TABLE 3.
Mutation frequency of RG/B4 and its high-fidelity variants in the presence or absence of 0.5 mM ribavirin after 12 passages in RD cells
| Condition and virus | Mutationa |
Total no. of mutations | No. of mutations per 104 nucleotides | |||||
|---|---|---|---|---|---|---|---|---|
| A→C | A→G | T→C | T→G | C→T | G→A | |||
| Ribavarin treatment | ||||||||
| RG/B4 | 1 | 6 | 3 | 2 | 25 | 17 | 54 | 16.93 |
| RG/B4-G64R | 2 | 1 | 9 | 5 | 17 | 5.33 | ||
| RG/B4-L123F | 1 | 1 | 7 | 5 | 14 | 4.39 | ||
| RG/B4-G64R,L123F | 1 | 6 | 2 | 9 | 2.82b | |||
| Untreated | ||||||||
| RG/B4 | 1 | 4 | 3 | 2 | 1 | 11 | 3.45 | |
| RG/B4-G64R | 3 | 1 | 2 | 6 | 1.88 | |||
| RG/B4-L123F | 2 | 2 | 1 | 1 | 6 | 1.88 | ||
| RG/B4-G64R.L123F | 2 | 2 | 1 | 5 | 1.57 | |||
Eight P1 gene (2586 nucleotides each) and eight RdRp gene (1398 nucleotides each) sequences (total of 31,872 nucleotides) were analyzed for each population. The results indicated that RG/B4 was highly diverse, having on average 3.45 and 16.93 mutations per 104 nucleotides in the absence and presence of ribavirin, respectively, while all high-fidelity variants had fewer mutations per 104 nucleotides in the absence and presence of ribavirin.
The double mutant RG/B4-G64R,L123F had a significantly lower mutation frequency (2.82) than RG/B4-G64R (5.33) and RG/B4-L123F (4.39) per 104 nucleotides in the presence of ribavirin, indicating that the fidelity of EV71 was further elevated by two mutations, G64R and L123F, in its RdRp.