We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05 existed between the results determined by our assay and ELISA respectively. Conclusion

  13. Improving Drug Design: An Update on Recent Applications of Efficiency Metrics, Strategies for Replacing Problematic Elements, and Compounds in Nontraditional Drug Space.

    Science.gov (United States)

    Meanwell, Nicholas A

    2016-04-18

    -based application. In this review, after describing the background literature behind the derivation of efficiency metrics and approaches to assessing compound aesthetics, synopses of some recent practical application in lead optimization campaigns are presented. However, molecules that fall into space beyond that associated with traditional drug-like properties are an important part of the current and future landscape, exemplified by the summary of direct acting hepatitis C virus NS3 and NS5A inhibitors that have transformed clinical therapy for this chronic disease. While drug development in nontraditional drug-like space is more challenging and the rules for compound quality will be different with much still to be understood, careful and disciplined drug design practices will be an essential element of success. PMID:26974882

  14. A five patient’s case study on the influence of two different probiotics on individual intestinal microbiota

    Directory of Open Access Journals (Sweden)

    Yoko Uchiyama-Tanaka

    2013-05-01

    Full Text Available ABSTRACTBackground: The composition and activities of indigenous intestinal microbiota are of paramount importance to human immunity, nutrition, and pathological processes, and hence, the health of the individual. It is well established that the intestine is an important site for local immunity. It is known that the effect of probiotics increases beneficial microbiota and improves chronic conditions such as atopic diseases, irritable bowel disease, and obesity. However, as there are so many probiotics, it is unknown which probiotics might have more of an impact upon intestinal microbiota.Objective: To understand how two different types of probiotics influence human intestinal microbiota, we analyzed human fecal microbiota after taking each of the probiotics.Methods: Five outpatients from Yoko Clinic (1 male and 4 females; aged between 34–46 years old were enrolled in this study. None of the subjects had cancer or any active inflammatory diseases. The five patients took Lactobacillus buchneri (SU for 4 weeks, no probiotics the following week, and mixed probiotics (NS which are Lactobacillus plantarum (NS-5, Lactobacillus rhamnosus (NS-11, Lactobacillus delbruekii (NS-12, Lactobacillus helveticus (NS-8, Lactobacillus fermentum (NS-9 for the following 4 weeks. Fecal samples were collected before and after the outpatients took each of the two probiotics, and were then analyzed using a kit from Techno Suruga Laboratory Co., Ltd. The analysis of the microbiota was performed by targeting bacterial 16S rRNA genes with a terminal restriction fragment length polymorphism analysis program (Nagashima method.Results: Three patients of the five patients decreased the percentage of beneficial bacteria(Lactobacillales, Bifidobacteria after taking SU (13.7 ± 7.1% to 4.0 ± 3.5%, whereas the remaining two patients showed an increased percentage of beneficial bacteria (16.8 ± 3.4% to 30.4 ± 4.6%. After taking NS, the three patients who decreased the

  15. HIV Drug Resistance Surveillance in Honduras after a Decade of Widespread Antiretroviral Therapy.

    Directory of Open Access Journals (Sweden)

    Santiago Avila-Ríos

    Full Text Available We assessed HIV drug resistance (DR in individuals failing ART (acquired DR, ADR and in ART-naïve individuals (pre-ART DR, PDR in Honduras, after 10 years of widespread availability of ART.365 HIV-infected, ART-naïve, and 381 ART-experienced Honduran individuals were enrolled in 5 reference centres in Tegucigalpa, San Pedro Sula, La Ceiba, and Choluteca between April 2013 and April 2015. Plasma HIV protease-RT sequences were obtained. HIVDR was assessed using the WHO HIVDR mutation list and the Stanford algorithm. Recently infected (RI individuals were identified using a multi-assay algorithm.PDR to any ARV drug was 11.5% (95% CI 8.4-15.2%. NNRTI PDR prevalence (8.2% was higher than NRTI (2.2% and PI (1.9%, p500 vs. <350 CD4+ T cells/μL. PDR in recently infected individuals was 13.6%, showing no significant difference with PDR in individuals with longstanding infection (10.7%. The most prevalent PDR mutations were M46IL (1.4%, T215 revertants (0.5%, and K103NS (5.5%. The overall ADR prevalence in individuals with <48 months on ART was 87.8% and for the ≥48 months on ART group 81.3%. ADR to three drug families increased in individuals with longer time on ART (p = 0.0343. M184V and K103N were the most frequent ADR mutations. PDR mutation frequency correlated with ADR mutation frequency for PI and NNRTI (p<0.01, but not for NRTI. Clusters of viruses were observed suggesting transmission of HIVDR both from ART-experienced to ART-naïve individuals and between ART-naïve individuals.The global PDR prevalence in Honduras remains at the intermediate level, after 10 years of widespread availability of ART. Evidence of ADR influencing the presence of PDR was observed by phylogenetic analyses and ADR/PDR mutation frequency correlations.

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

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

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

  17. Sofosbuvir for the treatment of chronic hepatitis C: between current evidence and future perspectives

    Directory of Open Access Journals (Sweden)

    Degasperi E

    2014-04-01

    Full Text Available Elisabetta Degasperi, Alessio AghemoDivision of Gastroenterology and Hepatology, AM and M Migliavacca Center, Fondazione IRCCS Ca' Granda Maggiore Hospital, University of Milan, Milan, ItalyAbstract: In recent years, clinical research in the field of new treatments for chronic hepatitis C (HCV has been devoted to developing regimens based on direct-acting antivirals (DAAs, with the goal of increasing treatment efficacy and improving tolerability and safety. This can be achieved by Peginterferon (PegIFN-free anti-HCV regimens, as PegIFN is responsible for many side effects and limits treatment access due to contraindications in some patient categories. Sofosbuvir (SOF is the first compound to enter the market with IFN-free combination regimens; it belongs to the nucleotide inhibitors of viral polymerase NS5B and acts as a chain terminator during the HCV replication process, exhibiting pan-genotypic antiviral activity with a high barrier to resistance. Clinical trials in HCV genotype 2/3 patients have demonstrated optimal efficacy in HCV-2, where the combination SOF/ribavirin (Rbv for 12 weeks resulted in >90% sustained virological response (SVR rates, while HCV-3 patients with advanced liver fibrosis and previous failure to PegIFN plus Rbv therapy still require individualized and optimized treatment strategies. Historically difficult-to-treat genotypes HCV-1, -4–6 can benefit from reduced duration of PegIFN plus SOF and Rbv, while IFN-free regimens in these patients will be based on SOF in combination with other DAA classes. Due to an optimal tolerability and safety profile with no significant drug-to-drug interactions, SOF is currently undergoing clinical trials in the setting of pre- and post-liver transplantation and HIV-coinfected patients, with the objective to address the until now unmet need for safe and efficient treatment in these populations. This article provides an overview of SOF features and the main clinical trials, discussing

