A Database-Derived Global Overview of HCV Resistance-Associated Substitutions: Characterizing Genotypic, Regional, and Temporal Heterogeneity.

Direct-acting antivirals (DAAs) have revolutionized hepatitis C virus (HCV) therapy, yet resistance-associated substitutions (RASs) remain a concern in specific clinical contexts. Here, we present a database-derived global overview of HCV RASs by analyzing 19,449 publicly available sequences across multiple genotypes and subtypes, encompassing both untreated and previously treated infections, in the NS3, NS5A, and NS5B genomic regions. We demonstrate a markedly heterogeneous distribution of RASs shaped by viral genotype, geographic origin, and treatment era. Importantly, RASs against pan-genotypic NS3 protease inhibitors (glecaprevir and voxilaprevir) were rare (generally <1% across genotypes). In contrast, NS5A inhibitors showed greater vulnerability, with the Y93H substitution detected at notable frequencies in major genotypes (3.2-7.0%) and near-universal resistance-associated substitutions (e.g., 100% Q30S) observed in the rare genotype 8. The NS5B nucleotide analogue sofosbuvir retained a high genetic barrier, with the canonical S282T substitution detected only sporadically (2.1% of genotype 4 sequences). At the population level, geographic heterogeneity was evident, with higher RAS frequencies observed in specific regions, alongside pronounced data gaps in high-prevalence areas of Africa. Temporal analyses revealed an increase in NS3 and NS5A RASs following the introduction of first-generation DAAs, with NS5A substitutions persisting into the current interferon-free era, whereas NS5B resistance remained consistently rare across all treatment periods. Together, these findings provide a global, population-level overview of resistance-associated HCV diversity and reinforce the durability of high-barrier regimens while highlighting persistent genotype-specific vulnerabilities with implications for antiviral resistance surveillance and HCV elimination efforts.