Clinical Stage-Specific Alteration of Hsa-miR-424-5p in Milk Small Extracellular Vesicles from Bovine Leukemia Virus Infection to Progression of Enzootic Bovine Leukosis

Bovine leukemia virus (BLV) is an oncogenic retrovirus responsible for enzootic bovine leukosis (EBL) in cattle. Previous studies indicated that increased levels of the miRNA hsa-miR-424-5p in milk small extracellular vesicles (sEVs) may serve as an EBL biomarker in cattle; however, its alteration across the clinical stages of BLV infection to the onset of EBL remains unclear. This study evaluated the prognostic potential of hsa-miR-424-5p in milk sEVs across distinct clinical stages of BLV infection to EBL onset. qPCR analysis showed that the abundance of hsa-miR-424-5p was lower in milk sEVs from BLV-infected cattle with low and high proviral load (LPVL and HPVL) compared to BLV-uninfected healthy cattle (Control), but was not significant (p > 0.05). In contrast, the abundance of hsa-miR-424-5p in milk sEVs was significantly higher in EBL cattle than in the Control and HPVL cattle (p < 0.05). Spearman’s correlation analysis revealed a significant inverse association between hsa-miR-424-5p and the percentage of lactate dehydrogenase 2 + 3 (r = −0.28, p < 0.001), whereas no significant correlations were observed with other parameters. hsa-miR-424-5p showed the highest discriminative performance in distinguishing BLV-infected cattle from EBL cattle (area under the curve [AUC] = 0.71), followed by Control vs. BLV (AUC = 0.63), indicating statistically meaningful biomarker utility across disease stages. These findings suggest that the amount of hsa-miR-424-5p in milk sEVs increases during the progression from the late stage of BLV infection to EBL onset, demonstrating that hsa-miR-424-5p may serve as a part of the biomarker panel for monitoring BLV progression and predicting EBL onset.