Functional relevance of naturally occurring single nucleotide polymorphisms in bovine leukemia virus-encoded microRNAs.

Background: Bovine leukemia virus (BLV) microRNAs (miRNAs) contribute to viral latency and immune evasion. Naturally occurring single-nucleotide polymorphisms (SNPs) within the BLV miRNA cluster have been associated with persistent lymphocytosis in cattle, yet their functional impact remains unclear. Aims: This study aims to (1) evaluate how SNPs in BLV miRNAs affect miRNA biogenesis and mature miRNA levels; (2) determine whether specific SNPs alter miRNA–mRNA interactions and identify affected targets; and (3) characterize transcriptomic changes induced by reference versus SNP-bearing miRNAs. Methods: BLV miRNA loci were PCR-amplified and sequenced from 53 blood samples of infected cattle. Both reference and SNP-containing precursors were cloned into expression vectors and co-transfected with an miRNA-deficient BLV clone into HEK293T cells. Mature miRNA levels were quantified via stem-loop RT-qPCR. Computational target prediction (miRanda) validated changes in mRNA targeting. Gene-expression effects were assessed using Agilent microarrays, with selected findings confirmed by RT-qPCR and subjected to pathway enrichment analysis (IPA). Significance: This work will elucidate how BLV miRNA polymorphisms modify miRNA maturation and target recognition, reshape host gene networks, and contribute to viral persistence and immune modulation. Fifty‐three blood samples were collected from cattle naturally infected with BLV. BLV miRNA coding regions were PCR‐amplified and sequenced to identify isolates carrying either the reference miRNA sequence or specific SNP variants. Selected miRNA precursors (both reference and SNP‐containing) were cloned into expression vectors and co‐transfected with an miRNA‐deficient BLV clone into HEK293T cells in three independent biological replicates. Mature miRNA levels were quantified by RT‐qPCR. For transcriptomic profiling, total RNA from the three independent transfections for selected miRNA variants (reference, V29, V90) and from two control groups (empty vector ± BLV) were pooled by group and analyzed using Agilent two‐color microarrays in six technical replicates. Differentially expressed genes were validated by RT‐qPCR and then subjected to pathway enrichment analysis (IPA).