Neli Montero Machuca
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Jorge Luis Tórtora Pérez
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Ana Silvia González Méndez
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Lucia Angelica García-Camacho
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Ernesto Marín Flamand
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Abstract
The pX genetic region of the 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 determine the pathogenic role associated with BLV genotype 1 pX region genetics in terms of lymphocytosis, lymphomas and proviral load. We screened 724 serological samples from mixed-age Holstein Friesian cattle from six states in Mexico. Once peripheral blood leukocytes were isolated from whole blood with anticoagulant, we extracted genomic DNA using a commercial kit. Then, we designed in silico primers that hybridize in conserved regions of the BLV pX region, which allowed for PCR standardization to detect proviral DNA in infected cells. Positive amplicons were sequenced using the Sanger method, obtaining 1156 nucleotide-long final sequences that included the four pX region genes. The 30 heads of cattle that formed the genetic study population included 12 with lymphocytosis, 12 without lymphocytosis and six with lymphoma. Lymphoma presence was determined in six bovine tumor tissues using histopathology, and we identified BLV presence with in situ hybridization.Phylogenetic analysis determined that the 30 sequences were associated with genotype 1, and genetic distance between the sequences ranged from 0.2% - 2.09%. We identified two sequences in the G4 gene, one with a three-nucleotide deletion (AGU_7488L, in a cow with lymphocytosis), and one with a nine nucleotide deletion (AGU_18A, in a cow without lymphocytosis). PX region analysis identified positive selection in the G4, rex and R3 genes, and we found no difference in proviral load between the studied groups. It was not possible to establish an association between pX region variability and the development of lymphocytosis, lymphoma, asymptomatic status and proviral load in BLV-infected cattle.
Genetic Analysis of the pX Region in Bovine Leukemia Virus genotype 1 in Holstein Friesian Cattle with Different Stages of Infection