Figure 2.

Substitution networks emerging in NS3P domain under protease inhibitor treatments. (A–C) ED43 full-length cultures were treated with protease inhibitors paritaprevir (A), grazoprevir (B) and glecaprevir (C) and analysed by NGS. The percentage of HCV-antigen positive cells was determined by immunostaining (line graph, left y-axis). The distribution of haplotypes (bar graphs, right y-axis) was determined by linkage analysis and indicated by different colours. Treatments were initiated with concentrations equivalent to 8x-EC₅₀, then concentrations were increased to 128x-EC₅₀ at day 16 (paritaprevir), 70 (grazoprevir) and 63 (glecaprevir). Only haplotypes constituting ≥2% of the viral population are shown. Supernatant samples from the last treatment time-points were used for passage. P1 and P2: the first and second passages. (D) Efficacy of glecaprevir against indicated ED43 full-length DAA escape viruses. Values are means of triplicates±SEM. (E) EC₅₀ values and 95% CI of indicated escape viruses were calculated from data shown in (D) and online supplemental figure 4. Fold changes in EC₅₀ values were compared with the original ED43. (F) Fitness of ED43 recombinant virus harbouring NS3P RASs. For details, see figure 1D, E legend. The corresponding drug targets and protein-specific numbers are indicated for RASs. Other substitutions are shown with polyprotein specific numbers. ^#The data were obtained from a separate experiment with similar titres of the original virus. NGS, next-generation sequencing; RAS, resistance-associated substitution; SEM, standard error of the mean.