  18. Pharmacogenetics of hepatitis C: transition from interferon-based therapies to direct-acting antiviral agents

    Directory of Open Access Journals (Sweden)

    Kamal SM

    2014-06-01

    Full Text Available Sanaa M Kamal1,21Department of Medicine, Division of Hepatology, Gastroenterology and Tropical Medicine, Ain Shams Faculty of Medicine, Cairo, Egypt, 2Department of Medicine, Salman Bin Abdul Aziz College of Medicine, Kingdom of Saudi ArabiaAbstract: Hepatitis C virus (HCV has emerged as a major viral pandemic over the past two decades, infecting 170 million individuals, which equates to approximately 3% of the world's population. The prevalence of HCV varies according to geographic region, being highest in developing countries such as Egypt. HCV has a high tendency to induce chronic progressive liver damage in the form of hepatic fibrosis, cirrhosis, or liver cancer. To date, there is no vaccine against HCV infection. Combination therapy comprising PEGylated interferon-alpha and ribavirin has been the standard of care for patients with chronic hepatitis C for more than a decade. However, many patients still do not respond to therapy or develop adverse events. Recently, direct antiviral agents such as protease inhibitors, polymerase inhibitors, or NS5A inhibitors have been used to augment PEGylated interferon and ribavirin, resulting in better efficacy, better tolerance, and a shorter treatment duration. However, most clinical trials have focused on assessing the efficacy and safety of direct antiviral agents in patients with genotype 1, and the response of other HCV genotypes has not been elucidated. Moreover, the prohibitive costs of such triple therapies will limit their use in patients in developing countries where most of the HCV infection exists. Understanding the host and viral factors associated with viral clearance is necessary for individualizing therapy to maximize sustained virologic response rates, prevent progression to liver disease, and increase the overall benefits of therapy with respect to its costs. Genome wide studies have shown significant associations between a set of polymorphisms in the region of the interleukin-28B (IL

  19. A multicenter evaluation of the Abbott RealTime HCV genotype II assay

    Directory of Open Access Journals (Sweden)

    Marco Ciotti

    2009-12-01

    Full Text Available Viral genotype is an important determinant of the therapeutical outcome of the chronic hepatitis C and is useful in clinical practice to determine the duration of treatment1.While the viral type shows a clear association with therapeutic success, there is currently no evidence to that effect for HCV subtype, whose value is thus confined to epidemiological studies.The Abbott RealTime HCV Genotype II assay, through the use of Minor Groove Binder probes (MGB is able to distinguish genotypes 1 to 6 (target 5’-UTR region and subtypes 1a and 1b (NS5B region. In four different Italian centers a comparison between the Abbott RealTime HCV Genotype II assay and the Versant HCV Genotype 2.0 (LIPA has been performed.A total of 143 non selected samples with the request of HCV genotyping have been analysed. 141/143 samples (98.6% have provided reportable results with both tests (2 indeterminates with LIPA. Concordance at the type level was 96.5% (136/141. Considering the 136 concordant samples, the distribution was as follows: type 1 = 61 (44.9%, 2 = 36 (26.5%, 3 = 21 (15.4%, 4 = 17 (12.5% 5 = 1 (0.7%. Both assays assigned subtype in 56/61 (91.8% samples of genotype 1 (3 and 2 samples only provided the type for LIPA and Abbott, respectively and 50/56 (89.3% had concordant subtype. It is worth to note that 4 of the 5 samples with discordant subtype Abbott 1a/LiPA 1b came from the only center that used for LIPA the 5-UTR amplicon, loosing the benefit of the core region which has been introduced in the version 2 of the test to improve the accuracy in distinguishing between 1a and 1b.There was only one discordant sample at type level (Abbott 4, LIPA 1b which after sequencing and phylogenetic analysis was resolved as type 4. Four mixed infections were detected, 3 with the Abbott test (two 1a+4 and one 1b+3 and 1 with the LIPA test (1a+3. In all cases the comparison test showed a single genotype 1 infection. The new Abbott RealTime HCV Genotype II assay showed a

  20. DCs pulsed with novel HLA-A2-restricted CTL epitopes against hepatitis C virus induced a broadly reactive anti-HCV-specific T lymphocyte response.

    Directory of Open Access Journals (Sweden)

    Zhongsheng Guo

    Full Text Available OBJECTIVE: To determine the capacity of dendritic cells (DCs loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL effector functions. METHODS: A bioinformatics approach was used to predict HLA-A2-restricted HCV-specific CTL epitopes, and the predicted peptides identified from this screen were synthesized. Subsequent IFN-γ ELISPOT analysis detected the stimulating function of these peptides in peripheral blood mononuclear cells (PBMCs from both chronic and self-limited HCV infected subjects (subjects exhibiting spontaneous HCV clearance. Mature DCs, derived in vitro from CD14(+ monocytes harvested from the study subjects by incubation with appropriate cytokine cocktails, were loaded with novel peptide or epitope peptide mixtures and co-cultured with autologous T lymphocytes. Granzyme B (GrB and IFN-γ ELISPOT analysis was used to test for epitope-specific CTL responses. T-cell-derived cytokines contained in the co-cultured supernatant were detected by flow cytometry. RESULTS: We identified 7 novel HLA-A2-restricted HCV-specific CTL epitopes that increased the frequency of IFN-γ-producing T cells compared to other epitopes, as assayed by measuring spot forming cells (SFCs. Two epitopes had the strongest stimulating capability in the self-limited subjects, one found in the E2 and one in the NS2 region of HCV; five epitopes had a strong stimulating capacity in both chronic and self-limited HCV infection, but were stronger in the self-limited subjects. They were distributed in E2, NS2, NS3, NS4, and NS5 regions of HCV, respectively. We also found that mDCs loaded with novel peptide mixtures could significantly increase GrB and IFN-γ SFCs as compared to single peptides, especially in chronic HCV infection subjects. Additionally, we found that DCs pulsed with multiple epitope peptide mixtures induced a Th1-biased immune response. CONCLUSIONS: Seven novel and

  1. Zika Virus Outbreak in Haiti in 2014: Molecular and Clinical Data.

    Directory of Open Access Journals (Sweden)

    John Lednicky

    2016-04-01

    Full Text Available Zika virus (ZIKV, first isolated in Uganda in 1947, is currently spreading rapidly through South America and the Caribbean. In Brazil, infection has been linked with microcephaly and other serious complications, leading to declaration of a public health emergency of international concern; however, there currently are only limited data on the virus (and its possible sources and manifestations in the Caribbean.From May, 2014-February, 2015, in conjunction with studies of chikungunya (CHIKV and dengue (DENV virus infections, blood samples were collected from children in the Gressier/Leogane region of Haiti who presented to a school clinic with undifferentiated febrile illness. Samples were initially screened by RT-PCR for CHIKV and DENV, with samples negative in these assays further screened by viral culture.Of 177 samples screened, three were positive for ZIKV, confirmed by viral sequencing; DENV-1 was also identified in culture from one of the three positive case patients. Patients were from two different schools and 3 different towns, with all three cases occurring within a single week, consistent with the occurrence of an outbreak in the region. Phylogenetic analysis of known full genome viral sequences demonstrated a close relationship with ZIKV from Brazil; additional analysis of the NS5 gene, for which more sequences are currently available, showed the Haitian strains clustering within a monophyletic clade distinct from Brazilian, Puerto Rican and Guatemalan sequences, with all part of a larger clade including isolates from Easter Island. Phylogeography also clarified that at least three major African sub-lineages exist, and confirmed that the South American epidemic is most likely to have originated from an initial ZIKV introduction from French Polynesia into Easter Island, and then to the remainder of the Americas.ZIKV epidemics in South America, as well as in Africa, show complex dissemination patterns. The virus appears to have been

  2. A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems.

    Science.gov (United States)

    Carta, A; Briguglio, I; Piras, S; Corona, P; Ibba, R; Laurini, E; Fermeglia, M; Pricl, S; Desideri, N; Atzori, E M; La Colla, P; Collu, G; Delogu, I; Loddo, R

    2016-07-19

    In this work, we present and discuss a comprehensive set of both newly and previously synthesized compounds belonging to 5 distinct molecular classes of linear aromatic N-polycyclic systems that efficiently inhibits bovine viral diarrhea virus (BVDV) infection. A coupled in silico/in vitro investigation was employed to formulate a molecular rationale explaining the notable affinity of all molecules to BVDV RNA dependent RNA polymerase (RdRp) NS5B. We initially developed a three-dimensional common-feature pharmacophore model according to which two hydrogen bond acceptors and one hydrophobic aromatic feature are shared by all molecular series in binding the viral polymerase. The pharmacophoric information was used to retrieve a putative binding site on the surface of the BVDV RdRp and to guide compound docking within the protein binding site. The affinity of all compounds towards the enzyme was scored via molecular dynamics-based simulations, showing high correlation with in vitro EC50 data. The determination of the interaction spectra of the protein residues involved in inhibitor binding highlighted amino acids R295 and Y674 as the two fundamental H-bond donors, while two hydrophobic cavities HC1 (residues A221, I261, I287, and Y289) and HC2 (residues V216, Y303, V306, K307, P408, and A412) fulfill the third pharmacophoric requirement. Three RdRp (K263, R295 and Y674) residues critical for drug binding were selected and mutagenized, both in silico and in vitro, into alanine, and the affinity of a set of selected compounds towards the mutant RdRp isoforms was determined accordingly. The agreement between predicted and experimental data confirmed the proposed common molecular rationale shared by molecules characterized by different chemical scaffolds in binding to the BVDV RdRp, ultimately yielding compound 6b (EC50 = 0.3 μM; IC50 = 0.48 μM) as a new, potent inhibitor of this Pestivirus. PMID:27161176

  3. Characteristics of HCV co-infection among HIV infected individuals from an area with high risk of blood-borne infections in central China.

    Directory of Open Access Journals (Sweden)

    Tiejun Zhang

    Full Text Available OBJECTIVE: Hepatitis C virus (HCV and human immunodeficiency virus (HIV co-infection has been proved to be a growing public health concern. The prevalence and genotypic pattern vary with geographic locations. Limited information is available to date with regard to HCV genotype and its clinical implications among those former commercial blood donor communities. The aims of this study were to genetically define the HCV genotype and associated clinical characteristics of HIV/HCV co-infected patients from a region with commercial blood donation history in central China. METHODS: A cross sectional study, including 164 HIV infected subjects, was conducted in Shanxi province central China. Serum samples were collected and HCV antibody testing, AST and ALT testing were performed. Seropositive samples were further subjected to RT-PCR followed by direct sequence coupled with phylogenetic analysis of Core-E1 and NS5B regions performed in comparison with known reference genotypes. FINDINGS: A total of 139 subjects were HCV antibody positive. Genotype could be determined for 88 isolates. Phylogenetic analysis revealed that the predominant circulating subtype was HCV 1b (65.9%, followed by HCV 2a (34.1%. The HCV viral load in the subjects infected with HIV1b was significantly higher than those infected with HCV 2a (P = 0.006. No significant difference for HCV RNA level was detected between ART status, CD4+ cell count level and HIV RNA level. Serum AST and ALT level were likely to increase with HCV RNA level, although no significance was observed. Those who had conducted commercial donation later than 1991 (OR 3.43, 95% CI: 1.12-10.48 and had a short duration of donation (OR 0.35, 95% CI: 0.13-0.96 were more likely to be infected with HCV 1b. CONCLUSION: These results suggest that HCV subtype 1b predominates in this population, and the impact of HIV status and ART on HCV disease progression is not significantly correlated.

  4. The evolution of the major hepatitis C genotypes correlates with clinical response to interferon therapy.

    Directory of Open Access Journals (Sweden)

    Phillip S Pang

    Full Text Available BACKGROUND: Patients chronically infected with hepatitis C virus (HCV require significantly different durations of therapy and achieve substantially different sustained virologic response rates to interferon-based therapies, depending on the HCV genotype with which they are infected. There currently exists no systematic framework that explains these genotype-specific response rates. Since humans are the only known natural hosts for HCV-a virus that is at least hundreds of years old-one possibility is that over the time frame of this relationship, HCV accumulated adaptive mutations that confer increasing resistance to the human immune system. Given that interferon therapy functions by triggering an immune response, we hypothesized that clinical response rates are a reflection of viral evolutionary adaptations to the immune system. METHODS AND FINDINGS: We have performed the first phylogenetic analysis to include all available full-length HCV genomic sequences (n = 345. This resulted in a new cladogram of HCV. This tree establishes for the first time the relative evolutionary ages of the major HCV genotypes. The outcome data from prospective clinical trials that studied interferon and ribavirin therapy was then mapped onto this new tree. This mapping revealed a correlation between genotype-specific responses to therapy and respective genotype age. This correlation allows us to predict that genotypes 5 and 6, for which there currently are no published prospective trials, will likely have intermediate response rates, similar to genotype 3. Ancestral protein sequence reconstruction was also performed, which identified the HCV proteins E2 and NS5A as potential determinants of genotype-specific clinical outcome. Biochemical studies have independently identified these same two proteins as having genotype-specific abilities to inhibit the innate immune factor double-stranded RNA-dependent protein kinase (PKR. CONCLUSION: An evolutionary analysis of all

  5. Zika Virus Outbreak in Haiti in 2014: Molecular and Clinical Data

    Science.gov (United States)

    Lednicky, John; Beau De Rochars, Valery Madsen; El Badry, Maha; Loeb, Julia; Telisma, Taina; Chavannes, Sonese; Anilis, Gina; Cella, Eleonora; Ciccozzi, Massimo; Rashid, Mohammed; Okech, Bernard; Salemi, Marco; Morris, J. Glenn

    2016-01-01

    Background Zika virus (ZIKV), first isolated in Uganda in 1947, is currently spreading rapidly through South America and the Caribbean. In Brazil, infection has been linked with microcephaly and other serious complications, leading to declaration of a public health emergency of international concern; however, there currently are only limited data on the virus (and its possible sources and manifestations) in the Caribbean. Methods From May, 2014-February, 2015, in conjunction with studies of chikungunya (CHIKV) and dengue (DENV) virus infections, blood samples were collected from children in the Gressier/Leogane region of Haiti who presented to a school clinic with undifferentiated febrile illness. Samples were initially screened by RT-PCR for CHIKV and DENV, with samples negative in these assays further screened by viral culture. Findings Of 177 samples screened, three were positive for ZIKV, confirmed by viral sequencing; DENV-1 was also identified in culture from one of the three positive case patients. Patients were from two different schools and 3 different towns, with all three cases occurring within a single week, consistent with the occurrence of an outbreak in the region. Phylogenetic analysis of known full genome viral sequences demonstrated a close relationship with ZIKV from Brazil; additional analysis of the NS5 gene, for which more sequences are currently available, showed the Haitian strains clustering within a monophyletic clade distinct from Brazilian, Puerto Rican and Guatemalan sequences, with all part of a larger clade including isolates from Easter Island. Phylogeography also clarified that at least three major African sub-lineages exist, and confirmed that the South American epidemic is most likely to have originated from an initial ZIKV introduction from French Polynesia into Easter Island, and then to the remainder of the Americas. Conclusions ZIKV epidemics in South America, as well as in Africa, show complex dissemination patterns. The

  6. Intradermal fractional booster dose of inactivated poliomyelitis vaccine with a jet injector in healthy adults.

    Science.gov (United States)

    Soonawala, Darius; Verdijk, Pauline; Wijmenga-Monsuur, Alienke J; Boog, Claire J; Koedam, Patrick; Visser, Leo G; Rots, Nynke Y

    2013-08-12

    For global eradication of poliomyelitis, inactivated poliovirus vaccine (IPV) needs to become available in all countries. Using fractional-doses (reduced-doses) may impact affordability and optimize the utilization of the production capacity. Intradermal administration has the potential to lower the dose without reducing immunogenicity. A needle-free jet injector may be a reliable way to administer vaccines intradermally. The primary objective of this randomized controlled trial was to compare the immunogenicity and tolerability of fractional-dose intradermal IPV (Netherlands Vaccine Institute, NVI) booster vaccination administered with a jet injector (PharmaJet) to full-dose and fractional-dose intramuscular vaccination with a needle and syringe. Immunogenicity was assessed by comparing the differences in the post-vaccination log2 geometric mean concentrations of neutralizing antibodies (GMC) between the study groups. A total of 125 Dutch adult volunteers with a well-documented vaccination history were randomized to one of four groups: full-dose intramuscular needle (IM-NS-0.5), full-dose intramuscular jet injector (IM-JI-0.5), 1/5th dose intramuscular needle (IM-NS-0.1), 1/5th dose intradermal jet injector (ID-JI-0.1). Vaccination with the JI was less painful (87% no pain) than vaccination with a NS (60% no pain), but caused more transient erythema (JI 85%, NS 24%) and swelling (JI 50%, NS 5%). Intradermal vaccination caused less vaccination site soreness (ID 16%, IM 52%). At baseline all subjects had seroprotective antibody concentrations. After 28 days, GMC were slightly lower in the ID-JI-0.1 group than in the reference group (IM-NS-0.5). The differences were not statistically significant, but the stringent non-inferiority criterion (i.e. a difference of 1 serum dilution in the microneutralization assay) was not met. After one year, differences in GMC were no longer apparent. In contrast, intramuscular vaccination with a fractional dose administered with a

  7. Antiviral therapies for chronic hepatitis C virus infectionwith cirrhosis

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Patients who are infected with hepatitis C virus (HCV)and also have advanced fibrosis or cirrhosis have beenrecognized as "difficult-to-treat" patients during an erawhen peginterferon and ribavirin combination therapy isthe standard of care. Recent guidelines have clearly statedthat treatment should be prioritized in this populationto prevent complications such as decompensationand hepatocellular carcinoma. Recent advances in thetreatment of chronic hepatitis C have been achievedthrough the development of direct-acting antiviral agents(DAAs). Boceprevir and telaprevir are first-generationDAAs that inhibit the HCV NS3/4A protease. Bocepreviror telaprevir, in combination with peginterferon andribavirin, improved the sustained virological responserates compared with peginterferon and ribavirin alone andwere tolerated in patients with HCV genotype 1 infectionwithout cirrhosis or compensated cirrhosis. However, theefficacy is lower especially in prior non-responders withor without cirrhosis. Furthermore, a high incidence ofadverse events was observed in patients with advancedliver disease, including cirrhosis, in real-life settings.Current guidelines in the United States and in someEuropean countries no longer recommend these regimensfor the treatment of HCV. Next-generation DAAs includesecond-generation HCV NS3/4A protease inhibitors, HCVNS5A inhibitors and HCV NS5B inhibitors, which have ahigh efficacy and a lower toxicity. These drugs are usedin interferon-free or in interferon-based regimens withor without ribavirin in combination with different classesof DAAs. Interferon-based regimens, such as simeprevirin combination with peginterferon and ribavirin, are welltolerated and are highly effective especially in treatmentna?vepatients and in patients who received treatmentbut who relapsed. The efficacy is less pronounced in nullrespondersand in patients with cirrhosis. Interferonfreeregimens in combination with ribavirin and/ortwo or more DAAs could be

  8. Full-length genomes of 16 hepatitis C virus genotype 1 isolates representing subtypes 1c, 1d, 1e, 1g, 1h, 1i, 1j and 1k, and two new subtypes 1m and 1n, and four unclassified variants reveal ancestral relationships among subtypes.

