Lymphoma cells are circulating in blood of Enzootic Bovine Leukosis and clonality value of virus-infected cells is a useful information for the diagnostic test

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.