Japanese encephalitis (JE) virus strain P3 was highly neurovirulent and neuroinvasive in weanling mice whereas two other JE virus strains, SA14/USA and S892, were only neurovirulent. Infectivity titrations of brains and sera showed that P3 virus multiplied faster and reached a higher infectivity titre than S892 virus following inoculation of viruses by the intraperitoneal (i.p.) route. The p.f.u./LD was 10 and 10 for P3 and S892 viruses respectively, following i.p. inoculation, while JE virus strain SA14/USA did not kill mice when inoculated by this route (i.e. ⩾ 10 p.f.u./LD). Nevertheless, the genomic similarity between P3 virus and strains SA14/USA and S892 was more than 97.8% at the nucleotide level and 99% at the amino acid level. Compared with S892 and SA14/USA viruses, P3 virus had 33 and 21 amino acid differences, respectively. The structural protein genes of P3 virus were more divergent than non-structural protein genes. Nine unique amino acids were found in the envelope protein gene. None of these amino acid differences were shared with other wild-type JE virus strains. Although we cannot identify the precise molecular determinants of virulence of JE virus, there were no unique amino acids in M, NS1, NS2A, NS3, NS4A and NS4B proteins of P3 virus compared with other wild-type viruses. Therefore, it appears that these proteins make no significant contribution to the high neuroinvasiveness of P3 virus. The structural proteins, and non-structural proteins NS2B and NS5 may be involved in increasing neurovirulence and neuroinvasiveness of P3 virus. P3 virus differed by several nucleotides in the 3′ non-coding region while no nucleotide difference was found in the 5′ non-coding region.


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