LAUSR

BACKGROUND AIMS The high hepatitis C virus (HCV) infection cure rates achieved with direct-acting antiviral (DAA) treatments could be compromised in the future by the emergence of antiviral resistance. Thus, it is essential to understand the viral determinants that influence DAA resistance, which is most prevalent in genotype 3. We aimed at studying how resistance to protease-, NS5A-, and NS5B-inhibitors influences the activity of glecaprevir/pibrentasvir, sofosbuvir/velpatasvir and sofosbuvir/velpatasvir/voxilaprevir in cell culture, and how the HCV genome adapts to selective pressure by successive rounds of treatment failure. APPROACH RESULTS A previously developed in vivo infectious cDNA clone of strain S52 (genotype 3a) was adapted to efficiently replicate and propagate in human hepatoma cells (Huh7.5) using 31 adaptive substitutions. DAA escape experiments resulted in the selection of S52 variants with decreased drug susceptibility (resistance), which was linked to the emergence of known resistance-associated substitutions (RAS). NS5A-inhibitor resistance was sufficient to promote treatment failure with double-DAA but not triple-DAA regimens. Enhanced viral fitness associated with the selection of sofosbuvir resistance accelerated escape from DAA. After serial DAA treatment failure, HCV genetic evolution led to a complex genome-wide network of substitutions, some of which co-evolved with known RAS. CONCLUSIONS Baseline NS5A-RAS can compromise the efficacy of double-DAA pangenotypic regimens for HCV genotype 3 and enhanced viral fitness can accelerate treatment failure. Persistence of RAS after successive treatment failure is facilitated by the remarkable evolutionary capacity and plasticity of the HCV genome. Proof-of-concept for the potential development of multi-DAA resistance is shown.