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Abstract Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T-lymphotropic virus. Bovine major histocompatibility complex (BoLAs) are used extensively as markers of disease and immunological traits in cattle. For BLV diagnosis, proviral load is a major diagnosis index for the determination of disease progression and transmission risk. Therefore, we investigated the frequency of BoLA-DRB3 alleles, BoLA-DQA1 alleles, and haplotypes of BoLA class II isolated from the heads of 910 BLV-infected cows out of 1290 cows assessed from BLV-positive farms, in a nationwide survey from 2011 to 2014 in Japan. Our aim was to identify BoLA class II polymorphisms associated with the BLV proviral load in the Holstein cow. The study examined 569 cows with a high proviral load and 341 cows with a low proviral load. Using the highest odds ratio (OR) as a comparison index, we confirmed that BoLA-DRB3 was the best marker for determining which cow spread the BLV (OR 13.9 for BoLA-DRB3, OR 11.5 for BoLA-DQA1, and OR 6.2 for BoLA class II haplotype). In addition, DRB3*002:01, *009:02, *012:01, *014:01, and *015:01 were determined as BLV provirus associated alleles. BoLA-DRB3*002:01, *009:02, and *014:01 were determined as resistant alleles (OR > 1), and BoLA-DRB3*012:01 and *015:01 were determined as susceptible alleles (OR |