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. 2005 Jun 28;102(26):9294-9.
doi: 10.1073/pnas.0503596102. Epub 2005 Jun 6.

Robust hepatitis C virus infection in vitro

Jin Zhong  1 , Pablo Gastaminza, Guofeng Cheng, Sharookh Kapadia, Takanobu Kato, Dennis R Burton, Stefan F Wieland, Susan L Uprichard, Takaji Wakita, Francis V Chisari
Affiliations

Robust hepatitis C virus infection in vitro

Jin Zhong et al. Proc Natl Acad Sci U S A. .

Abstract

The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.

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Figures

Fig. 1.
Fig. 1.
Production of infectious HCV after transfection of genomic JFH-1 RNA. Ten micrograms of in vitro transcribed JFH-1 RNA was electroporated into 4 × 106 Huh-7.5.1 cells. Transfected cells and supernatant were harvested at the indicated time points posttransfection. Intracellular HCV RNA was analyzed by RT-QPCR and displayed as genome equivalents (GE)/μg total RNA (line). Supernatant infectivity titers were determined in naïve Huh-7.5.1 cells and are expressed as ffu/ml (bars).
Fig. 2.
Fig. 2.
Detection of infected cells by NS5A immunofluorescence. (Upper) Immunofluorescent detection of NS5A in transfected cells: (A) day 5 and (B) day 24 posttransfection. (Lower) Infectivity titration of transfected cell supernatant on naïve Huh-7.5.1 cells; (C) undiluted supernatant; (D) 10-fold diluted supernatant. NS5A staining in red; nuclei stained with Hoechst (blue) (×50).
Fig. 3.
Fig. 3.
HCV infection kinetics and passage in tissue culture cells. Naïve Huh 7.5.1 cells were inoculated with culture supernatants at an moi of 0.01. Supernatants from the inoculated cells were collected at the indicated times p.i. and evaluated for infectivity (ffu/ml). Data represent the average of two or more experiments with error bars. (A) Huh-7.5.1 cells inoculated with supernatant harvested at day 19 after transfection of Huh-7.5.1 cells with JFH-1 genomic RNA by electroporation (dashed line) or day 24 after lipofection (solid line). (B) Huh-7.5.1 cells inoculated with supernatant collected at day 5 from the infection shown as a solid line in A; (C) Increasing NS5A immunostaining in Huh-7.5.1 cells between days 5 and 7 p.i. in the experiment shown in B.
Fig. 4.
Fig. 4.
Inhibition of HCV infection by anti-E2 and anti-CD81 antibodies. (A) JFH-1 virus was preincubated with the indicated concentrations of anti-E2 antibody or irrelevant human IgG1 antibody for 1 h at 37°C before inoculating Huh-7.5.1 cells. Total cellular RNA was analyzed by RT-QPCR at day 3 p.i. (B) Huh-7.5.1 cells were preincubated with the indicated concentrations of anti-human CD81 or control mouse IgG1 antibody for 1 h at 37°C before inoculation with JFH-1 virus at an moi of 0.3. Total cellular RNA was analyzed by RT-QPCR at day 3 p.i.
Fig. 5.
Fig. 5.
Sucrose gradient sedimentation of infectious HCV. Supernatant from infected Huh-7.5.1 cells was fractionated as described in Materials and Methods. Fractions (1-9) were collected from the top of the gradient and analyzed by RT-QPCR for HCV RNA (line). The infectivity of each fraction was determined (bars) by titration. Fraction densities are expressed as g/ml.
Fig. 6.
Fig. 6.
Kinetics of JFH-1 HCV infection in Huh-7.5.1 and Huh-7 cells. A virus stock generated in Huh-7.5.1 was diluted to infect Huh-7.5.1 and Huh-7 cells at an moi of 0.01. Culture supernatant was collected at the indicated times and titrated. Infectious titers in Huh-7.5.1 (solid lines) and Huh-7 cells (dashed lines) are expressed as ffu/ml. Average values of two independent infection experiments are shown.
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