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J. Virol. doi:10.1128/JVI.01362-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant

Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany; Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185, 9000 Ghent, Belgium

^* To whom correspondence should be addressed. Email: Ralf_Bartenschlager{at}med.uni-heidelberg.de.

	Abstract

Production of infectious hepatitis C virus (HCV) in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titres are rather low limiting the utility of this system. Here we describe the generation of cell culture adapted JFH1 variants yielding higher titres of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed between two independent experiments. Adaptive mutations were observed both in the structural and in the non-structural region with the latter having the highest impact on enhancement of virus titres. The major adaptive mutation was identified in NS5A and enhanced titres of 3 intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows-down processing implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying 6 mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titres in cell culture the most, had reverted to wild type in nearly half of the viral genomes isolated from these animals 15 weeks post inoculation. These results argue for some level of impaired fitness of this mutant in vivo.

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