Bovine leukemia virus (BLV) infects cattle and integrates into host DNA, causing enzootic bovine leukosis (EBL), an aggressive B-cell lymphoma. Here, we developed a novel proviral DNA-capture sequencing (proviral DNA-capture-seq) method investigating BLV proviral integration in two B-cell lymphoma lines, BLSC-KU1 and BLSC-KU17, derived from BLV-infected cattle with EBL. We designed BLV-specific biotinylated probes to capture the provirus genome and enrich libraries for next-generation sequencing. Validation showed high specificity and efficient enrichment of target sequence reads as well as identification of three BLV proviral integration sites on BLV persistently infected FLK-BLV cells as a positive control. We successfully detected a single BLV proviral integration site on chromosome 19 of BLSC-KU1 and chromosome 9 of BLSC-KU17, which were confirmed by standard PCR and Sanger sequencing. Further, a defective provirus in BLSC-KU1 and complete BLV proviral sequence in BLSC-KU17 were confirmed using long PCR and sequencing. This is the first study to provide comprehensive information on BLV proviral structure and viral integration in BLSC-KU1 and BLSC-KU17. Moreover, the proposed method can facilitate understanding of the detailed mechanisms underlying BLV-induced leukemogenesis and may be used as an innovative tool to screen BLV-infected cattle at risk at an earlier stage than those that have already developed lymphoma.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), yet its pathogenic mechanisms remain largely unclear. In particular, the role of BLV genomic integration sites (IS(s)) in BLV-induced leukemogenesis has not been fully elucidated. Here, we identified a total of 235 ISs from 99 BLV-infected cattle with lymphoma, of which 4.3% and 46.8% were located within exon and intron, respectively, while no preferential integration into CpG islands or repetitive regions was observed. All identified ISs were distinct, and no identical sites were detected among the samples. We identified 246 genes related with these ISs and protein–protein interaction analysis of these genes demonstrated one “IS-Clustered genes” composed of 85 among 246 genes. This “IS-Clustered genes” contains 12 cancer genes (14.1%) with high significantly proportion. Notably, with 55 among 99 cattle tested (55.6%) harboring ISs within this cluster, suggesting its crucial involvement in BLV-induced pathogenesis. Furthermore, integrated analysis of known lymphoma- and IS-related genes and the 85 “IS-Clustered genes” showed that key genes formed a shared cluster, indicating a potential “common EBL-associated cluster.” These findings provide important insights into the role of BLV integration in EBL development and may contribute to elucidating its molecular mechanisms underlying onset of EBL. In addition, these findings may also aid in the development of therapeutic strategies and facilitate early diagnosis.