Replicase complex genes of Semliki Forest virus confer lethal neurovirulence

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rode nts, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is un able to form virions and hence does not reach a critical threshold of neuro nal damage. To elucidate the molecular mechanisms of neurovirulence, we hav e cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and co mpared it to the highly neurovirulent SFV4 virus, We found several sequence differences and sought to localize determinants conferring the neuropathog enicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural regi on by showing that rA774 structural genes combined with the SFV4 nonstructu ral genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonst ructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replace ment in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the v irulent phenotype, whereas an arginine at the opal position significantly i ncreased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the op al codon and amino acid mutations other than the deleted residues are mainl y responsible for the attenuation of A7(74) and that the attenuating determ inants reside entirely in the nonstructural region.