Multiple defects in the genome of pseudorabies virus can affect virulence without detectably affecting replication in cell culture

Several independently isolated vaccine strains of pseudorabies virus were studied to identify the functions that play a role in the expression of virulence of this virus. All the strains that were studied grew well in three different cell types. No differences that could be correlated with avirulence could be detected either in the virus yield produced by the cells or in the length of the eclipse phases. All the attenuated strains, however, had lost their ability to replicate efficiently in the brains of day-old chickens. The defects leading to the decrease in the virulence for day-old chickens varied in the different vaccine strains. The Tatarov vaccine strain is defective in the thymidine kinase (TK) gene; restoration of a functional TK gene restores to this strain its virulence for day-old chickens and for pigs. Three out of four different, independently isolated avirulent strains were found to be defective in different loci, as determined by their ability to generate virulent recombinants. Two strains, Bartha and Buk Z300, however, yielded few virulent recombinants, indicating that they were defective in at least one closely linked function. Furthermore, all the virulent recombinants obtained from cells coinfected with different pairwise combinations of the vaccine strains had higher LD 50 values than virulent wild-type virus, indicating that the recombinants had not acquired all the functions necessary for optimum expression of virulence. Partial virulence was also restored to Buk Z900 by marker rescue with sequences originating from three different regions of the wild-type pseudorabies virus genome. All three of these regions were different from those that had previously been shown to recue virulence of the Bartha strain (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1987, J. Virol. 61, 796–801) . Our results thus show that (1) defects in several different loci of the pseudorabies virus genome can affect virulence without detectably affecting growth in cell culture and (2) most vaccine strains have multiple defects contributing to their lack of virulence.