Laboratory of Global Infectious Diseases Control Science, Department of Global Agricultural Science, Graduate School of Agriculture and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan.
Viruses. 2022 May 7;14(5):995. doi: 10.3390/v14050995.
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.
牛白血病病毒 (BLV) 感染牛并整合到宿主 DNA 中，导致地方性牛白血病 (EBL)，这是一种侵袭性 B 细胞淋巴瘤。在这里，我们开发了一种新的前病毒 DNA 捕获测序 (proviral DNA-capture-seq) 方法，用于研究源自 EBL 感染 BLV 的牛的两种 B 细胞淋巴瘤系 BLSC-KU1 和 BLSC-KU17 中的 BLV 前病毒整合。我们设计了 BLV 特异性生物素标记探针来捕获前病毒基因组，并富集文库进行下一代测序。验证表明，目标序列读取具有很高的特异性和高效的富集，以及鉴定 BLV 持续感染 FLK-BLV 细胞作为阳性对照的三个 BLV 前病毒整合位点。我们成功地在 BLSC-KU1 的 19 号染色体和 BLSC-KU17 的 9 号染色体上检测到单个 BLV 前病毒整合位点，这通过标准 PCR 和 Sanger 测序得到了证实。此外，使用长 PCR 和测序进一步证实了 BLSC-KU1 中的缺陷前病毒和 BLSC-KU17 中的完整 BLV 前病毒序列。这是首次提供有关 BLSC-KU1 和 BLSC-KU17 中 BLV 前病毒结构和病毒整合的全面信息的研究。此外，所提出的方法可以促进对 BLV 诱导的白血病发生的详细机制的理解，并可用作一种创新工具，以便比已经发展为淋巴瘤的牛更早地筛选处于风险中的 BLV 感染牛。
