Laboratory of Animal Health, Department of Animal Science, Faculty of Agriculture, Tokyo University of Agriculture, Atsugi, Kanagawa, 243-0034, Japan.
Division of Genomics and Transcriptomics, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, 860-8556, Japan.
Sci Rep. 2021 Feb 25;11(1):4521. doi: 10.1038/s41598-021-83909-3.
Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5' end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.
牛白血病病毒（BLV）是一种致癌逆转录病毒，在较长的潜伏期后会引发称为地方性牛白血病（EBL）的恶性淋巴瘤。BLV 前病毒基因组的插入位点以及病毒和宿主基因组之间的多态性与疾病进展之间的关联尚未完全阐明。为了阐明病毒与宿主之间的生物学相关性，我们开发了一种 DNA 捕获测序（DNA-capture-seq）方法，该方法使用 DNA 探针从下一代测序（NGS）文库中有效地富集靶序列读段。此外，由于读段包含宿主和前病毒基因组的嵌合读段，因此还可以对富集的读段进行分析，以检测前病毒整合位点和感染细胞的克隆扩增。为了验证这种 DNA 捕获测序方法，我们分析了持续 BLV 感染的胎牛肾细胞系（FLK-BLV）、4 个 EBL 肿瘤样本和 4 个非 EBL 血液样本，以鉴定 BLV 整合位点。结果表明，该方法能够有效地富集靶序列读段和 BLV 前病毒基因组的寡克隆整合。此外，4 个 EBL 肿瘤样本中有 3 个显示 BLV 前病毒基因组的多个整合位点，而 1 个样本显示单个整合位点。在本研究中，我们首次发现持续性淋巴细胞增多症牛中存在 5'端缺陷的整合前病毒。本研究中描述的 BLV 变异性、整合位点和克隆扩增的高效和敏感鉴定为使用这种创新工具来理解疾病进展过程中 BLV 感染的详细机制提供了支持。
