Mutagenesis of a bovine herpesvirus type 1 genome cloned as an infectious bacterial artificial chromosome: analysis of glycoprotein E and G double deletion mutants.

The genome of bovine herpesvirus type 1 Schönböken was cloned as a bacterial artificial chromosome (BAC) by inserting mini F plasmid sequences into the glycoprotein (g) E gene. The resulting BAC clone, pBHV-1DeltagE, was transfected into bovine kidney cells and viable gE-negative BHV-1 (BHV-1DeltagE) was recovered. By RecE/T mutagenesis in Escherichia coli, the gG open reading frame was deleted from pBHV-1DeltagE. From the mutated BAC, double negative BHV-1DeltagE-gG was reconstituted and its growth properties were compared to those of rescuant viruses in which the gE gene was restored (BHV-1rev, BHV-1DeltagG). The mutant viruses did not exhibit markedly lowered virus titres. Plaque sizes of BHV-1DeltagE, BHV-1DeltagE-gG and BHV-1DeltagG, however, were reduced by 19 to 55 % compared to parental strain Schönböken or BHV-1rev. Our results suggested that gE and gG function independently from each other in cell-to-cell spread, because an additive effect on plaque formation was observed in the gE/gG double deletion mutant.