Characterization of the bovine leukocyte antigen (BoLA) DRB3 gene has shown that specific alleles associate with susceptibility or resilience to the progression of bovine leukemia virus (BLV), measured by proviral load (PVL). Through surveillance of multi-farm BLV eradication field trials, we observed differential phenotypes within seropositive cows that persist from months to years. We sought to develop a multiplex next-generation sequencing workflow (NGS-SBT) capable of genotyping 384 samples per run to assess the relationship between BLV phenotype and two BoLA genes. We utilized longitudinal results from milk ELISA screening and subsequent blood collections on seropositive cows for PVL determination using a novel BLV proviral load multiplex qPCR assay to phenotype the cows. Repeated diagnostic observations defined two distinct phenotypes in our study population, ELISA-positive cows that do not harbor detectable levels of provirus and those who do have persistent proviral loads. In total, 565 cows from nine Midwest dairy farms were selected for NGS-SBT, with 558 cows: 168 BLV susceptible (ELISA-positive/PVL-positive) and 390 BLV resilient (ELISA-positive/PVL-negative) successfully genotyped. Three BoLA-DRB3 alleles, including one novel allele, were shown to associate with disease resilience, *009:02, *044:01, and *048:02 were found at rates of 97.5%, 86.5%, and 90.3%, respectively, within the phenotypically resilient population. Alternatively, DRB3*015:01 and *027:03, both known to associate with disease progression, were found at rates of 81.1% and 92.3%, respectively, within the susceptible population. This study helps solidify the immunogenetic relationship between BoLA-DRB3 alleles and BLV infection status of these two phenotypic groupings of US dairy cattle.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease in cattle. Previous work estimates that 78% of US beef operations and 38% of US beef cattle are seropositive for BLV. Infection by BLV in a herd is an economic concern for producers as evidence suggests that it causes an increase in cost and a subsequent decrease in profit to producers. Studies investigating BLV in dairy cattle have noted disease resistance or susceptibility, measured by a proviral load (PVL) associated with specific alleles of the bovine leukocyte antigen (BoLA) DRB3 gene. This study aims to investigate the associations between BoLA DRB3 alleles and BLV PVL in beef cattle. Samples were collected from 157 Midwest beef cows. BoLA DRB3 alleles were identified and compared with BLV PVL. One BoLA DRB3 allele, *026:01, was found to be associated with high PVL in relation to the average of the sampled population. In contrast, two alleles, *033:01 and *002:01, were found to be associated with low PVL. This study provides evidence of a relationship between BoLA DRB3 alleles and BLV PVL in US beef cows.