Efficient infection of tree shrew (Tupaia belangeri) with hepatitis C virus grown in cell culture or from patient plasma

The generation of a new, cost-effective, non-primate, small-animal model would greatly facilitate research into hepatitis C virus (HCV) pathogenesis and the development of novel therapeutic and preventative technologies to control the increasing HCV threat to public health. Native HCV from patient plasma and HCV grown in cell culture (HCVcc) were used to inoculate adult tree shrews. Each animal was inoculated with one HCV genotype. Alanine aminotransferase (ALT) levels, HCV RNA and viral load were determined in the animals before and after inoculation. For native HCV, 16/18 inoculated tree shrews (89 %) became infected; 12/16 (75 %) of these animals became chronically infected, whilst infection was resolved in the remaining four (25 %). For HCVcc, infection occurred in 10/12 inoculated tree shrews (83 %) and chronic infection was observed in two of these animals. HCVcc from Huh7 cells showed a higher infectivity than that from HeLa cells. The animals inoculated with inadequate amounts of HCV were not infected in either native HCV or HCVcc experiments. Peak viral loads reached 103–105 international units ml−1 in chronically infected animals. ALT level changes reflected the normal fluctuation range in most animals. Thus, tree shrews without immunosuppression can be infected efficiently by native HCV and HCVcc when the animal is inoculated with an adequate amount of single-genotype HCV.

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Reduction of Hepatitis C Virus NS5A Hyperphosphorylation by Selective Inhibition of Cellular Kinases Activates Viral RNA Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.78.23.13306-13314.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13306-13314 ◽

Cited By ~ 102

Author(s):

Petra Neddermann ◽

Manuela Quintavalle ◽

Chiara Di Pietro ◽

Angelica Clementi ◽

Mauro Cerretani ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Kinase Inhibitors ◽

Rna Replication ◽

Hcv Rna ◽

Adaptive Mutations ◽

Direct Demonstration ◽

Huh7 Cells ◽

Efficient Replication

ABSTRACT Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.

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Replicon Cell Culture System as a Valuable Tool in Antiviral Drug Discovery against Hepatitis C Virus

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020501600101 ◽

2005 ◽

Vol 16 (1) ◽

pp. 1-12 ◽

Cited By ~ 28

Author(s):

Nigel Horscroft ◽

Vicky CH Lai ◽

Wayne Cheney ◽

Nanhua Yao ◽

Jim Z Wu ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Drug Discovery ◽

Culture System ◽

Antiviral Drug ◽

Small Animal ◽

Hcv Rna ◽

Cell Culture System ◽

Replicon Cell

Discovery of potential therapeutics against hepatitis C virus (HCV) infection has been hampered in the past decade by the inability to grow this virus in tissue culture and by the lack of robust small animal models. This situation has been improved by the recent development of a selectable HCV replicon cell culture system. For the first time, drug discovery scientists are able to screen large compound collections using the replicon cell culture system to identify small molecules with the potential to inhibit HCV RNA replication. The replicon system has also been used to elucidate inhibitors' antiviral mechanism of action and to optimize antiviral potency. In this review, we will summarize the recent development of HCV replicon cell culture system and its use in anti-HCV drug discovery. The antiviral activities of promising lead compounds are also reviewed.

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Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

Journal of Virology ◽

10.1128/jvi.00526-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 10999-11009 ◽

Cited By ~ 149

Author(s):

Pablo Gastaminza ◽

Kelly A. Dryden ◽

Bryan Boyd ◽

Malcolm R. Wood ◽

Mansun Law ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Structural Characteristics ◽

Buoyant Density ◽

Hcv Rna ◽

Membrane Bilayer ◽

Biophysical Characterization ◽

Negative Stain ◽

A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

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The presence of hepatitis C virus (HCV) antibody in human immunodeficiency virus-positive hemophilic men undergoing HCV "seroreversion"

