The degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Va silchenko (Vs) was constructed previously. Virus recovered from pGGVs produ ced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3' untranslated region (3'UTR). Two m utations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the co nserved regions of the genome, R-496 --> H in the envelope (E) gene and C-1 0884 --> T in the 3'UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was eva luated. The engineered viruses with R-496 in the E protein produced plaques of smaller size than viruses with H-496 at this position. This mutation al so affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T-10884 --> C substitution within the 3'UTR was noticeabl e only in cytotoxicity and neuroinvasiveness tests. However, all virus muta nts engineered by modification of the infectious clone, including one with two wild-type mutations, H-496 and T-10884, showed reduced neuroinvasivenes s in comparison with the Vs parental virus. Therefore, although the H-496 - -> R and T-10884 -->C substitutions clearly reduce virus virulence, the oth er mutations within the variable regions of the capsid (I-45 --> F) and the NS5 (T-2688 --> A and M-3385 --> I) genes also contribute to the process o f attenuation. In terms of developing flavivirus vaccines, the impact of ac cumulating apparently minor mutations should be assessed in detail.