Figure 2. Applying polymerase-based mini-genome assay to identify mutation(s) conferring reduced sensitivity to ribavirin.

(a) Effect of the gene segment conferring to ribavirin resistance were evaluated by substituting wild-type Wuhan95 polymerase (RNP^wild-type), with PB1, PB2, PA, or NP genes from the ribavirin resistant variant (N4) harboring all 10 mutations (RNP^rib-resist). The experiment was performed two times with similar trends; (b) mapping of the PB1 mutation conferring to ribavirin resistance was performed by substituting each of the five PB1 mutations identified from the ribavirin-resistant clones N4 and N2. The ratio of firefly luciferase and renilla luciferase derived for each reaction were normalized to that of the ratio derived at 0 μM ribavirin (retained polymerase activity %). Three independent experiments were performed with one representative result shown. The error bars represents the S.D. of triplicates (N=3). The error bars were shown only for RNP^wild-type, PB1-V43I, PB1-S678N, and RNP^rib-resist where statistical tests (t test) were performed. (c) Ribavirin dose-response curve in MDCK cells using recombinant Wuhan95 viruses each carrying a single-point mutation (V43I, V191I, A661T, S678N) in PB1. Cells were infected at MOI=0.1 and the supernatant were harvested at 48h post-infection for titration. The error bars in represents the S.D. determined for triplicates (N=3) for one representative experiment. The experiment was performed two times. One representative result of two independent experiments is shown. (d) Mini-genome assays for polymerase activity were performed in the presence of increasing concentrations of ribavirin to compare the sensitivities of the wild-type with the PB1-V43I counterparts using H3N2 or H5N1 polymerase complexes. The error bars represent samples prepared in triplicates (N=3). The experiment was performed three times (e) Ribavirin dose-response curve in MDCK cells using the recombinant H3N2 and H5N1 viruses with or without the PB1-V43I mutation. P values are based on Student’s t-test. N.S. denotes not statistically significant. The error bar represents the S.D. determined from samples prepared in triplicates (N=3). The ribavirin dose response curve with Wuhan95 (H3N2) wild-type and PB1-V43I mutant viruses were repeated five times.