Blood ◽

10.1182/blood.v82.3.1010.1010 ◽

1993 ◽

Vol 82 (3) ◽

pp. 1010-1015 ◽

Cited By ~ 36

Author(s):

MV Ragni ◽

OK Ndimbie ◽

EO Rice ◽

FA Bontempo ◽

S Nedjar

Keyword(s):

Human Immunodeficiency Virus ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Second Generation ◽

Hcv Rna ◽

Negative State ◽

Immunodeficiency Virus ◽

Before And After ◽

Hcv Antibody ◽

Cd4 Lymphocyte

Abstract Hepatitis C virus (HCV) is a major cause of transfusion-induced chronic liver disease in hemophiliacs, with 70% to 90% being anti-HCV positive. Seroreversion or loss of antibody response to HCV has been observed in a small proportion of human immunodeficiency virus-positive [HIV(+)] anti-HCV(+) hemophilic men. Despite the seroreversion to an anti-HCV- negative state, such patients continue to show serum alanine aminotransferase (ALT) elevations and biopsy evidence of cirrhosis and/or chronic active hepatitis. To determine the cause for the loss of anti-HCV antibody, we compared first- and second-generation anti-HCV enzyme immunosorbent assay (EIA 1.0 and 2.0), second-generation recombinant immunoblot (RIBA 2.0), and HCV-RNA amplification using polymerase chain reaction (PCR) in 19 “seroreverters” before and after seroreversion. There was no difference between 19 seroreverters and 59 persistently anti-HCV-positive hemophiliacs in mean ALT (1.1 +/- 0.1 XUL v 2.0 +/- 0.2 XUL; chi 2 = 1.80, P > .05), in mean CD4 (188 +/- 36/microL v 232 +/- 28/microL; t = 0.965, P > .05), or in the rate of progression to acquired immunodeficiency syndrome (13 of 19 [68.4%] v 30 of 59 [50.9%]; chi 2 = .987, P > .05, respectively). Before seroreversion, all 19 seroreverters (100%) were positive for EIA 1.0 and 2.0 and PCR, and all but 2 of 19 (89.5%) were RIBA 2.0 positive, whereas, after seroreversion, none were positive for EIA 1.0, 15 of 19 (78.9%) were positive for EIA 2.0, 8 of 18 (44.4%) were positive for RIBA 2.0, and 18 of 19 (94.7%) were positive for PCR. There was a lower CD4 lymphocyte number after seroreversion in those who were RIBA 2.0 negative as compared with those who were RIBA 2.0 positive (32 +/- 10/microL v 171 +/- 52/microL; t = 2.638, P > .05). These results indicate that HIV(+) anti-HCV(+) hemophilic men who undergo “HCV seroreversion” are truly infectious and anti-HCV positive by second- generation tests. Anti-HCV detection in immunosuppressed hosts is significantly improved by second-generation EIA and RIBA assays.

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Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01227-07 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2097-2110 ◽

Cited By ~ 22

Author(s):

Pantxika Bellecave ◽

Christian Cazenave ◽

Julie Rumi ◽

Cathy Staedel ◽

Ophélie Cosnefroy ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Selection Procedure ◽

Inhibitory Effect ◽

Hcv Rna ◽

Dna Aptamers ◽

Huh7 Cells

ABSTRACT We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

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Novel Robust Hepatitis C Virus Mouse Efficacy Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00413-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3260-3268 ◽

Cited By ~ 22

Author(s):

Qing Zhu ◽

Yoko Oei ◽

Dirk B. Mendel ◽

Evelyn N. Garrett ◽

Montesa B. Patawaran ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

Small Animal ◽

Rna Replication ◽

Treatment Withdrawal ◽

Whole Body ◽

Hcv Rna ◽

Whole Body Imaging

ABSTRACT The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-α) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-α combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

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Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

Journal of General Virology ◽

10.1099/vir.0.040725-0 ◽

2012 ◽

Vol 93 (7) ◽

pp. 1422-1431 ◽

Cited By ~ 8

Author(s):

