Comprehensive Comparison of Novel Bovine Leukemia Virus (BLV) Integration Sites between B-Cell Lymphoma Lines BLSC-KU1 and BLSC-KU17 Using the Viral DNA Capture High-Throughput Sequencing Method

Abstract

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.

Visualization of clonal expansion after massive depletion of cells carrying the bovine leukemia virus (BLV) integration sites during the course of disease progression in a BLV naturally-infected cow: a case report

“…We clarified the BLV integration sites from Stages I-III using BLV proviral DNA-capture-seq, which is a targetenrichment high-throughput sequencing system for characterization of BLV integration sites [14]. The DNAs molecules were fragmented to an average length of approximately 600 bp to generate DNA libraries.…”

“…In this study, we monitored the sequential alternation of BLV integration sites in a BLV naturally-infected Holstein cow from Stages I to III, using proviral DNA-captureseq previously developed by us [14]. In this study, we identified 16 distinct proviral integration sites in Stage I.…”

“…Figure 4B shows a schematic diagram to detect both the Chr 1/BLV-5′LTR Table 1 Clinical information used in this study a The proviral load (expressed as the copy number per 10 5 cells) was evaluated by BLV-CoCoMo-qPCR-2 b ELISA was performed using an anti-BLV ELISA kit, according to the manufacturer's instructions (JNC Inc., Tokyo, Japan). +: Positive for anti-BLV antibodies c The clinical stage of BLV infection was evaluated according to the lymphocyte count (per 1 µL) and age of the animal [14] 4C, D). After sequencing both the 5′ and 3′ sites, we confirmed six nucleotides (GTA AAA ) (Fig.…”

“…Like human retroviruses, BLV preferentially integrates into transcriptionally active genomic regions near transcriptional start sites and transcription factor-binding sites, and BLV integration accelerates the proliferation of infected cells [10,13]. Indeed, the BLV provirus in BLV-induced B-cell lymphoma line BLSC-KU1 is integrated at a single site in the intron of the regulatory-associated protein of mTOR (RPTOR) gene, whereas in other B-cell lymphoma line BLSC-KU17, it is integrated at a single site in the intergenic region between RTN4IP1 and ATG5 [14]. Interestingly, previous research revealed that BLV provirus is preferentially integrated in the vicinity of cancer drivers that are affected via either premature transcription interruption or antisense dependent cis-perturbation.…”

“…from asymptomatic to the terminal stages of leukemia/lymphoma. In this study, we collected blood samples classified into three stages such as (Stage I, polyclonal stage; Stage II, polyclonal toward oligoclonal stage; Stage III, oligoclonal stage) from a naturally-infected Holstein dairy cow at three time points, and successfully monitored the alternation of the BLV integration sites using our BLV proviral DNA-capture sequencing method, which is a target enrichment high-throughput sequencing system for characterization of BLV integration sites [14]. This is the first report to illustrate the time course of clonal expansion after massive depletion of cells carrying BLV proviral clones and sequential changes of integration sites in naturally infected BLV individuals progressing from the premalignant stage to the terminal disease.…”