Clone Dynamics and Its Application for the Diagnosis of Enzootic Bovine Leukosis.

Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset.