Comparison of integration sites of bovine leukemia virus-induced lymphoma between blood and lymphoma tissues using a high-density BLV-proviral capture sequencing method.

Bovine leukemia virus (BLV) infects cattle, integrates into the host genome as a provirus, and induces leukemia/lymphoma. We previously developed a BLV proviral DNA capture sequencing (cap-seq) method to analyze BLV integration sites using next-generation sequencing. However, the previous five-probe method provided only partial coverage of the BLV genome and did not allow simultaneous determination of the complete proviral sequence. Herein, we modified the cap-seq method by designing a high-density probe panel to capture the full-length BLV sequence, and analyzed the integration sites using blood and matched lymphoma tissues from six cattle with lymphoma. The 12 integration sites were distinct from each other and from previously reported sites. Protein-protein interaction analysis showed that the six genes near integration sites formed a single cluster with genes in the "common Enzootic bovine leukosis (EBL)-related clusters," including eight oncogenes, suggesting their potential involvement in EBL onset. The clonal distribution of integration sites was similar between the blood and lymphoma tissues of five animals; however, one animal showed higher heterogeneity in blood than in tumor tissues, suggesting that blood might serve as a primary site of lymphoma. Five animals harbored the full-length proviral genome, while one harbored two genome types: one partial and one with a large deletion in the 5' sequence. Phylogenetic tree analysis classified all proviral genomes as genotype 1, the most prevalent in Japan. The high-density probe panel enabled simultaneous analysis of integration sites and BLV sequence variations. This modified method is expected to facilitate understanding of EBL pathogenesis.