Multiple determinants contribute to the virulence of HSV ocular and CNS infection and identification of serine 34 of the US1 gene as an ocular disease determinant

PURPOSE. The virulence of any given strain of herpes simplex virus (HSV) is probably due to the effects of the constellation of genes in that strain and how they act in concert to promote disease. The goal of this work was to develop a system to identify and study the role of multiple genes in HSV disease. METHODS. Mixed ocular infection with HSV-1 strains CJ394 and OD4 yield recombinants with increased ocular and central nervous system (CNS) virulence. Clones and subclones of the CJ394 genome were cotransfected with intact OD4 DNA into Vero cells, the transfection pools were inoculated into BALB/c mouse eyes, and disease severity was scored. Fragments transferring increased ocular or CNS disease were sequenced. Site-directed mutagenesis was used to revert one mutation to wild type. RESULTS. Five of the determinants (UL9, -33, -41, and -42 and US1) increased ocular disease when transferred singly. Transfer of the UL36/37 determinant increased both ocular and CNS disease. Transfer of the UL41 and -42 genes increased mortality and a combination of the UL36/37, -41, and -42 determinants increased virulence further. Reversion of the S34A change in the OD4 US1 gene to wild type restored ocular virulence. CONCLUSIONS. Multiple HSV genes can operate to increase virulence. The UL9, -33, -36/37, and -42 genes have not previously been identified as virulence determinants. The UL41 and US1 genes are known to affect disease, but the changes identified had not been described. Multiple novel mutations were found in the OD4, UL9, UL36, and US1 genes, and we showed that S34 in the US1 gene is essential in ocular disease.