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JVI Accepts, published online ahead of print on 14 February 2007
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J. Virol. doi:10.1128/JVI.02334-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Production of Infectious Hepatitis C Virus of Various Genotypes in Cell Culture

Takanobu Kato, Takuya Matsumura, Theo Heller, Satoru Saito, Ronda K. Sapp, Krishna Murthy, Takaji Wakita, and T. Jake Liang*

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney, Diseases, National Institutes of Health, Bethesda, MD, 20892; Southwest Foundation for Biomedical Research, San Antonio, TX, 78227, USA.; Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan

* To whom correspondence should be addressed. Email: JakeL{at}bdg10.niddk.nih.gov.


  Abstract

A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes including 1a (H77), 1b (CG1b) and 2a (J6 and JFH-1) in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and effects of culture conditions. After transfection into Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells were inoculated to naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had identical sequences as the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.




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