Strategy of PCR-RFLP genotyping of BLV and its correspondence to phylogenetic classification

The current classification of Bovine leukemia virus includes 12 genotypes that can be identified by phylogenetic analysis of sequenced nucleotide sequences of the BLV env gene locus, as well as by updated PCR-RFLP genotyping strategies. The purpose of this study was to systematize knowledge about the genetic diversity of Bovine leukemia virus with typing of BLV isolates using the previously developed PCR-RFLP genotyping strategy and assess its consistency with the phylogenetic classification of the studied viral pathogen. The study was carried out in the laboratory of leukemia of the FSBSI FSC VIEV RAS. A bioinformatic search in GenBank NCBI indicates the deposition of nucleotide sequences of the env gene locus from at least 1110 BLV isolates, the genotypic affiliation of which was established by alignment of the analyzed DNA and subsequent phylogenetic analysis. Interpretation of calculated PCR-RFLP profiles of 1110 BLV isolates generated by restriction mapping of the env gene locus for 5 restriction endonucleases (PvuII, SspI, HphI (AsuHPI isoschizomer), HaeIII, and BstYI (BstX2I isoschizomer)) led to an extension of the previously updated genotyping strategy ten new combinations of PCR-RFLP profiles. At the same time, 4 out of 10 new and 2 out of 58 previously described combinations of PCR-RFLP profiles no longer had a genotype-associated status. Thus, the total proportion of genotype-associated combinations of PCR-RFLP profiles was 91.2%, and the proportion of isolates identified by the proposed genotyping strategy with predominantly deposited sequences of the BLV env gene locus in GenBank NCBI was 90,3%. The addition of the previously developed PCR-RFLP genotyping strategy for BLV with two new restriction endonucleases DdeI (BstDEI isoschizomer) and HpyCH4III (Bst4CI isoschizomer) opens the prospect for its full consistency with the phylogenetic classification, which is dynamically developing due to new knowledge about the genetic diversity of Bovine leukemia virus. Additionally, it should be noted that the nucleotide sequences of the env gene locus of 68 of the listed type isolates of 12 known BLV genotypes can be considered as reference in the phylogenetic analysis of the genetic diversity of the representatives of the studied viral pathogen in terms of their genotypic affiliation.