Selection of hepatitis C virus resistant to ribavirin

BACKGROUND: Given the side effects associated with intravenous injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable. Recently published clinical studies show that interferon-free combination therapies containing ribavirin are efficacious, suggesting that an interferon-free therapy could be adopted in the near future. Therefore, understanding HCV resistance to ribavirin could be of major importance. In an approach to understand the effect of ribavirin on HCV replication and HCV resistance, we have selected a ribavirin resistant mutant of HCV in vitro. METHODS: We serially passed the J6/JFH1 strain of HCV in Huh7D cells (a Huh7 cell derivative more permissive to HCV replication) in the presence of different concentrations of ribavirin. Virus replication was assessed by detection of HCV antigens by immunfluorscence of infected cells and titration of recovered virus present in the supernatant. cDNAs from virus RNA grown in 0 or 250 uM concentrations of ribavirin were synthesized by RT-PCR, and sequenced. RESULTS: A concentration of 125 uM of ribavirin did not have a dramatic effect on HCV replication, while 500 uM of ribavirin lead to viral extinction. Concentrations of 250 uM of ribavirin dramatically reduced virus replication which was sustained over six passages. At passage seven viral resurgence began and over two passages the level of virus reached that of the wild type virus grown without ribavirin. Virus recovered from these cultures were more resistant to 250 uM ribavirin than wild type virus, and showed no difference in replication relative to wild type virus when grown in the absence of ribavirin. The ribavirin resistant virus accumulated multiple synonymous and non-synonymous mutations that are presently being analyzed for their relationship to ribavirin resistance. CONCLUSIONS: It is possible to select a ribavirin resistant mutant of HCV that can replicate to levels similar to wild type virus grown without ribavirin. Analysis of the mutations responsible for the ribavirin resistance may aid in understanding the mechanism of action of ribavirin.