    Science.gov (United States)

    Lu, Ling; Li, Chunhua; Xu, Yan; Murphy, Donald G

    2014-07-01

    We characterized the full-length genomes of 16 distinct hepatitis C virus genotype 1 (HCV-1) isolates. Among them, four represented the first full-length genomes for subtypes 1d (QC103), 1i (QC181), 1j (QC329) and 1k (QC82), and another four corresponded to subtypes 1c (QC165), 1g (QC78), 1h (QC156) and 1e (QC172). Both QC196 and QC87 were assigned into a new subtype 1m, and QC113 and QC74 into another new subtype 1n. The remaining four (QC60, QC316, QC152 and QC180) did not classify among the established subtypes and corresponded to four new lineages. Subtypes 1j, 1k, 1m, 1n and the unclassified isolate QC60 were identified in Haitian immigrants. In the updated HCV nomenclature of 2005, a total of 12 subtypes of HCV-1 were designated. Including the data from the present study, all but subtype 1f now have their full-length genomes defined. Further analysis of partial NS5B sequences available in GenBank denoted a total of 21 unclassified lineages, indicating the taxonomic complexity of HCV-1. Among them, six have had their full-length genomes characterized. Based on the available full-length genome sequences, a timescale phylogenetic tree was reconstructed which estimated important time points in the evolution of HCV-1. It revealed that subtype 1a diverged from its nearest relatives 135 years ago and subtype 1b diverged from its nearest relatives 112 years ago. When subtypes 1a, 1j, 1k, 1m, 1n and six close relatives (all but one from Haitian immigrants) were considered as a whole, the divergence time was 176 years ago. This diversification was concurrent with the time period when the transatlantic slave trade was active. When taking all the HCV-1 isolates as a single lineage, the divergence time was 326 years ago. This analysis suggested the existence of a recent common ancestor for subtype 1a and the Haitian variants; a co-origin for subtypes 1b, 1i and 1d was also implied. PMID:24718832

  9. Correlação e correspondência topográfica entre espessura da camada de fibras nervosas da retina e campo visual no glaucoma primário de ângulo aberto Correlation and topographic match between nerve fiber layer thickness and visual field in primary open angle glaucoma

    Directory of Open Access Journals (Sweden)

    Paula Boturão de Almeida

    2001-04-01

    classificados como discretos: SF e SN. Nos olhos classificados como moderados: SF e SI. Nos olhos classificados como graves: MD e SN; MD e EM; MD e S; CPSD e EM. 4- DN t e TD t; DN s e TD ni; DN i e TD ns. 5- Encontramos correspondência positiva (+ em 36 olhos (51,43% dos casos e correspondência negativa (- em 34 olhos (48,57% dos casos. Conclusões: Concluiu-se que houve poucas correlações significantes entre esses dois exames, e que as existentes foram muito fracas. Conclui-se, também, que houve correspondência topográfica, na análise dos setores mais comprometidos, em 51,43% dos casos.Purpose: To determine the relationship between nerve fiber layer thickness detected by scanning laser polarimetry (GDx and visual field function measured by automated conventional white-on-white perimetry. Methods: 82 eyes of 48 open angle glaucoma patients were studied. The following correlations were inves-tigated: 1- Correlation between mean sensitivity of 4 quadrants plus the fixation point, in dBs, and the mean of the nerve fiber layer thickness of the correspondent retina, in micra. 2- Correlation between mean sensitivity of 4 quadrants plus the fixation point, in dBs, and the mean of the nerve fiber layer thickness of the correspondent retina, in micra, in the patients classified according to the amount of visual field loss. 3- Correlation between the global indices of visual field and the numeric indices of GDx. 4- Correlation between the mean of "total deviation" of visual field and the "deviation from normal" of GDx. 5- Topographic match between visual field and retinal nerve fiber layer thickness changes. Statistical analysis was performed using the Spearmann coeficient correlation test. Results: We observed a very poor correlation regarding: 1- total GDx and VF total; superior GDx and nasal inferior VF; inferior GDx and nasal superior VF; nasal GDx and temporal VF; superior GDx and inferior VF; inferior GDx and superior VF. 2- inferior GDx and nasal superior VF (severe eyes

  10. Molecular basis of hepatitis C virus -associated hepatocarcinogenesis

    Directory of Open Access Journals (Sweden)

    Isabelle Chemin

    2007-02-01

    Full Text Available

    In areas with an intermediate rate of Hepatocellular Carcinoma (HCC such as Western Europe and Japan, hepatitis C is the predominant cause, whereas in low rate areas such as Western Europe and North USA, HCC is often related to other factors as alcoholic liver disease. There is a rising incidence in HCC in developed countries during the last two decades, due to the increasing rate of hepatitis C infection and improvement of the clinical management of cirrhosis (most of the time appear after cirrhosis, the total number of HCC cases annually registered should be multiplied by 3 until 2020.

    The pathogenesis of HCC in HCV infection has extensively been analysed. Hepatitis C virus-induced chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation are considered as one of the major pathogenic mechanisms. Increasing experimental evidence suggests that HCV contributes to HCC by directly modulating pathways that promote the malignant transformation of hepatocytes. Hepatitis C virus is an RNA virus that does not integrate into the host genome but HCV proteins interact with many host-cell factors well beyond their roles in the viral life cycle and are involved in a wide range of activities at least in vitro, including cell signalling, transcription, cell proliferation, apoptosis, membrane rearrangements, vesicular trafficking and translational regulation. At least four of the HCV gene products, namely HCV core, NS3, NS4B and NS5A, have been shown to exhibit transformation potential in tissue culture and several potentially oncogenic pathways have been shown to be altered by the expression of HCV proteins. Both HCV core and NS5A induce the accumulation of wild-type betacatenin and the Wnt-beta-catenin pathway emerges as a common target

  11. Design, synthesis and conversion evaluation in rats of PSI-6130 prodrugs%PSI-6130前药的设计、合成及其在大鼠体内的转化评价

    Institute of Scientific and Technical Information of China (English)

    张明; 顾为; 张天宏; 张振清; 聂爱华

    2011-01-01

    Hepatitis C virus (HCV) ,is one of the major causes of cirrhosis, hepatocellular carcinoma. HCV infection affects 180 million people worldwide,about 3% of the world's population. Novel treatment options now in development are focused on inhibitors of HCV specific enzymes,NS3 protease and NS5B polymera-se.PSI-6130 (also called R-l 656 ), chemical name β-D-(2'R)-2'-methyl-2'-fluorocytidine, was developed through a partnership formed by Roche and Pharmasset to synthesize nucleoside polymerase NS5B inhibitors for HCV treatment. The compound potently inhibited HCV replication in subgenomic HCV replicon cells (EC90 =4.6 (xmol·L-1). Pharmacokinetic studies in rhesus monkeys,however,revealed slow and incomplete absorption and an oral bioavailability of 24. 0%. To improve absorption and drug exposure and to limit or eliminate the formation of the uridine metabolite led to the identification of PSI-6130 prodrug,R-7128. R-7128 is ongoing a phase lib study. To improve the drug metabolic activity of PSI-6130,five new PSI-6130 prodrugs( I - V) were designed and synthesized in this research. In the course of synthesizing these compounds,the synthesis for PSI-6130 and condensation reaction of valine and PSI-6310 were emphatically explored. The structures of these target compounds were identified by 13C-NMR,MS and "H-NMR. The purity of these compounds was determined by HPLC. Their synthetic routes were investigated and optimized. The conversion efficiency of these new PSI-6130 prodrugs was evaluated by oral bioavailability test in rats. The concentration of PSI-6130 in rat plasma was detected after PSI-6130 I. V. Administration[2(g·L-1 )·kg-1, 0. 00772(mol·L-1 )·kg-1 ]and PSI-6130,prodrugs p. O. Administration[0. 019 mmol·kg-1 ,n =3]respec-tively. The results show that the oral bioavailability of these five prodrugs had greatly improvement against PSI-6130. The conversion efficiencies of prodrugs I ,1V, II , 1 and V were 129% ,88% ,77. 8% ,63. 7% and 58. 1% respectively. The

