FIG 1.

Isolation of ribavirin-resistant FMDV mutants. (A) Schematic of the FMDV RNA genome. The 3Dpol region is shaded in gray. A 500-bp section of the viral capsid gene was RT-PCR amplified and cloned for sequencing. Individual clones were used to determine the mutation frequencies presented throughout this study. (B) Generation of ribavirin-resistant FMDV mutants. The WT virus was serially passaged in the presence (gray) or absence (black) of 50 μM ribavirin at an MOI of 0.01. p0 is the initial WT stock titer. The mean titers of harvests ± standard deviations are shown (n = 3) (**, P < 0.01 by Student's t test). (C) Resistance of the D5N, A38V, and DAMM mutants to ribavirin. Matched titers of each mutant, the p18 population, or the WT were inoculated into triplicate wells with BHK-21 cells and 50 μM ribavirin. The mean titers ± standard deviations are shown (n = 3) (*, P < 0.05; **, P < 0.01 [determined by Student's t test]). (D and E) None of the mutants were resistant to 5-FU (D) or AZC (E). BHK-21 cells pretreated with 1,200 μM 5-FU or 1,000 μM AZC were infected with each mutant; WT FMDV and the ribavirin-selected p18 population at an MOI of 0.01 were used as controls. Within 72 h after infection, the titer of progeny virus was determined by a TCID₅₀ assay using BHK-21 cells. The mean virus titers ± standard deviations are shown (n = 3) (no significant differences were identified by Student's t test [ns]).