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

Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK¹

Author for correspondence: E. A. Gould Fax +44 1865 281696. e-mail eag{at}ceh.ac.uk

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced 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 mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R₄₉₆H in the envelope (E) gene and C₁₀₈₈₄T in the 3'UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R₄₉₆ in the E protein produced plaques of smaller size than viruses with H₄₉₆ at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T₁₀₈₈₄C substitution within the 3'UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H₄₉₆ and T₁₀₈₈₄, showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H₄₉₆R and T₁₀₈₈₄C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I₄₅F) and the NS5 (T₂₆₈₈A and M₃₃₈₅I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.

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