Combinations of long terminal repeat and tax protein substitutions influence bovine leukemia virus transmission through altered viral productivity

Bovine leukemia virus (BLV) infection imposes a substantial economic burden on the global cattle industry. Although proviral load (PVL) in blood influences transmission, the effects of viral genetic variations, including the high viral production-associated long terminal repeat (LTR) substitution at nucleotide position 175 (LTR175), on herd-level dynamics remain unclear. This study investigated the effects of specific substitutions on the regulation of viral productivity and transmission. Longitudinal monitoring of a single farm (herd size: 50–68 cows; 438 samples collected) over a 7-year period revealed that viral strains carrying cytosine at LTR175 (LTR175C) significantly increased in frequency, becoming dominant over wild-type strains carrying thymidine. In contrast, amino acid substitutions in the Tax protein at positions 69 (Tax69) and 73 (Tax73) modified viral transactivation independent of LTR175. Structural modeling via AlphaFold2 predicted that Tax69 and Tax73 mutations alter protein properties, suggesting modulation of transactivation activity. Consistent with this prediction, in vitro assays confirmed that the substitutions modified viral production by regulating transactivation independent of LTR175. Moreover, high viral-productivity haplotypes containing LTR175C and Tax73Q (glutamine at amino acid residue 73) exhibited a transmission advantage within the herd, even when host PVL levels were not significantly elevated. These findings suggest that cellular-level viral productivity is one of the key determinants of transmission fitness. Overall, this study revealed a regulatory interaction between the LTR and Tax protein that optimizes viral spread and highlights the need for control strategies that account for viral genetic substitutions. IMPORTANCE Controlling the spread of bovine leukemia virus (BLV) remains a major challenge for the livestock industry. This study identifies viral genetic mechanisms that enable certain strains to spread more efficiently than others. By integrating in silico analyses, in vitro experiments, and a 7-year field study on a working farm, we showed that three mutations in viral regulatory regions, i.e., in the long terminal repeat and Tax protein, strongly influence viral success. These substitutions modify viral protein properties and promoter activity, increasing production of new virus particles. Our findings suggested that high-viral-productivity genetic profiles enhance transmission between cattle, allowing specific strains to become dominant within herds over time. This research highlights the importance of monitoring key viral genetic markers to identify high-risk strains and improve strategies to limit the spread of BLV.