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.

No abstract

Bovine leukemia virus (BLV) is a causative agent of enzootic bovine lymphoma (EBL). BLV is prevalent worldwide, and ten genotypes have been classified based on the sequence of the envelope glycoprotein (gp51) gene. In this study, we present a simple and generally applicable PCR restriction fragment length polymorphism (PCR-RFLP) method to identify BLV genotypes. While the genotyping results obtained by previously described PCR-RFLP methods matched only 78.96% to the results of phylogenetic analysis, we demonstrated that our PCR-RFLP method can identify 90.4% of the sequences available in the database in silico . The method was validated with 20 BLV sequences from EBL tumor tissues and 3 BLV sequences from blood of BLV infected cattle, and was found to show high specificity. We utilized this method to determine genotypes of blood samples from 18 BLV seropositive cattle in Kanagawa and Niigata, as well as 12 EBL cattle in Chiba, Japan. Our analysis with the modified PCR-RFLP detected two genotypes, Genotypes 1 and 3. Genotype 1 was detected as the main genotype, while Genotype 3 was sporadically observed. This technique can be used as a reliable system for screening a large number of epidemiological samples.

Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry.

Bovine leukemia virus (BLV), a retrovirus, causes Enzootic Bovine Leukosis (EBL) in cattle following a latent infection period. The BLV infection results in polyclonal expansion of infected B-lymphocytes and ∼5% of infected cattle develop monoclonal leukosis. Since the clonal expansion of virus-infected cell is a key in the pathogenesis of EBL, assessing the clonality of malignant cells is crucial for both understanding viral pathogenesis, which might be useful for EBL diagnosis.For the investigation of clonality of BLV-infected cells in non-EBL and EBL cattle, two methods were used to evaluate the status of EBL; BLV-DNA-capture-seq method with high sensitivity and specificity and simple and cost-effective Rapid Amplification of Integration Site for BLV (BLV-RAIS) method. We found that the RAIS method efficiently detect expanded clone in EBL tissue sample as BLV-DNA-capture-seq method. Taking advantage of high frequency of BLV-infected cells in blood, we simplified RAIS method and showed that similar to BLV-DNA-capture-seq, this method could reliably provide quantitative value about clonal abundance of BLV-infected cells.Next, we aimed to establish a diagnostic blood test for EBL by using the clonality information. First, we compared clonality of BLV-infected cells in blood with that in tumor tissue in EBL cattle. There was a remarkably similar clonality between blood and tissue in each animal. Furthermore, BLV integration site information clearly showed that the same clone was the most expanded in both blood and tumor tissue, indicating that tumor cells were circulating in blood in the disease cattle. We also analyzed tumor tissue at two independent anatomical regions and found the same clones was most expanded in both regions, supporting the idea that tumor cells are systemically circulating in the diseased cattle. Finally, we compared clonality value between non-EBL and EBL cattle by using BLV-RAIS method and found that there was clear difference between non-EBL and EBL. More importantly, we found that clonality value was low in asymptomatic phase but high in EBL phase in the longitudinal cohort study.These findings have demonstrated that BLV integration site and clonality value are is a useful information to establish diagnostic blood test for EBL. That would contribute to reduction of economic damage caused by EBL and improvement of productivity in cattle industry.