Attenuation of bovine herpesvirus type 1 by deletion of its glycoprotein G and tk genes and protection against virulent viral challenge.

To develop a novel vaccine against infectious bovine rhinotracheitis (IBR), a bovine herpesvirus-1 (BoHV-1) mutant was constructed by deleting the genes for glycoprotein G (gG) and thymidine kinase (tk) through homologous recombination. The resulting sequences for both genes were shown to be correct and a gG expression defect was also confirmed. A parallel study of the BoHV-1 gG(-)/tk(-), gE(-)/tk(-) mutants and wild type (wt) in 31 calves was performed at three different doses, 4×10(5)PFU, 4×10(6)PFU and 4×10(7)PFU. Compared to wt BoHV-1, inoculation of BoHV-1 gG(-)/tk(-) and gE(-)/tk(-) produced no clinical signs and the virus was not reactivated by dexamethasone (dex). Inoculation of BoHV-1 gG(-)/tk(-) at the doses of 4×10(6) and 4×10(7)PFU provided full clinical protection for the cattle against wt BoHV-1 challenge at 4×10(7)PFU/calf. Although the mutants were associated with significantly lower levels of serum neutralizing antibody, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) than wt BoHV-1 on days 3, 5 and 7 after immunization, stimulation of IFN-β by BoHV-1 gG(-)/tk(-) was significantly higher than that of wt BoHV-1 and gE(-)/tk(-) on days 3 and 5. We conclude that BoHV-1 gG(-)/tk(-) was attenuated adequately and that it maintains the ability to stimulate immune protection. Therefore, it may be a promising candidate for a marker vaccine against IBR.