Genetic Heterogeneity of Hepatitis C Virus in Association with Antiviral Therapy Determined by Ultra-Deep Sequencing

Background and Aims: The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail. Methods: Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) alpha 2b plus ribavirin therapy. Results: Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naive patients, suggesting the risk of the development of drug resistance against these agents. Conclusion: Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.