Randomized trial shows ribavirin increases mutational load and specific transition signatures in HCV.

Background: Ribavirin is a guanosine analogue with clinical antiviral activity against a range of RNA viruses including hepatitis C virus (HCV), respiratory syncytial virus and Lassa virus. Several potential mechanisms of action have been proposed, but there is limited data supporting them clinically. Methods: We studied 196 HCV-infected participants from a trial of short-course directly antiviral therapy (STOPHCV-1) which included a factorial randomisation to ribavirin versus no ribavirin. Deep sequencing of the HCV genome was performed on samples with detectable viremia from three time-points: baseline (n = 191), day 3 of treatment (n = 25) and post-treatment failure (n = 47). Results: Ribavirin exposure significantly increased total mutational load at treatment failure (P = 0.0065) and enriched classical ribavirin-associated transitions, including G->A (P = 0.026) and C[->]U (P = 0.004), along with other key changes including A->G (P = 0.005), U->C (P = 0.023), C->G (P = 0.010), and U->A (P = 0.026). The resulting mutational signature was broad, not dominated by G-related changes. Region-specific analyses demonstrated this increase was broadly distributed across the viral genome, without strong evidence for protection of specific regions. Non-synonymous to synonymous mutation ratios (dN/dS) rose at day 3 (P = 5.5e-5) before declining at failure (P = 8.5e-7), with trends toward higher dN/dS in the ribavirin group at day 3 (P = 0.06). Conclusions: Ribavirin acts as a potent in vivo mutagen, driving viral populations toward genome-wide diversity rather than selecting a few highly fit drug-resistant clones. These findings support an error-catastrophe model.