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. 2017 May 1;9(5):1212–1228. doi: 10.1093/gbe/evx075

Fig. 1.—

Fig. 1.—

Passage history, acquisition of mutations in the polymerase (3D)-coding region, and fitness of FMDV-3D(V173I) relative to FMDV-wt in the presence of different FU concentrations. (A) FMDV-RP (its origin is described in Materials and Methods) was subjected to 15 passages in the absence or presence of 5-fluorouracil (FU) (50 µg/ml or 200 µg/ml added to culture medium) to yield populations RP-15, RP(FU50)-15 and RP(FU200)-15, respectively. (B) Amino acid substitutions in 3D of populations depicted in A, deduced from the corresponding consensus sequence of the 3Dcoding region; +, presence of mutation; −, absence of mutation; +/ −, presence of mutation in ∼50% of the RNA population, according to the corresponding nucleotide peak in the sequence. Numbering of genomic and amino acid residues is according to Escarmís et al. (1999). (C) To determine the relative fitness of FMDV-3D(V173I) and FMDV-wt, BHK-21 cells were infected with a mixture of FMDV-3D(V173I) and FMDV-wt; the RNA ratio of the two viruses ranged between 1:1 and 0.1:1, at a total initial MOI of 0.1 PFU/cell. Passages were performed in the presence of the concentration of FU (µg/ml) indicated below each bar. RNA of the two competing viruses was measured in triplicate. Fitness values were calculated as described in Materials and Methods; the fitness determination plots are given in supplementary figure S2, Supplementary Material online. The error bars indicate the error of the fit of each individual fitness value. Statistical significances are computed through the t-test, to evaluate if the slope of the ratio of RNA of the two competing viruses versus the passage number (nine points) is different from zero; significances are represented as (*P < 0.05; **P < 0.01). The statistical significance of the difference between pairs of fitness values is indicated on the lines linking two bars (*P < 0.05; **P < 0.005; Welch’s two-tailed test); P values above 0.05 were obtained in the comparison of fitness values obtained with 400 µM FU versus 200 µM FU, and 200 µM FU versus 100 µM FU. Procedures are further detailed in Materials and Methods.