  12. Gas source molecular beam epitaxy of scandium nitride on silicon carbide and gallium nitride surfaces

    International Nuclear Information System (INIS)

    Scandium nitride (ScN) is a group IIIB transition metal nitride semiconductor with numerous potential applications in electronic and optoelectronic devices due to close lattice matching with gallium nitride (GaN). However, prior investigations of ScN have focused primarily on heteroepitaxial growth on substrates with a high lattice mismatch of 7%–20%. In this study, the authors have investigated ammonia (NH3) gas source molecular beam epitaxy (NH3-GSMBE) of ScN on more closely lattice matched silicon carbide (SiC) and GaN surfaces (3-GSMBE conditions of 10−5–10−4 Torr NH3 and 800–1050 °C where selected for initial investigation. In-situ x-ray photoelectron spectroscopy (XPS) and ex-situ Rutherford backscattering measurements showed all ScN films grown using these conditions were stoichiometric. For ScN growth on 3C-SiC (111)-(√3 × √3)R30° carbon rich surfaces, the observed attenuation of the XPS Si 2p and C 1s substrate core levels with increasing ScN thickness indicated growth initiated in a layer-by-layer fashion. This was consistent with scanning electron microscopy (SEM) images of 100–200 nm thick films that revealed featureless surfaces. In contrast, ScN films grown on 3C-SiC (111)-(3 × 3) and 3C-SiC (100)-(3 × 2) silicon rich surfaces were found to exhibit extremely rough surfaces in SEM. ScN films grown on both 3C-SiC (111)-(√3 × √3)R30° and 2H-GaN (0001)-(1 × 1) epilayer surfaces exhibited hexagonal (1 × 1) low energy electron diffraction patterns indicative of (111) oriented ScN. X-ray diffraction ω-2θ rocking curve scans for these same films showed a large full width half maximum of 0.29° (1047 arc sec) consistent with transmission electron microscopy images that revealed the films to be poly-crystalline with columnar grains oriented at ≈15° to the [0001] direction of the 6H-SiC (0001) substrate. In-situ reflection electron energy loss spectroscopy measurements determined the band-gap for

  13. Gas source molecular beam epitaxy of scandium nitride on silicon carbide and gallium nitride surfaces

    Energy Technology Data Exchange (ETDEWEB)

    King, Sean W., E-mail: sean.king@intel.com; Davis, Robert F. [Department of Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina 27695 (United States); Nemanich, Robert J. [Department of Physics, North Carolina State University, Raleigh, North Carolina 27695 (United States)

    2014-11-01

    Scandium nitride (ScN) is a group IIIB transition metal nitride semiconductor with numerous potential applications in electronic and optoelectronic devices due to close lattice matching with gallium nitride (GaN). However, prior investigations of ScN have focused primarily on heteroepitaxial growth on substrates with a high lattice mismatch of 7%–20%. In this study, the authors have investigated ammonia (NH{sub 3}) gas source molecular beam epitaxy (NH{sub 3}-GSMBE) of ScN on more closely lattice matched silicon carbide (SiC) and GaN surfaces (<3% mismatch). Based on a thermodynamic analysis of the ScN phase stability window, NH{sub 3}-GSMBE conditions of 10{sup −5}–10{sup −4} Torr NH{sub 3} and 800–1050 °C where selected for initial investigation. In-situ x-ray photoelectron spectroscopy (XPS) and ex-situ Rutherford backscattering measurements showed all ScN films grown using these conditions were stoichiometric. For ScN growth on 3C-SiC (111)-(√3 × √3)R30° carbon rich surfaces, the observed attenuation of the XPS Si 2p and C 1s substrate core levels with increasing ScN thickness indicated growth initiated in a layer-by-layer fashion. This was consistent with scanning electron microscopy (SEM) images of 100–200 nm thick films that revealed featureless surfaces. In contrast, ScN films grown on 3C-SiC (111)-(3 × 3) and 3C-SiC (100)-(3 × 2) silicon rich surfaces were found to exhibit extremely rough surfaces in SEM. ScN films grown on both 3C-SiC (111)-(√3 × √3)R30° and 2H-GaN (0001)-(1 × 1) epilayer surfaces exhibited hexagonal (1 × 1) low energy electron diffraction patterns indicative of (111) oriented ScN. X-ray diffraction ω-2θ rocking curve scans for these same films showed a large full width half maximum of 0.29° (1047 arc sec) consistent with transmission electron microscopy images that revealed the films to be poly-crystalline with columnar grains oriented at ≈15° to the [0001] direction of the

  14. Survey on natural infection of Japanese encephalitis virus in mosquitoes in Longgang District of Shenzhen City%深圳市龙岗区蚊虫自然感染流行性乙型脑炎病毒状况调查

    Institute of Scientific and Technical Information of China (English)

    邹志辉; 周健明; 林琳; 刘渠; 张起文; 王德全

    2012-01-01

    目的 研究深圳市龙岗区蚊虫流行性乙型脑炎病毒自然感染状况,为预测该地区乙脑发病趋势及流行强度,制定有效预防控制措施提供科学依据.方法 采用灯诱法捕捉蚊虫标本并进行分类鉴定,以JEV的NS3基因区段设计特异引物,通过实时荧光RT-PCR (real-time RT-PCR)对蚊虫中流行性乙型脑炎病毒进行检测,阳性标本经测序后再在GenBank中进行核苷酸序列的同源性比对.结果 捕获的蚊虫隶属3属5种,致倦库蚊是优势蚊种;1 533只蚊虫分成25批,从3批致倦库蚊标本中检出JEV核酸阳性,阳性率为12%.阳性标本与GenBank中报道的JEV参考株间相应的核苷酸序列同源性达99%以上,均属于JEV Ⅰ型病毒.结论 龙岗区存在着蚊虫自然感染JEV,致倦库蚊是区内乙脑传播的主要蚊媒,有流行人间和畜间流行性乙型脑炎的潜在危险,提示应进一步完善疫苗接种和免疫监测,积极开展消除蚊虫孳生地,灭蚊、防蚊工作,以控制和切断乙脑传播途径.%OBJECTIVE To explore JEV natural infection of captured mosquitoes in Longgang District of Shenzhen City and provide reference for prediction of the prevalence trend and intension of the epidemic encephalitis B. METHODS Lamp baited trap was used to capture mosquitoes. JEV specific primers in NS5 were designed and the Japanese encephalitis virus was identified with real-time RT-PCR. The nucleonic acid detection of positive samples was confirmed by sequencing and the NS3 gene sequence was compared with that published in GenBank. Then the genotype of Japanese encephalitis virus was determined. RESULTS Mosquitoes collected were subjected to 3 genera 5 species and Culex fatigans was dominant population. A total of 1 533 mosquitoes were collected and divided into 25 groups, and 12% were identified positive for viral RNA. The real-time RT-PCR assay showed that 3 positive samples were all Culex fatigans. Aligning the sequence with the Gen

