Differential protein abundance in colostrum-derived extracellular vesicles from bovine leukemia virus-infected cattle are implicated in immune regulation and inflammation.

INTRODUCTION: Bovine leukemia virus (BLV) is an oncogenic retrovirus that naturally infects cattle. Once infected, the virus persists indefinitely throughout life at a transcriptionally silent stage. Soon after infection, humoral and cytotoxic activities efficiently abolish the viral replicative cycle, allowing only mitotic expansion of provirus-carrying cells. These anti-viral activities persist throughout the animal's life indicating that the immune system is permanently stimulated by BLV antigens. Although protein production is abolished during the chronic stage, viral transcription is not completely shut down. Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released from most cells and play multiple roles in cell-to-cell communication, including immune modulation, angiogenesis, and transformation of cells by transferring genetic material and functional proteins. Colostrum is rich in EVs that carry bioactive proteins and RNAs reflecting maternal physiology and immune status. In this study, we aimed to characterize how BLV infection influences the protein cargo of colostrum-derived EVs. METHODS: Colostral EVs were isolated using a protocol that combines acid pre-treatment with ultracentrifugation on sucrose gradients. RESULTS: Transmission electron microscopy and nanoparticle tracking analysis revealed a heterogeneous population of vesicles ranging from 40 to 120 nm in diameter, exhibiting the characteristic morphology of EVs. Flow cytometry analysis confirmed the presence of the EV marker CD9. In addition, the presence of CD63, CD81, and TSG101 was also detected by Western blot. Quantitative proteomic analysis using a mass spectrometer coupled to liquid chromatography (LC/MS) revealed a total of 13 bovine differentially abundant proteins (FDR < 0.05 and FC ≥ |1.5|) from a total of 1,082 proteins identified. Among them, 9 (69.2%) showed decreased abundance (e,g., ANXA1, CLU, DMBT1, LRRC32), whereas 4 (30.8%) showed an increased abundance (e.g., IDO1, MARCKS, LCP1) in the group of BLV-infected animals. Enrichment analysis showed that differentially abundant proteins were primarily associated with immune-related processes, including complement activation and immunoglobulin-mediated immunity, as well as cellular metabolic processes, cytoskeletal organization, intracellular signaling and vesicle trafficking. DISCUSSION: Overall, the results observed in colostrum-derived EVs from BLV-infected cows provide compelling evidence that BLV infection induced systemic molecular alterations that may modulate inflammation and the immune response in calves.