Detection and genetic characterization of circulating bovine leukemia virus in dairy cattle from Punjab, Pakistan

Bovine leukemia virus (BLV), the causative agent of enzootic bovine leukosis (EBL), is a globally distributed deltaretrovirus that causes significant economic losses in the dairy sector. In Pakistan, BLV sero-prevalence is increasing; however, molecular data on circulating strains remain limited. These studies focused on characterize the env gene fragment encoding the envelope glycoprotein gp51 in BLV-positive cattle from Punjab and to evaluate the evolutionary relationships and amino acid substitutions of local BLV isolates. This study aimed to investigate the genetic diversity of BLV in naturally infected cattle from Punjab Province by analyzing the env gene segment encoding the envelope glycoprotein gp51. A total of 1080 blood samples were randomly collected from nine divisions and screened using ELISA, identifying 367 seropositive animals. Molecular confirmation was conducted using nested PCR targeting both 444 bp and 903 bp regions of the env-gp51 gene. Three PCR-positive samples from each amplicon size were subjected for Sanger sequencing. Phylogenetic analysis was performed using sequences from both regions, while nucleotide and amino acid variation analyses were based on the 903 bp fragments. Comparative analysis included 23 reference sequences representing the global diversity of BLV. Punjab isolates showed 98.65-99.66 % nucleotide similarity with worldwide distributed strains. Phylogenetic analysis revealed that all local isolates grouped within genotype G1, demonstrating limited genetic divergence. Amino acid analysis revealed five substitutions localized within known gp51 epitopes; however, the zinc-binding motif (Cys-X₂-Cys-X₄-His-X₄-Cys) remained fully conserved, indicating strong purifying selection. The results indicate that BLV strains prevalent in Punjab are genetically conserved and closely related to worldwide BLV strains. The high degree of conservation observed in the gp51 gene supports its long-term reliability as a molecular target for diagnostic applications and immunological interventions.