Hepatitis C virus (HCV) is an important human pathogen that previously could be studied only in the chimpanzee model because the virus could not be grown in cultured cells. Growth of the virus in cultured hepatoma cells was recently achieved by Wakita et al. but even now growth is strain-specific and inefficient and infectious titers rarely reach even 100,000 viruses per ml of culture medium. Prior to discovery of this culture system, a retrovirus pseudotyped particle system was the only means to study neutralization of HCV and to perform mutational analysis of the two glycoproteins of HCV. Cell entry by enveloped viruses is mediated by a heterodimer formed of E1 and E2 glycoproteins. Since HCV infections commonly progress to chronicity, it is believed that HCV evades the immune system by existing as a complex quasispecies. The hypervariable 1 region (HVR1) in the E2 glycoprotein of HCV is a target of neutralizing antibodies and is implicated in entry into cells. In order to study viral neutralization and infectivity in vitro, it would be most useful to begin with a quasispecies that mimics an in vivo situation. For that reason, in FY2009 we showed that we could reliably reproduce in complexity and distribution, the glycoprotein quasispecies circulating in a patient. This was accomplished by shotgun cloning E1E2 pairs amplified by RT-PCR as a cassette and cloned into a modified cDNA clone of HCV that could replicate in cultured cells. In FY2010 we used this replicating quasispecies to compare parameters leading to dominance of one species in cell culture in the absence of immune pressure as well as in the presence of neutralizing antibody raised against a peptide specific for the originally dominant species.Mutants engineered by swapping different regions between the two species which dominated at various times in culture, indicated that the HVR1 region does not function as an independent domain either in cell entry or in neutralization. We also participated in microarray studies comparing hepatitis C and E infections In experimentally infected chimpanzees.
