Wakita, T, Pietschmann, T, Kato, T, Date, T, Miyamoto, M, Zhao, Z et al.. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11: 791-796

Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan.
Nature Medicine (Impact Factor: 27.36). 08/2005; 11(7):791-6. DOI: 10.1038/nm1268
Source: PubMed


Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.

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Available from: Thomas Pietschmann, Dec 20, 2013
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    • "Boc: Boceprevir. entry has been firmly established in HuH-7 cells (Evans et al., 2007), an experimental system that has been instrumental in the exploration of the viral infectious cycle (Wakita et al., 2005). In HuH-7 cells exposed to sorafenib, we found that CLDN1 subcellular localization and expression levels were altered. "
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    ABSTRACT: Hepatitis C virus (HCV) chronic infection is a major cause of hepatocellular carcinoma. Sorafenib is the only medical treatment that has been approved for the treatment of this cancer. It is a multikinase inhibitor with anti-tumor activity against a wide variety of cancers. Sorafenib blocks angiogenesis and tumor cell proliferation through inhibition of kinases, such as VEGFR2, PDGFR, or the serine/threonine kinases RAF. Previous studies have reported an anti-HCV effect of sorafenib in vitro, but various mechanisms of action have been described. The aim of this study was to clarify the action of sorafenib on the complete HCV infectious cycle. In order to examine the action of sorafenib on all steps of the HCV infectious cycle, we used a combination of validated cell culture models, based on the HuH-7 reference cell line and primary human hepatocytes. We found that sorafenib blocks HCV infection by altering the viral entry step and the production of viral particles. Moreover, we observed that treatment with sorafenib lead to a modification of Claudin-1 expression and localization, which could partly be responsible for the anti-HCV effect. Collectively, our findings confirm the anti-HCV effect of sorafenib in vitro, while highlighting the complexity of the action of sorafenib on the HCV infectious cycle. Copyright © 2015. Published by Elsevier B.V.
    Antiviral research 03/2015; 24. DOI:10.1016/j.antiviral.2015.03.012 · 3.94 Impact Factor
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    • "The initial studies to define the virology of HCV took time to develop because HCV is very difficult to grow in cell culture. For example, it took 10 years after the discovery of HCV to be able to study replicating HCV RNA in a cell culture system [10], and a fully infectious clone of HCV to be used in cell culture was only developed within the last 10 years [11] [12]. Current systems for studying HCV have expanded from studying the virus in Huh7 human hepatoma cell lines to using primary human hepatocytes, mice with a chimeric human liver, or mice engineered with various human factors that promote HCV infection [13] [14]. "
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    ABSTRACT: Experimental studies on the interactions of the positive strand RNA virus hepatitis C virus (HCV) with the host have contributed to several discoveries in the field of antiviral innate immunity. These include revealing the antiviral sensing pathways that lead to the induction of type I interferon (IFN) during HCV infection and also the importance of type III IFNs in the antiviral immune response to HCV. These studies on HCV/host interactions have contributed to our overall understanding of viral sensing and viral evasion of the antiviral intracellular innate immune response. In this review, I will highlight how these studies of HCV/host interactions have led to new insights into antiviral innate immunity. Overall, I hope to emphasize that studying antiviral immunity in the context of virus infection is necessary to fully understand antiviral immunity and how it controls the outcome of viral infection. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Cytokine 03/2015; 74(2). DOI:10.1016/j.cyto.2015.03.007 · 2.66 Impact Factor
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    • "Genotype 1b HCV subgenomic replicon cell lines, R6FLR-N (R6, genotype 1b, strain N) (Watanabe et al., 2006), FLR3-1 (genotype 1b, Con-1) (Sakamoto et al., 2005) and Rep JFH Luc3-13 genotype 2a (Takano et al., 2011), strain JFH-1 (Wakita et al., 2005) (Supplementary Fig. 1) were cultured at 37 °C (5% CO 2 ) in Dulbecco's modified Eagle medium-GlutaMAX-I (DMEM-GlutaMAX-I; Invitrogen, Carlsbad, CA, USA) containing 10% foetal bovine serum and 0.5 mg/mL G418 (Invitrogen, Carlsbad, CA, USA) (Sakamoto et al., 2005; Watanabe et al., 2006). The JFH-1/K4 cell line, which comprises HuH-7 cells persistently infected with the HCV JFH-1 strain, was maintained in DMEM with 10% FCS (Takano et al., 2011). "
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    ABSTRACT: Chronic hepatitis C virus (HCV) infection increases the risk of liver cirrhosis and hepatocellular carcinoma. In the last decade, the current standard HCV treatment, pegylated interferon and ribavirin, have limited efficacy and significant side effects. Novel direct acting antivirals show promise, but escape mutants are expected, along with potential side effects. Pycnogenol®, a French maritime pine extract, has been reported to have antioxidant and antiviral effects. Here, we evaluated the effect of Pycnogenol® on HCV replication. Wild-type and protease inhibitor (VX-950; telaprevir)-resistant HCV replicon cells were treated with Pycnogenol®, Pycnogenol® and interferon-alpha, and ribavirin and telaprevir. Pycnogenol® effects on replication were also evaluated in HCV-infected chimeric mice. Pycnogenol® treatment showed antiviral effects without cytotoxicity at doses up to 50 μg/mL. Pycnogenol® in combination with interferon-alpha or ribavirin showed synergistic effects. Moreover, Pycnogenol® inhibited HCV replication in telaprevir-resistant replicon cells; telaprevir and Pycnogenol® acted additively to reduce HCV RNA levels in wild-type HCV replicon cells without significantly increasing cytotoxicity. Pycnogenol® antiviral activity was higher than its components procyanidin and taxifolin. Further, treatment of infected chimeric mice with Pycnogenol® suppressed HCV replication and showed a synergistic effect with interferon-alpha. In addition, Pycnogenol® treatment resulted in dose-dependent reduction of reactive oxygen species in HCV replicon cell lines. Pycnogenol® is a natural product that may be used to improve the efficacy of the current standard antiviral agents and even to eliminate resistant HCV mutants.
    Antiviral Research 01/2015; 113:93-102. DOI:10.1016/j.antiviral.2014.10.017 · 3.94 Impact Factor
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