13th Conference on Retroviruses and Opportunistic Infections Denver, Colorado - February 5-8, 2006

Conf Retrovir Opportunistic Infect 2006 Feb 5-8;13:abstract no. 15

Takaji Wakita
Tokyo Metropolitan Inst for Neurosci, Japan

BACKGROUND: Hepatitis C virus (HCV), one of the plus-strand RNA viruses, is a primary cause of post-transfusion and sporadic acute hepatitis. An estimated 170 million people worldwide are chronically infected with HCV. Infection leads to chronic liver diseases, including cirrhosis and hepatocellular carcinoma, because most patients fail to clear the virus, and persistent infection follows. Thus, the development of adequate treatment for HCV infection is important. However, this development has been hampered by the lack of a robust viral culture system. It has been reported that subgenomic, dicistronic, and neomycin-selectable HCV RNA replicons are capable of autonomously replicating in Huh7 cells. Although this offers a powerful tool for studying HCV replication and searching for potential antiviral agents, infectious viral particles were not produced from replicon cells even though structural genes were included in replicon constructs. Analysis of HCV replication capacities has revealed that wild type viral genomes display low replication capacities. Adaptive mutations have substantially enhanced the replication of subgenomic and full-length HCV RNA, but introduction of these mutations into the full-length genome reduces viral infectivity in vivo.

METHODS: The JFH-1 strain of HCV was cloned from a patient with fulminant hepatitis. By phylogenetic analysis, the JFH-1strain fit into the cluster of genotype 2a with notable deviations in the 5'UTR, core, NS3 and NS5A regions, and monoclonality of the hyper-variable region sequence. A subgenomic replicon using JFH-1 cDNA replicated in cultured cells with high colony formation efficiency without adaptive mutations. Wild type JFH-1 strain HCV thus appears to possess higher replication capacities than other reported HCV strains. Testing of the JFH-1 strain for full-length viral RNA replication and viral particle formation was thus considered worthwhile. Into Huh7 cells in vitro transcribed full-length JFH-1 RNA was transfected.

RESULTS: Subsequently, viral RNA efficiently replicated in transfected cells and viral particles were secreted into culture medium. Importantly, secreted viral particles were infectious for both cultured cells and a chimpanzee. Furthermore, infectivity for cultured cells was improved by using permissive cell lines.

CONCLUSIONS: This infectious HCV system provides for the first time a powerful tool with which to study the viral life cycle, to construct anti-viral strategies and to develop effective vaccines.

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2006-02-05
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