Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients.

The preeminent mode of action of the broad-spectrum antiviral nucleoside ribavirin in the therapy of chronic hepatitis C is currently unresolved. Particularly under contest are possible mutagenic effects of ribavirin that may lead to viral extinction by lethal mutagenesis of the hepatitis C virus (HCV) genome. We applied ultradeep sequencing to determine ribavirin-induced sequence changes in the HCV coding region (nucleotides [nt] 330 to 9351) of patients treated with 6-week ribavirin monotherapy (n = 6) in comparison to placebo (n = 6). Baseline HCV RNA levels maximally declined on average by -0.8 or -0.1 log10 IU/ml in ribavirin- versus placebo-treated patients. No general increase in rates of nucleotide substitutions in ribavirin-treated patients was observed. However, more HCV genome positions with high G-to-A and C-to-U transition rates were detected between baseline and treatment week 6 in ribavirin-treated patients in comparison to placebo-treated patients (rate of 0.0041 transitions per base pair versus rate of 0.0022 transitions per base pair; P = 0.049). Similarly, the sensitive detection of low-frequency minority variants by statistical filtering indicated significantly more positions with G-to-A and C-to-U transitions in ribavirin-treated patients than in placebo-treated patients (rate of 0.0331 transitions versus rate of 0.0186 transitions per G/C-containing position at baseline; P = 0.018). In contrast, non-ribavirin-associated A-to-G and U-to-C transitions were not enriched in the ribavirin group (P = 0.152). We conclude that ribavirin exerts a mutagenic effect on the virus in patients with chronic hepatitis C by facilitating G-to-A and C-to-U nucleotide transitions.

Analysis of long-term persistence of resistance mutations within the hepatitis C virus NS3 protease after treatment with telaprevir or boceprevir.

BACKGROUND: Telaprevir and boceprevir are highly selective hepatitis C virus (HCV) NS3/4A proteaseinhibitors in phase 3 development. Viral breakthrough during mono- and triple-therapies with PEG-interferon and ribavirin and relapse is associated with resistance. OBJECTIVES: Potential persistence of resistance mutations during long-term follow-up should be analyzed. STUDY DESIGN: Clonal sequence analysis of the NS3-protease gene was performed at long-term follow-up in HCV genotyp-1 infected patients who received telaprevir or boceprevir within phase-1b studies for comparison with resistant variants present directly after the end-of-treatment. RESULTS: After a median follow-up of 4.2 years in 28 of 82 patients HCV-RNA was still detectable. Resistance variants were detected in two of 14 telaprevir- and in four of 14 boceprevir-treated patients. For telaprevir patients two low-level (V36M, V36A) and one high-level (A156T) mutation associated with resistance were detected at low frequencies (4-9% of the clones). In five boceprevir-treated patients four low level mutations (V36A, T54A/S, V55A) were observed at low frequencies (1-10%) while in one patient additionally a combined variant (T54S+R155K) was detected at 94%. Presence of resistant variants at long-term follow-up was not predictable by variants detected at the end-of-treatment. In one patient a V55A variant which was dominant already at baseline was still detectable at long-term follow-up. CONCLUSIONS: In the majority of patients after short-term treatment with telaprevir or boceprevir wild-type NS3-protease isolates are detectable by clonal sequencing at long-term follow-up. Detectable resistance mutations in single patients are not predictable by initial frequencies of variants.