Recombinants are isolated at high frequency following in vivo mixed ocular infection with two avirulent herpes simplex virus type 1 strains.

Mixed infections with different strains of herpes simplex virus type 1 (HSV-1) may result in more severe disease than infection with either strain alone. This phenomenon is important because it may facilitate the identification of virulence genes through the transfer of virulence determinants between complementing strains, and it may pose a problem in the use of attenuated HSV strains for vaccines and gene delivery vectors. In this study, we have compared the percentage of recombinants present after mixed infection with HSV-1 strains OD4 and 994 in vitro and in vivo. After corneal inoculation, we found that 74% of randomly picked isolates from the trigeminal ganglia were recombinants, compared with 59% from the cornea. Twenty-six percent of randomly picked isolates were recombinant following mixed infection of Vero cells in vitro. Seventeen recombinant strains isolated from the in vivo mixed infections were assayed for ocular virulence, and they were found to exhibit a wide range of virulence phenotypes. The presence of virulent recombinants suggests that recombination plays a role in the increased disease observed in this mixed infection, and the broad range of virulence indicates that there may be multiple genetic factors involved in the increased virulence observed after mixed infection with these two strains. The recombinants were also tested for their ability to grow in NIH 3T3 fibroblasts, and though some correlation was observed between growth in vitro and ability to cause ocular disease, improved growth in murine cells does not sufficiently explain the increased virulence observed in some recombinants.