A simple selection system for construction of recombinant gD-negative pseudorabies virus as a vaccine vector

We describe a simple, efficient two-step method for construction of glycoprotein D (gD)-negative pseudorabies virus (PrV) carrying transgenes inserted in place of the gD gene. The first step was the use of the thymidine kinase (TK) gene of herpes simplex virus (HSV) for insertional inactivation of the gD gene in a PrV mutant deficient in both TK and glycoprotein E (gE). The gD-negative, HSV-TK-positive mutant could be selected in HAT medium. The second step was substitution of HSV-TK with other genes of interest. The resultant gD/gE/TK-negative mutant was easily isolated by acyclovir selection. The expression of the transgene was detectable in vivo and the antibody responses against both inserted antigens and PrV were induced. The protective efficacy of the gD/gE/TK-negative PrV against lethal PrV challenge was also demonstrated. This PrV mutant carrying immunogenic proteins from unrelated porcine pathogens may be tested as a multivalent vaccine candidate for swine.