Midori Takeda ◽

Masanori Ikeda ◽

Yasuo Ariumi ◽

Takaji Wakita ◽

Nobuyuki Kato

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Hepatoma Cell ◽

Rna Replication ◽

Hcv Rna ◽

Reporter Assay ◽

Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

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Cytoskeletal Requirements for Hepatitis C Virus (HCV) RNA Synthesis in the HCV Replicon Cell Culture System

Journal of Virology ◽

10.1128/jvi.77.7.4401-4408.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4401-4408 ◽

Cited By ~ 52

Author(s):

Anne G. Bost ◽

Daryl Venable ◽

Lifei Liu ◽

Beverly A. Heinz

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Actin Polymerization ◽

Rna Synthesis ◽

Hcv Rna ◽

Cell Culture System ◽

Replicon Cell ◽

Replication Complex ◽

Huh7 Cells ◽

Cytoskeletal Elements

ABSTRACT Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.

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Genetic Interactions between Hepatitis C Virus Replicons

Journal of Virology ◽

10.1128/jvi.78.21.12085-12089.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 12085-12089 ◽

Cited By ~ 28

Author(s):

Matthew J. Evans ◽

Charles M. Rice ◽

Stephen P. Goff

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Genetic Interactions ◽

Hcv Rna ◽

Replication Complex ◽

Huh7 Cells ◽

High Level

ABSTRACT To investigate interactions between hepatitis C virus (HCV) RNA replication complexes, a system was developed to simultaneously select different HCV subgenomic replicons within the same cell. Transcomplementation of defective replicons was not observed, suggesting an isolated and independent nature of the HCV RNA replication complex. In contrast, a high level of competition between replicons was observed, such that the presence and increased fitness of one replicon reduced the capacity of a second one to stably replicate. These results suggest that at least one factor in Huh7 cells required for HCV RNA replication is limiting and saturable.

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Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01426-06 ◽

2007 ◽

Vol 51 (6) ◽

pp. 2016-2027 ◽

Cited By ~ 57

Author(s):

Masahiko Yano ◽

Masanori Ikeda ◽

Ken-ichi Abe ◽

Hiromichi Dansako ◽

Shogo Ohkoshi ◽

...

Keyword(s):

Cell Culture ◽

Vitamin D ◽

Hepatitis C Virus ◽

Linoleic Acid ◽

Hepatitis C ◽

Combined Treatment ◽

Rna Replication ◽

Assay System ◽

Hcv Rna ◽

Β Carotene

ABSTRACT To date, only a limited number of studies have reported finding an influence of ordinary nutrients on hepatitis C virus (HCV) RNA replication. However, the effects of other nutrients on HCV RNA replication remain largely unknown. We recently developed a reporter assay system for genome-length HCV RNA replication in hepatoma-derived HuH-7 cells (OR6). Here, using this OR6 assay system, we comprehensively examined 46 nutrients from four nutrient groups: vitamins, amino acids, fatty acids, and salts. We found that three nutrients—β-carotene, vitamin D2, and linoleic acid—inhibited HCV RNA replication and that their combination caused additive and/or synergistic effects on HCV RNA replication. In addition, combined treatment with each of the three nutrients and interferon alpha or beta or fluvastatin inhibited HCV RNA replication in an additive manner, while combined treatment with cyclosporine synergistically inhibited HCV RNA replication. In contrast, we found that vitamin E enhanced HCV RNA replication and negated the effects of the three anti-HCV nutrients and cyclosporine but not those of interferon or fluvastatin. These results will provide useful information for the treatment of chronic hepatitis C patients who also take anti-HCV nutrients as an adjunctive therapy in combination with interferon. In conclusion, among the ordinary nutrients tested, β-carotene, vitamin D2, and linoleic acid possessed anti-HCV activity in a cell culture system, and these nutrients are therefore considered to be potential candidates for enhancing the effects of interferon therapy.

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