Background: Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in BLV-infected animals. Indeed, the assay was highly effective in detecting BLV in cattle from a range of international locations. This assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. In this study, we compared the sensitivity of our BLV-CoCoMo-qPCR method for detecting BLV proviruses with the sensitivities of two real-time PCR systems, and also determined the differences of proviral load with serotests.Results: BLV-CoCoMo-qPCR was found to be highly sensitive when compared with the real-time PCR-based TaqMan MGB assay developed by Lewet al. and the commercial TaKaRa cycleave PCR system. The BLV copy number determined by BLV-CoCoMo-qPCR was only partially correlated with the positive rate for anti-BLV antibody as determined by the enzyme-linked immunosorbent assay, passive hemagglutination reaction, or agar gel immunodiffusion. This result indicates that, although serotests are widely used for the diagnosis of BLV infection, it is difficult to detect BLV infection with confidence by using serological tests alone. Two cattle were experimentally infected with BLV. The kinetics of the provirus did not precisely correlate with the change in anti-BLV antibody production. Moreover, both reactions were different in cattle that carried different bovine leukocyte antigen (BoLA)-DRB3 genotypes.Conclusions: Our results suggest that the quantitative measurement of proviral load by BLV-CoCoMo-qPCR is useful tool for evaluating the progression of BLV-induced disease. BLV-CoCoMo-qPCR allows us to monitor the spread of BLV infection in different viewpoint compared with classical serotest.

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Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of PD-1+LAG-3+ heavily exhausted T cells among CD4+ and CD8+ T cells was higher in the blood of cattle with B-cell lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1 and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3 cooperatively mediate the functional exhaustion of CD4+ and CD8+ T cells and are associated with the development of B-cell lymphoma in BLV-infected cattle.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. BLV is closely related to human T cell leukemia virus. B cell epitopes are important for the use of antibodies as therapeutic agents, the epitope-driven vaccine design, and immunological assays. A common B cell epitope for BLV has not yet been found due to individual differences in disease susceptibility. We used a peptide microarray with 156 synthetic 15-mer peptides covering the envelope glycoprotein gp51 and the Gag proteins p15, p24, and p12 to map B cell epitope and one B cell epitope, gp51p16, was recognized by all four cattle experimentally infected with BLV. A newly developed high-throughput peptide ELISA system revealed 590 (91.2 %) of 647 cattle naturally infected with BLV, carrying 25 different bovine leukocyte antigen class II DRB3 (BoLA-DRB3) alleles, responded to a 20-mer gp51p16-C peptide containing a C-terminal cysteine and gp51p16. Alanine mutation and comparison of the sequences at 17 amino acid positions within gp51p16-C revealed that R7, R9, F10, V16, and Y18 were the common binding sites to BLV antibodies, and two of these sites were found to be highly conserved. Transient expression in the cells of five infectious molecular clones of BLV with a single alanine mutation at five common antibody binding sites had no effect syncytia formation of the gp51 protein. In addition, the mutant proteins, R7A and R9A had no effect on the expression of gp51 protein; the gp51 protein expressions of F10A, V16A and Y18A were lower than that of the wild type protein. This is the first report to identify a common B cell epitope in BLV by comprehensive screening of BLV-infected cattle with varied genetic backgrounds in BoLA-DRB3. Our results have important implications for disease control and diagnosis.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis. However, the propagation and distribution of BLV after primary infection still need to be fully elucidated. Here, we experimentally infected seven cattle with BLV and analyzed the BLV proviral load (PVL) in the blood and various organs. BLV was first detected in the blood of the cattle after one week, and the blood PVL increased for three weeks after infection. The PVL was maintained at a high level in five cattle, while it decreased to a low or medium level in two cattle. BLV was distributed in various organs, such as the heart, lung, liver, kidney, abomasum, and thymus, and, notably, in the spleen and lymph nodes. In cattle with a high blood PVL, BLV was detected in organs other than the spleen and lymph nodes, whereas in those with a low blood PVL, BLV was only detected in the spleen and lymph nodes. The amount of BLV in the organs was comparable to that in the blood. Our findings point to the possibility of estimating the distribution of BLV provirus in organs, lymph nodes, and body fluids by measuring the blood PVL, as it was positively correlated with the biodistribution of BLV provirus in the body of BLV infection during early stages.