  15. 白纹伊蚊感染登革病毒后病毒特异性piRNAs分析%Dengue virus-derived piRNAs in Aedes albopictus

    Institute of Scientific and Technical Information of China (English)

    赖植发; 王衍海; 顾金保; 陈晓光

    2014-01-01

    也具有偏向性,最高峰位于病毒非结构蛋白NS5编码区域的3'末端.第10位碱基具有较强的腺嘌呤偏好性,但第1位碱基不具有尿嘧啶偏好性;也无典型“乒乓”扩增循环的特征.结论 白纹伊蚊感染登革病毒后,体内出现病毒特异性vsRNAs与vpiRNAs,对其鉴定与分析将为在白纹伊蚊抗病毒免疫研究提供理论基础.

  16. 硫辛酸注射液与木糖醇和果糖注射液的配伍稳定性考察%Compatible stability of thioctic aciol injection(ALA) in xylitol injection and fructose injection

    Institute of Scientific and Technical Information of China (English)

    甘惠贞; 林淑瑜; 潘丹婷; 杨昌云

    2014-01-01

    目的:探讨硫辛酸注射液的高效液相色谱法检测条件以及在木糖醇和果糖注射液中的稳定性,为临床提供参考。方法将600 mg硫辛酸注射液与250 mL的木糖醇注射液及果糖注射液配伍,测定溶液的pH值、硫辛酸的含量,定时观察溶液的颜色及澄明度。结果本实验所建立的色谱条件对硫辛酸注射液的检测专属性好,硫辛酸在0.06~2.0 mg/mL浓度范围内,线性关系良好( r=0.9997)。硫辛酸注射液与木糖醇配伍后,在避光条件下硫辛酸含量、溶液的pH值、澄清度均无明显的变化,24 h内稳定性好。而与果糖注射液配伍后溶液的颜色澄清度有明显的变化,且硫辛酸降解快,4 h后的含量为89.89%,配伍稳定性不好。结论硫辛酸注射液可以与木糖醇注射液配伍,不宜与果糖注射液配伍。%Objective To investigate a appropriate chromatography method for ALA and the stability of ALA in xylitol injection and fructose injection. Methods 600 mg ALA was added into 250 mL xylitol injection and fructose injection respectively at room temperature and dark conditions,pH value and visual appearance of the admixture were observed at immediate 1,2,4,6,8,12,24 h after ALA adding to the solvent. ALA concentrations were determined by the method of HPLC. Results The chromatography method established in this study was specific,ALA showed a good linear relationship within the range of 0. 06 ~2. 0 mg/mL ( r =0. 999 7 ) . The admixture showed clear and achromatic color in 24 h after adding into the 0. 9%NS,5%xylitol injection,and the pH value and ALA content showed no great change. pH value,colour,visual appearance of the admixture changed apparently after ALA adding into fructose injec-tion,and the degradation was rapid,the content of ALA was 89. 89%after 4 h. Conclusion ALA can be mixed with xylitol injection in clinical practice,but cannot be mixed with fructose injection.

  17. Иммуногенные свойства ДНК-конструкций, содержащих структурную и неструктурную области вируса гепатита С

    OpenAIRE

    ПЕРМЯКОВА К.Ю.; ЛЕСНОВА Е.И.; МАСАЛОВА ОЛЬГА ВЛАДИМИРОВНА; А. В. Иванов; АТАУЛЛАХАНОВ Р.И.; Кущ, А. А.

    2015-01-01

    Создание вакцины против гепатита С приоритетная задача современной биотехнологии и медицины. Одним из перспективных подходов является разработка ДНК-конструкций, содержащих гены вируса гепатита С (ВГС). Цель настоящей работы состояла в сравнительном анализе иммунного ответа мышей на дНК-иммунизацию плазмидой pcE1-E2, содержащей гены поверхностных белков ВГС, с иммунным ответом на плазмиду pcNS3-NS5B, включающую гены неструктурной области вируса, и оценке эффективности их совместного введения....

  18. St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil Vírus da encefalite São Luis: primeiro isolamento de humano no Estado de São Paulo, Brasil

    Directory of Open Access Journals (Sweden)

    Iray M. Rocco

    2005-10-01

    Full Text Available This paper reports the isolation of St. Louis encephalitis virus (SLEV from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7, confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.O presente estudo relata o isolamento do vírus da encefalite São Luis (SLEV de um caso febril humano suspeito de dengue, em São Pedro, Estado de São Paulo. MAC-ELISA realizado com soros das fases aguda e convalescente foi inconclusivo e anticorpos IgG foram detectados por inibição da hemaglutinação para flavivirus. Imunofluorescência indireta com cultura de células C6/36 inoculadas com soro da fase aguda foi positivo para flavivirus mas negativo quando testado com anticorpos monoclonais para dengue. O RNA extraído de cultura de células infectadas foi amplificado na presença de primers universais para o gênero Flavivirus, deduzidos de uma regi

  19. Trace Gas Measurements on Mars and Earth Using Optical Parametric Generation

    Science.gov (United States)

    Numata, Kenji; Haris, Riris; Li, Steve; Sun, Xiaoli; Abshire, James Brice

    2010-01-01

    for more detailed in-situ analysis. In this paper we report on remote sensing measurements of methane using a high peak power, widely tunable optical parametric generator (OPG) operating at 3.3 micron and 1.65 micron. The OPG is pumped by a passively q-switched single frequency laser (3ns, 5KHz, 50uJ) and seeded by a diode laser. The spectral width of both signal and idler of seeded OPG is nearly Fourier transform limited. The output of seeded OPG is single frequency with high spectral purity and is widely tunable. Both 1650 nm and 3300 nm can be generated with a conversion efficiency of more than 30%. We have demonstrated detection of methane at 3274 nm and 1650 nm in a cell and also performed open path atmospheric measurements of methane at the same wavelengths. Finally, we were able to demonstrate simultaneous detection of methane at 3270.4 nm and CO2 at 1578.2 nm. In this paper we will discuss the OPG performance and atmospheric open path measurement results.

