| Japanese encephalitis (JE) is transmitted by mosquitoes and it is the most importantcause of viral encephalitis, with fatality rates averaging30%.The live attenuated Japaneseencephalitis (JE) vaccine SA14-14-2was licensed in1989and now approved for clinical usein most JE endemic countries.The SA14-14-2vaccine was derived from a virulent strainSA14after hundreds of serial passaging in cells and animal, concern about virulencereversion remains exist.In the present study, to study the in vitro and in vivo genetic and attenuation stability ofthe vaccine virus, SA14-14-2was serially passaged in Vero cells and mouse brain followed bysequence comparison and attenuation phenotype analysis. The results showed that nosignificant mutation was acquired after serial passaging in Vero cells except a single Ser66Leumutation within capsid protein, which had no effect on viral virulence in mice. Importantly,serial passaging of SA14-14-2in suckling mouse brain resulted in emergence of adaptivemutations and increased virulence in mice. Population and plaque-purified clone consensussequence analysis showed four adaptive mutations in envelope (E) protein, F107L, K138E,T226R and I270T, sequentially occurred and become predominant during serial passaging insuckling mouse brain. Especially, these adaptive mutations were close related with theenhanced neurovirulence and neuroinvasiveness in mice. Our results provide experimentalevidence of highly genetic and attenuation stability of SA14-14-2following passaging in Verocells, and reveal the potential virulence reversion during passaging in mouse brain inassociation with critical adaptive mutations in E protein. These findings are important forquality control and evaluation of live JE vaccines and will help understand the attenuationmechanism of flavivirus vaccine. |
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