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Background and Aims: The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Conventional sequencing analyses for massive amounts of genetic information are limited due to the abundance of HCV quasispecies. 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) α2b 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 early virologic responders and non-responders at baseline (p=0.39). Early virologic responders 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 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 directly acting anti-HCV agents, such as viral protease and polymerase inhibitors, preexist with various abundances in treatment-naïve 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. none provided |