BLV-CoCoMo-qPCR: Estimation of Bovine Leukemia Virus (BLV) harbored by lymphocyte subpopulation in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis

YOKO AIDA; SHIN-NOSUKETAKESHIMA; CARLOS JAVIER PANEI; TAKASHI OMORI; TETSUO NUNOYA; WILLIAM C. DAVIS; HIROSHI ISHIZAKI; KAZUHIRO MATOBA

Montréal

Congreso; 16th International Conference on Human Retrovirology HTLV and Related Viruses; 2013

HTLV and Related Viruses

Background: Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. BLV infection may remain clinically silent at an aleukemic (AL) stage, it may emerge as persistent lymphocytosis (PL), or, more rarely, as B cell lymphoma. BLV has been found not only in B cells but also in CD2+ T cells, monocytes, and granulocytes in infected cattle without tumor, although the most consistent tumor cell phenotype is CD5+ B cell. The mechanism by which BLV causes CD5+ B cell proliferation is unknown. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method, BLV-CoCoMo-qPCR, which 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 determined the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations taken from the BLV-infected cows at subclinical stage of EBL using cell sorting and our BLV-CoCoMo-qPCR. Results: Phenotypic characterization by flow cytometry showed a high percentage of CD5+ IgM+ cells, but not CD5- IgM+ B cells, CD4+ T cells, or CD8+T cells, in five BLV-infected but clinically normal cattle with > 100 proviral load per 1 x 105 cells. These lymphocyte subpopulations were purified from three out of five cattle using cell sorting and the BLV proviral load was estimated by BLV-CoCoMo-qPCR. CD5+ IgM+ B cells showed a higher BLV proviral load in all animals, than the other cell populations: The extent of the copy number of proviruses infected in CD5- IgM+ B cells, CD4+ and CD8+ T cells per 1 ml of blood were 1/34 to 1/4, 1/22 to 1/3, 1/31 to 1/3, respectively, as compared with CD5+ IgM+ B cells. Moreover, our study demonstrated that, BLV provirus remains integrated in CD5+ IgM+ B cells, CD5- IgM+ B cells, CD4+ T cells and CD8+ T cells, even in BLV infected cattle with a proviral load