Tissue homogenates were obtained from swine co-infected with 2 vaccine strains of pseudorabies virus (PRV). Viral isolates derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures, were characterized as recombinant and parental PRV genotypes on the basis of thymidine kinase and glycoprotein X gene combinations. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant PRV. The thymidine kinase and glycoprotein X gene sequences were classified as wildtype or deleted, using a battery of polymerase chain reaction assays. Results substantiate the observation that PRV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection.

Pseudorabies virus (PRV), an alpha-herpesvirus, causes substantial economic losses in the swine industry and is currently the focus of eradication and control programs. Some of these programs rely on the ability of veterinarians to differentiate animals exposed to virulent strains of PRV from animals exposed to avirulent vaccine strains of PRV on the basis of a serologic response to nonessential glycoproteins that are deleted in some vaccine strains of PRV. Genetic recombination resulting in the creation of virulent strains of PRV with the same negative immunologic markers as vaccine strains could disrupt these programs. Two strains of PRV were coinoculated either into tissue culture or into sheep to facilitate recombination. Progeny viruses were selected to detect a specific recombinant phenotype. We were able to detect genetic recombination between vaccine strains of PRV following in vitro or in vivo coinoculation of 2 strains of PRV. The selected recombinants had marker-deleted phenotypes in strains with restored virulence genes. Increased virulence was observed in sheep after coinoculation of 2 avirulent vaccine strains of PRV.

We report here genetic recombination between 2USDA-licensed vaccine strains of pseudorabies virus co-inoculated into swine. The vaccine strains, one of which was a conventionally attenuated strain and the other, a genetically engineered deleted strain containing a negative immunologic marker, had complementary genomes. Coinoculation resulted in the creation of novel strains of pseudorabies virus containing negative immunologic markers with restored virulence genes. Plaque-purified recombinant progeny viruses were found in 2 litters of pigs in which both strains were co-inoculated IM, a litter in which both strains were co-inoculated oronasally, and a litter in which the conventionally attenuated strain was inoculated oronasally and the genetically engineered strain was inoculated IM. Recombinant phenotypes and recombinant restriction fragment patterns were observed. The creation, spread, and potential misdiagnosis of these types of recombinant strains could disrupt control and eradication programs that are based on the serologic identification of swine infected with potentially virulent strains of pseudorabies virus.