Lineage-aware evolutionary analysis of hepatitis C virus within-host dynamics

Analysis of viral genetic data has previously revealed distinct within-host population structures in both untreated and interferon-treated chronic hepatitis C virus (HCV) infections. While multiple subpopulations persisted during the infection, each subpopulation was observed only intermittently. However, it was unknown whether similar patterns were also present after Direct Acting Antiviral (DAA) treatment, where viral populations were often assumed to go through narrow bottlenecks. Here we tested for the maintenance of population structure after DAA treatment failure. We analysed whole-genome next-generation sequencing data generated from a randomised study using DAAs (the BOSON study). We focused on samples collected from patients (N=84) who did not achieve sustained virological response (i.e. treatment failure) and had sequenced virus from multiple timepoints. For each individual, we tracked concordance in nucleotide variant frequencies through time. Using a sliding window approach, we applied sequenced-based and tree-based clustering algorithms across the entire HCV genome. Finally, we reconstructed viral haplotypes and estimated lineage specific within-host divergence rates from the haplotype phylogenies. Distinct viral subpopulations were maintained among a high proportion of individuals post DAA treatment failure. Using maximum likelihood modelling and model comparison, we found an overdispersion of viral evolutionary rates among individuals, and significant differences in evolutionary rates between lineages within individuals. These results suggest the virus is compartmentalised within individuals, with the varying evolutionary rates due to different viral replication rates or different selection pressures. We propose lineage awareness in future analyses of HCV evolution and infections to avoid conflating patterns from distinct lineages, and to recognise the likely existence of unsampled subpopulations.