TABLE 1.
Quantification of genetic heterogeneity in the mutant spectrum of FMDV progeny in the absence or presence of the mutagenic base analog FU or AZCa
| Proteinb | Mutagen | Passage | No. of
|
Mutation frequencye | Shannon entropyf | |
|---|---|---|---|---|---|---|
| Clonesc | Nucleotidesd | |||||
| VP1 | None | 1 | 25 | 16,925 | 4.7 × 10−4 | 0.38 |
| 25 | 5 | 3,385 | 5.9 × 10−4 | 0.42 | ||
| FU | 1 | 25 | 16,925 | 8.3 × 10−4 | 0.65 | |
| 25 | 5 | 3,385 | 2.4 × 10−3 | 1.00 | ||
| AZC | 1 | 5 | 3,385 | 8.9 × 10−4 | 0.83 | |
| 10 | 5 | 3,385 | 2.1 × 10−3 | 0.83 | ||
| 3D | None | 1 | 5 | 7,025 | 2.8 × 10−4 | 0.59 |
| 25 | 5 | 7,025 | 2.8 × 10−4 | 0.59 | ||
| FU | 1 | 5 | 7,025 | 2.8 × 10−4 | 0.59 | |
| 25 | 5 | 7,025 | 1.8 × 10−3 | 1.00 | ||
| AZC | 1 | 5 | 7,025 | 1.4 × 10−4 | 0.31 | |
| 10 | 5 | 7,025 | 8.5 × 10−4 | 1.00 | ||
Populations analyzed and conditions of chemical mutagenesis are described in Materials and Methods and in the legend to Fig. 2. FU passage 25 is the undiluted population shown in Fig. 2. AZC passage 10 is the preextinction population indicated with an arrow in Fig. 2 (right panel). Changes in the consensus nucleotide sequences (relative to the sequence of C-S8c1) were observed in passage 25 with no mutagen (one replacement—C-3656 →A—which leads to T-150 → K in VP1) and in passage 25 with FU (six replacements—A-3328 → G and K-41 → E [VP1], A-3530 → G and H-108 → R [VP1], G-3706 → A and E-167 → K [VP1], A-6734 → G and K-42 → R [3D], T-7275 → C [synonymous], and T-6777 → C and T [mixture, synonymous]). The list of mutations observed in the different populations is given in Table 2, and nonsynonymous replacements are given in Table 3.
VP1 and 3D, products of genomic regions at positions 3193 to 3869 and 6609 to 8035, respectively.
Number of cDNA clones in E. coli (see Materials and Methods) analyzed.
Total number of nucleotides sequenced for each genomic region.
Number of different mutations found divided by the number of nucleotides sequenced (given for each population and genomic region), expressed as substitutions per nucleotide. Mutations were counted by comparing sequence of genomes of a mutant spectrum with the consensus sequence of the corresponding population.
The normalized Shannon entropy is calculated as −Σi[(pi × lnpi)/lnN] in which pi is the frequency of each sequence in the quasispecies and N is the total number of sequences compared (60).