Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

• DOI: 10.1128/jvi.74.10.4579-4589.2000
• Publication Date: 2002-07-27T10:06:23Z
• Authors: Minna T. Tuittila; Maria G. Santagati; Matias Röyttä; Jorma A. Määttä; Ari E. Hinkkanen
• Keywords: Neurons; Mice, Inbred BALB C; 0303 health sciences; Base Sequence; Genes, Viral; Alphavirus Infections; Molecular Sequence Data; Brain; Genome, Viral; Viral Nonstructural Proteins; RNA-Dependent RNA Polymerase; Semliki forest virus; Rats; 3. Good health; Mice; Viral Proteins; 03 medical and health sciences; Mutagenesis; Animals; RNA, Viral; Female; Codon; Gene Deletion

ABSTRACTSemliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, 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 unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, thensp3gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to thensp4junction. Replacement in rA774 of the entirensp3gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of thensp3deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.