Hepatitis C virus: propagation, quantification, and storage

Hepatitis C virus (HCV) is a leading cause of chronic liver diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is well known for its restricted tropism and it does not replicate well in animal species other than humans and chimpanzees. Since classical in vitro propagation of natural HCV isolates is not possible, a protocol for the rescue of infectious virus from cDNA clones (genotype 1a pH77S and genotype 2a pJFH-1) transfected as RNA into permissive cells is described here. Because these two molecular clones behave differently in their ability to propagate and produce infectious virus, different methods for propagation of these two viral strains are described. Methods for infectious virus titration, which can be accomplished by counting foci of infected cells following immunostaining for viral antigen expression in cells infected with serial dilutions of a virus harvest (focus forming unit, or FFU, assay), are also provided.