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.

Signature patterns in region V4 of small ruminant lentivirus surface protein in sheep and goats.

The env gene in Small Ruminant Lentiviruses (SRLV) encodes the surface glycoprotein (SU) that divides into conserved (C1-C4) and variable regions (V1-V5). SRLV region V4 has been found to be homologous to the V3 region of human lentivirus (HIV). HIV V3 is responsible for tropism and the development of nervous clinical patterns when there is a tendency to conserve amino acids in specific "signature pattern" positions. The goal of this study was to identify signature patterns in the V4 region of the SU, which is encoded by the SRLV env gene. Secondarily, to understand how these signature patterns are associated with different clinical status in naturally infected sheep and goats. Starting with 244 samples from seropositive animals from nine Mexican states, we amplified the V4 region using nested PCR and obtained 49 SRLV sequences from peripheral blood leukocytes. Based on phylogenetic analysis results, we identified three groups: asymptomatic genotypes A (Ssx GA) and B (Ssx GB), as well as animals with arthritic presentation, genotype B (A GB). Similarity levels between group sequences ranged from 67.9%-86.7%, with a genetic diversity ranging from 12.7%-29.5% and a dN / dS ratio that indicated negative selection. Analyses using Vespa and Entropy programs identified four residues at positions 54, 78, 79 and 82 in SU region V4 as possible signature patterns, although with variable statistical significance. However, position 54 residues "N" (p = 0.017), "T" (p = 0.001) and "G" (p = 0.024) in groups A GB, Ssx GA and Ssx GB respectively, best characterized the signature patterns. The results obtained identified a signature pattern related to different genotypes and clinical status by SRLV in sheep and goats.

Lack of association between amino acid sequences of the bovine leukemia virus envelope and varying stages of infection in dairy cattle.

We collected 724 blood samples from dairy cattle from six Mexican states, and tested them for the presence of antibodies against BLV using a commercial ELISA test. Our study groups consisted of 32 samples: 12 asymptomatic cows, 12 cows with lymphocytosis and 8 samples of tumor tissue of the abomasum and heart of cattle with lymphoma. We designed three pairs of primers to amplify the complete BLV env gene, and obtained a fragment of 1548 nucleotides in length with the sequenced products. According to the phylogenetic tree we constructed to identify the viral genotype, 96.87 % of the sequences grouped into genotype 1, while a single sample from a cow with lymphocytosis (3.13 %) was associated with genotype 3 sequences. The similarity between the Mexican BLV sequences ranged from 0.985-1.00. In addition, the proportion of non-synonymous and synonymous mutations indicated negative selection. We did not identify any conserved residues in the viral protein sequences that could be related to BLV infection stage in cattle. Proviral quantification was performed using quantitative polymerase chain reaction, and we used Mood´s median test as statistical analysis. We found no significant association between proviral load and phase of infection. The sequences showed high similarity without any association between BLV surface glycoprotein and the different infection stages, nor differences in the proviral load. BLV genotype 1 was identified as prevalent in the studied samples, and for the first time in Mexico, we identified BLV genotype 3 in cattle.