Genome-Wide Identification and KASP Validation of Two Host Genetic Loci Associated with Bovine Leukemia Virus Proviral Load

The proviral load (PVL) of bovine leukemia virus (BLV) is associated with viral transmission and disease progression to BLV-induced enzootic bovine leukosis and is influenced by genetic polymorphisms of the bovine leukocyte antigen (BoLA)-DRB3 gene. However, its host genetic determinants remain partially defined because additional host events are necessary for BLV infection outcomes. We performed a genome-wide association study of 113 BLV-positive Holstein cattle to identify novel single-nucleotide polymorphisms (SNPs). We first compared 33 high-PVL and 37 low-PVL cattle using 582,324 quality-controlled SNPs. Two SNPs, rs110742604 and rs137368875, exceeding the Bonferroni-corrected significance threshold, were identified in the BoLA class I region of chromosome 23. Kompetitive allele-specific PCR (KASP) assays were developed for both SNPs, and three genotype calls (TT, TC, and CC) were concordant with Sanger sequencing in all samples. Further, genotyping was performed for rs110742604 and rs137368875 in 364 and 362 cattle, respectively. At both loci, the C allele was associated with a lower PVL, with the lowest PVL observed in CC animals. In a combined genotype of three resistance markers, BoLA-DRB3 resistance-associated alleles, rs110742604-CC and rs137368875-CC, stepwise enrichment in cattle with low PVL was dependent on the increasing number of candidate resistance markers; further, 11 of 12 cattle carrying all three markers showed a tendency toward low PVL, and none had high PVL. These findings identify candidate host biomarkers for BLV PVL and establish rapid assays for large-scale validation. Replication across breeds and functional characterization are required before incorporation into genomic selection or integrated control programs for BLV.