Using high-throughput sequencing for investigating intra-host hepatitis C evolution over long retrospective periods.

Collections of biological samples held by hospitals represent invaluable resources for conducting retrospective evolutionary studies of chronic infections. Using high-throughput sequencing, those collections permit analysis of within-host genetic diversity over long follow-up periods, and allow a better understanding of resistance to treatment regimes during disease evolution. Here, we studied the evolution of hepatitis C virus (HCV) populations in two patients with an absence of response to dual therapies. We implemented amplicon sequencing to survey genomic variation at the Core and NS5B regions of HCV over a period of 13 years from blood samples obtained at multiple time points. We observed mixed infection by multiple HCV genotypes in both patients. Genetic heterogeneity and sample composition analysis provided information about the changes in viral population over the course of clinical treatment, with NS5B experiencing an increase in diversity after treatment initiation. Secondary infections were estimated to predate treatment year, and our results pointed towards diversifying selection occurring post-treatment, acting on standing genomic variation and maintaining high genetic heterogeneity during infection. For these two patients infected with multiple HCV genotypes, the maintenance of viral diversity was explained with the hypothesis of soft selective sweep started at the same time as antiviral treatment was initiated.