Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Cell culture and infection system for hepatitis C virus

Nature Protocols volume 1, pages 2334–2339 (2006)Cite this article

Abstract

Hepatitis C virus (HCV) infection causes chronic liver disease and is a worldwide health problem. Despite ever-increasing demand for knowledge on viral replication and pathogenesis, detailed analysis has been hampered by a lack of efficient viral culture systems. We isolated HCV genotype 2a strain JFH-1 from a patient with fulminant hepatitis. This strain replicates efficiently in Huh7 cells. Efficient replication and secretion of recombinant viral particles can be obtained in cell culture by transfection of in vitro–transcribed full-length JFH-1 RNA into Huh7 cells. JFH-1 virus generated in cell culture is infectious for both naive Huh7 cells and chimpanzees. The efficiency of viral production and infectivity of generated virus is substantially improved with permissive cell lines. This protocol describes how to use this system, which provides a powerful tool for studying viral life cycle and for the construction of antiviral strategies and the development of effective vaccines. Viral particles can be obtained in 12 days with this protocol.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Infected foci of cell culture–generated HCV-infected cells.
Figure 2
Figure 3: Immunofluorescence microscopy of HCV proteins in Huh7 cells transfected with JFH-1 RNA.
Figure 4

Similar content being viewed by others

References

  1. Kato, T. et al. Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J. Med. Virol. 64, 334–339 (2001).

    Article  CAS  Google Scholar 

  2. Kato, T. et al. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125, 1808–1817 (2003).

    Article  CAS  Google Scholar 

  3. Wakita, T. et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11, 791–796 (2005).

    Article  CAS  Google Scholar 

  4. Zhong, J. et al. Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. USA 102, 9294–9299 (2005).

    Article  CAS  Google Scholar 

  5. Date, T. et al. Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells. J. Biol. Chem. 279, 22371–22376 (2004).

    Article  CAS  Google Scholar 

  6. Kato, T. et al. Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon. J. Virol. 79, 592–596 (2005).

    Article  CAS  Google Scholar 

  7. Bukh, J. et al. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl. Acad. Sci. USA 99, 14416–14421 (2002).

    Article  CAS  Google Scholar 

  8. Lindenbach, B.D. et al. Complete replication of hepatitis C virus in cell culture. Science 309, 623–626 (2005).

    Article  CAS  Google Scholar 

  9. Blight, K.J., McKeating, J.A. & Rice, C.M. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J. Virol. 76, 13001–13014 (2002).

    Article  CAS  Google Scholar 

  10. World Health Organization. Laboratory biosafety manual. 3rd edn. (World Health Organization, Geneva, Switzerland, 2004).

  11. van den Hoff, M.J.B., Moorman, A.F.M. & Lamers, W.H. Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res. 20, 2902 (1992).

    Article  CAS  Google Scholar 

  12. Takeuchi, T. et al. Real-time detection system for quantification of hepatitis C virus genome. Gastroenterology 116, 636–642 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Partially supported by a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science, from the Ministry of Health, Labour and Welfare of Japan and from the Ministry of Education, Culture, Sports, Science and Technology, and by the Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation.

Author information

Authors and Affiliations

  1. Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Takanobu Kato

  2. Department of Virology II, National Institute of Infectious Diseases, Shinjuku, 162-8640, Tokyo, Japan

    Tomoko Date, Asako Murayama, Kenichi Morikawa, Daisuke Akazawa & Takaji Wakita

  3. Pharmaceutical Research Lab, Toray Industries, Kanagawa, 248-8555, Japan

    Daisuke Akazawa

Authors
  1. Takanobu Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomoko Date
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Asako Murayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kenichi Morikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daisuke Akazawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Takaji Wakita
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takaji Wakita.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kato, T., Date, T., Murayama, A. et al. Cell culture and infection system for hepatitis C virus. Nat Protoc 1, 2334–2339 (2006). https://doi.org/10.1038/nprot.2006.395

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.395

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing