Genome-Wide Characterization of a Recombinant BoAHV-1 ΔgE/β-gal Vaccine Strain Reveals an Unintended Multi-Gene Deletion

Bovine alphaherpesvirus 1 (BoAHV-1) is a major cattle pathogen, and glycoprotein E (gE)-negative marker vaccines are widely used for disease control and to differentiate infected from vaccinated animals. The genomic architecture of this previously developed recombinant strain beyond the intended gE/lacZ locus had not been characterized at the genome-wide level. Here, we performed whole-genome sequencing of the parental Los Angeles (LA) strain and two passages of the recombinant BoAHV-1ΔgEβgal strain. In addition, plaque area was descriptively assessed in primary fetal bovine kidney cells using measurements from a single experimental run. In addition to the expected replacement of the US8/gE locus with a β-galactosidase cassette, we identified a previously undescribed 3704 nt deletion in the US region. This deletion removed the complete US1.67, US2, and US3 coding sequences, as well as the 5' portion of US4/gG. PCR assays and Sanger sequencing of the amplicon spanning the breakpoint precisely mapped the junction. Comparative annotation showed that the parental genome contained 74 viral CDSs, whereas both vaccine passage genomes contained 69. In the plaque assay, the mean plaque area of p11 was 68.8% lower than that of the parental LA strain. These findings document an unintended structural alteration in a previously evaluated recombinant vaccine strain and illustrate how genome-wide sequencing can complement targeted molecular characterization by identifying changes outside the intended engineered locus.