  20. Phenotypic characterization of patient dengue virus isolates in BALB/c mice differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome

    Directory of Open Access Journals (Sweden)

    Buchy Philippe

    2011-08-01

    unique for the DSS isolates that were located both in structural genes (M and E and in non-structural genes (NS1, NS3, and NS5. The characterization of these clinically distinct DENV-1 isolates highlight that DENVs within the same genotype may have different in vivo phenotypes. Highlights • Clinical DENV-1 isolates have different organ tropism in BALB/c mice. • The isolate from a DSS patient is primarily neurotropic compared to the other isolates. • The DENV-1 isolates have different in vivo replication kinetics. • The isolate from a DSS patient persists longer compared to the other isolates. • These phenotypic differences confirm our earlier in vitro findings with the same DENV-1 isolates. Thus, DENVs within the same serotype and genotype may differ enough to affect clinical conditions in vivo.

  1. Specific genetic markers for detecting subtypes of dengue virus serotype-2 in isolates from the states of Oaxaca and Veracruz, Mexico

    Directory of Open Access Journals (Sweden)

    Camacho-Nuez Minerva

    2008-07-01

    Full Text Available Abstract Background Dengue (DEN is an infectious disease caused by the DEN virus (DENV, which belongs to the Flavivirus genus in the family Flaviviridae. It has a (+ sense RNA genome and is mainly transmitted to humans by the vector mosquito Aedes aegypti. Dengue fever (DF and dengue hemorrhagic fever (DHF are caused by one of four closely related virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4. Epidemiological and evolutionary studies have indicated that host and viral factors are involved in determining disease outcome and have proved the importance of viral genotype in causing severe epidemics. Host immune status and mosquito vectorial capacity are also important influences on the severity of infection. Therefore, an understanding of the relationship between virus variants with altered amino acids and high pathogenicity will provide more information on the molecular epidemiology of DEN. Accordingly, knowledge of the DENV serotypes and genotypes circulating in the latest DEN outbreaks around the world, including Mexico, will contribute to understanding DEN infections. Results 1. We obtained 88 isolates of DENV, 27 from Oaxaca and 61 from Veracruz. 2. Of these 88 isolates, 16 were serotype 1; 62 serotype 2; 7 serotype 3; and 2 serotype 4. One isolate had 2 serotypes (DENV-2 and -1. 3. Partial nucleotide sequences of the genes encoding C- prM (14 sequences, the NS3 helicase domain (7 sequences, the NS5 S-adenosyl methionine transferase domain (7 sequences and the RNA-dependent RNA polymerase (RdRp domain (18 sequences were obtained. Phylogenetic analysis showed that DENV-2 isolates belonged to the Asian/American genotype. In addition, the Asian/American genotype was divided into two clusters, one containing the isolates from 2001 and the other the isolates from 2005–2006 with high bootstrap support of 94%. Conclusion DENV-2 was the predominant serotype in the DF and DHF outbreak from 2005 to 2006 in Oaxaca State as well as in the 2006

  2. 拉科酰胺对神经病理性疼痛大鼠的镇痛作用%Analgesiac effects of lacosamide on neuropathic pain and its mechanism

    Institute of Scientific and Technical Information of China (English)

    姜迎海; 夏令杰; 陶熔; 刘琳

    2016-01-01

    目的 观察拉科酰胺在大鼠神经病理性疼痛中的镇痛作用.方法 制备神经病理性疼痛(DNP)模型大鼠27只,随机分为3组(n=9):A组为腹腔内注射生理盐水(NS)5 ml;B组为腹腔内注射拉科酰胺60 mg;C组为腹腔内注射拉科酰胺120 mg.测定给药前、给药后10、20、30、40、50 min大鼠机械性缩足反射阈值(MWT)和热缩足潜伏期(TWL).结果 各时间点B组大鼠MWT[10 min(17.46±0.71) g;20 min(18.17±0.61) g;30 min(17.21±0.41) g;40 min(17.82±0.53)g;50 min(18.17±0.37)g],TWL[10 min(9.21±0.37) s;20 min(8.90±0.62) s;30 min(9.46±0.29)s;40 min(9.28±0.73) s;50min(9.41±0.32)s],均较A组明显增加(P<0.05);各时间点C组大鼠MWT[10 min(21.02±0.29) g;20 min(20.89±0.35) g;30 min(20.24±0.62) g;40 min(21.71±0.36) g;50 min(21.07±0.74)g],TWL[10 min(10.46±0.23) s;20 min(11.21±0.74) s;30 min(10.97±0.27) s;40 min(10.62±0.46) s;50 min(10.71±0.39)s],均较A组明显增加(P<0.01).结论 腹腔内注射拉科酰胺对大鼠神经病理性疼痛有较强的镇痛作用.%Objective To evaluate the analgesic effect of lacosamide on neuropathic pain in rats.Methods Twenty-seven rats were used to establish the models of neuropathic pain.The rats were randomly divided into 3 groups (n =9):group A,5 ml normal saline was intraperitoneally injected;group B,60 mg lacosamide was intraperitoneally given;group C,120 mg lacosarnide was intraperitoneally administered.The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured before and 10,20,30,40 and 50 min after administraton respectirely.Results In group B,MWT at each time point [10 min:(17.46±0.71) g;20 min:(18.17 ±0.61) g;30 min:(17.21 ±0.41) g;40 min:(17.82±0.53) g;and 50 min:(18.17 ±0.37) g],and TWL [10 min:(9.21 ±0.37) s;20min:(8.90 ±0.62) s;30 min:(9.46 ±0.29) s;40 min:(9.28 ±0.73) s;50 min:(9.41 ± 0.32) s] were significantly higher than in group A (P < 0.05).In group C,MWT at each time point [10 min:(21.02 ±0

  3. Are the Real HCV Infection Features in Iranian Patients the Same As What Is Expected?

    Directory of Open Access Journals (Sweden)

    Seyed-Moayed Alavian

    2005-03-01

    analysis? Survey of published randomised controlled trials. BMJ 1999; 319:670-46. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: A randomised trial. Lancet 2001; 358:958-657. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL Jr, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347:975-828. Samimi-Rad K, Nategh R, Malekzadeh R, Norder H, Magnius L. Molecular epidemiology of hepatitis C virus in Iran as reflected by phylogenetic analysis of the NS5B region. J Med Virol 2004; 74:246-529. Ahmadipour MH, Keivani H. Sabahi F, Alavian SM. Determination of HCV genotypes in Iranian isolated by PCR-RFLP [Abstract]. J Gastroenterol Hepatol 2004; 19 (Suppl:A71210. Copegus™ [package insert]. Nutley, NJ: Hoffman-La Roche Inc; December 2002