Genetic analysis of the pX region of bovine leukemia virus genotype 1 in Holstein Friesian cattle with different stages of infection.

The pX genetic region of bovine leukemia virus (BLV) includes four genes with overlapping reading frames that code for the Tax, Rex, R3, and G4 proteins. These proteins are involved in the regulation of transcriptional and post-transcriptional viral expression, as well as having oncogenic potential. Our goal was to investigate the pathogenicity of the pX region of BLV genotype 1 in terms of lymphocytosis, lymphomas, and proviral DNA load. We screened 724 serological samples from mixed-age Holstein Friesian cattle from six states in Mexico. Peripheral blood leukocytes (PBLs) were isolated from whole blood with anticoagulant, and genomic DNA was extracted from the PBLs using a commercial kit. Then, a set of primers that hybridize in conserved regions of the BLV pX region were used, which allowed for PCR standardization to detect proviral DNA in infected cells. Positive amplicons were sequenced using the Sanger method, resulting in 1156-nucleotide-long final sequences that included the four pX region genes. The experimental group consisted of 30 animals. Twelve of these had lymphocytosis, six had lymphoma, and 12 were apparently healthy cattle without any signs of lymphocytosis or lymphoma. The presence of lymphoma was detected in six bovine tumor tissues using histopathology, and the presence of BLV was detected by in situ hybridization. Phylogenetic analysis demonstrated that the 30 sequences were associated with genotype 1, and the genetic distance between the sequences ranged from 0.2% to 2.09%. We identified two sequences in the G4 gene: one with a three-nucleotide deletion resulting in the loss of a leucine (AGU_7488L, in a cow with lymphocytosis), and one with a nine-nucleotide deletion resulting in the loss of leucine, proline, and leucine (AGU_18A, in a cow without lymphocytosis). Analysis of the PX region indicated that positive selection had occurred in the G4, rex, and R3 genes, and we found no difference in proviral DNA load between the studied groups. We were unable to establish an association between variations in the pX region and the development of lymphocytosis, lymphoma, asymptomatic status, or proviral DNA load in BLV-infected cattle.