Cell culture and infection system for hepatitis C virus (original) (raw)

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

Subscribe to this journal

Receive 12 print issues and online access

$259.00 per year

only $21.58 per issue

Buy this article

USD 39.95

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

Additional access options:

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
  2. Tomoko Date
  3. Asako Murayama
  4. Kenichi Morikawa
  5. Daisuke Akazawa
  6. Takaji Wakita

Corresponding author

Correspondence toTakaji Wakita.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights 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

This article is cited by