Virology, Genetics and Molecular Biology Laboratory, Faculty of Higher Education, Cuautitlan, Veterinary Medicine, Campus 4, FES-Cuautitlán, National Autonomous University of Mexico, Km. 2.5 Carretera Cuautitlán-Teoloyucan San Sebastián Xhala, 54714, Cuautitlan Izcalli, Estado de México, México.
Department of Biological Sciences, Faculty of Higher Education, Cuautitlan, National Autonomous University of Mexico, Cuautitlán Izcalli, México.
Arch Virol. 2022 Jan;167(1):45-56. doi: 10.1007/s00705-021-05252-2. Epub 2021 Oct 14.
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.
牛白血病病毒（BLV）的 pX 基因区域包括四个重叠阅读框的基因，它们编码 Tax、Rex、R3 和 G4 蛋白。这些蛋白参与病毒转录和转录后表达的调控，同时具有致癌潜能。我们的目标是研究 BLV 基因型 1 的 pX 区域在淋巴细胞增多症、淋巴瘤和前病毒 DNA 载量方面的致病性。我们从墨西哥六个州的混合年龄荷斯坦弗里森奶牛中筛选了 724 份血清学样本。用抗凝剂从全血中分离外周血白细胞（PBL），并使用商业试剂盒从 PBL 中提取基因组 DNA。然后，使用一组在 BLV pX 区域保守区域杂交的引物，允许 PCR 标准化以检测感染细胞中的前病毒 DNA。使用 Sanger 法对阳性扩增子进行测序，得到包括四个 pX 区域基因的 1156 个核苷酸长的最终序列。实验组由 30 只动物组成。其中 12 只动物患有淋巴细胞增多症，6 只动物患有淋巴瘤，12 只动物显然没有淋巴细胞增多症或淋巴瘤的迹象。使用组织病理学在 6 个牛肿瘤组织中检测到淋巴瘤的存在，并用原位杂交检测 BLV 的存在。系统发育分析表明，30 个序列与基因型 1 相关，序列之间的遗传距离在 0.2%至 2.09%之间。我们在 G4 基因中发现了两个序列：一个序列缺失三个核苷酸导致亮氨酸丢失（AGU_7488L，在患有淋巴细胞增多症的牛中），另一个序列缺失九个核苷酸导致亮氨酸、脯氨酸和亮氨酸丢失（AGU_18A，在没有淋巴细胞增多症的牛中）。对 PX 区域的分析表明，G4、rex 和 R3 基因发生了正选择，我们没有发现研究组之间前病毒 DNA 载量的差异。我们无法确定 pX 区域的变异与 BLV 感染牛的淋巴细胞增多症、淋巴瘤、无症状状态或前病毒 DNA 载量的发展之间存在关联。
