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Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Nature Medicine volume 11, pages 791–796 (2005)Cite this article

A Corrigendum to this article was published on 01 August 2005

Abstract

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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Figure 1: Transient replication of JFH1 RNA in transfected Huh7 cells.
Figure 2: Time course of HCV RNA replication and core protein expression in transfected cells.
Figure 3: Density gradient and electron microscope analysis of recombinant HCV particles.
Figure 4: Infectivity of viral particles and neutralization of infection.
Figure 5: In vivo infectivity of JFH1 virus produced in tissue culture.

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Acknowledgements

This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, by a Grant from Toray Industries, Inc., by the Program for Promotion of Fundamental Studies in Health Sciences of the Pharmaceuticals and Medical Devices Agency (PMDA), by the Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation, by the National Heart, Lung and Blood Institute contract N01-HB-27091 for the use of chimpanzees, by a grant from the European Community (QLK2-CT-2002-01329), and by a grant from the Deutsche Forschungsgemeinschaft (SFB 638; Teilprojekt A5). T.K. was partially supported by Hepatitis Virus Research Foundation of Japan. The authors are grateful to J. Bukh for providing the pJ6CF plasmid, to S. Foung for provision of the antibody CBH5, to S. Koike for his comments, to K. Yasui, S. Sone and J.-I. Tanabe for their support, to G. Koutsoudakis and E. Steinmann, and to S. Kallis and U. Herian for technical assistance. We are also grateful to K. Takagi, K. Hiramatsu and members of Central Clinical Laboratory in Nagoya City University Hospital, R. Sapp of the Liver Diseases Branch, H. McClure of the Southwest Foundation for Biomedical Research for their technical assistance, H. Barth, B. Bartosch, T. Baumert, J. Encke and C. Sarrazin for providing human sera.

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Author notes

  1. Takaji Wakita, Thomas Pietschmann and Ralf Bartenschlager: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, 183-8526, Japan

    Takaji Wakita, Takanobu Kato, Tomoko Date, Michiko Miyamoto & Zijiang Zhao

  2. Department of Molecular Virology, University Heidelberg, Im Neuenheimer Feld 345, Heidelberg, 69120, Germany

    Thomas Pietschmann & Ralf Bartenschlager

  3. Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, 467-8601, Japan

    Takanobu Kato & Masashi Mizokami

  4. Liver Disease Branch, NIDDK, National Institute of Health, Bethesda, 20892, Maryland, USA

    Takanobu Kato & T Jake Liang

  5. Southwest Foundation for Biomedical Research, San Antonio, 78227, Texas, USA

    Krishna Murthy

  6. Department of Virology, University of Heidelberg, Im Neuenheimer Feld 324, Heidelberg, 69120, Germany

    Anja Habermann & Hans-Georg Kräusslich

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  1. Takaji Wakita
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Corresponding author

Correspondence to Takaji Wakita.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Efficient replication of JFH1 HCV RNA in transiently transfected Huh7 cells as determined by western blot analysis for HCV proteins E2 and E1. (PDF 158 kb)

Supplementary Fig. 2

HCV core protein secreted from passaged transfected cells. (PDF 129 kb)

Supplementary Fig. 3

Density gradient analysis of culture supernatants of transfected Huh7 cells. (PDF 254 kb)

Supplementary Fig. 4

Virus particle release depends on envelope glycoproteins. (PDF 262 kb)

Supplementary Fig. 5

CD81-dependent infection of Huh7 cells with cell culture–derived HCV. (PDF 333 kb)

Supplementary Fig. 6

Production of infectious HCV particles carrying the firefly luciferase reporter gene. (PDF 207 kb)

Supplementary Methods (PDF 57 kb)

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Wakita, T., Pietschmann, T., Kato, T. et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11, 791–796 (2005). https://doi.org/10.1038/nm1268

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