Estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV-CoCoMo-qPCR

Abstract

BackgroundBovine 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 the aleukemic (AL) stage, cause persistent lymphocytosis (PL), or, more rarely, B cell lymphoma. BLV has been identified in B cells, CD2+ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, γ/δ T cells, monocytes, and granulocytes in infected cattle that do not have tumors, although the most consistently infected cell is the CD5+ B cell. The mechanism by which BLV causes uncontrolled 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 the proviral load correlates not only with BLV infection, as assessed by syncytium formation, but also with BLV disease progression. The present study reports the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations isolated from BLV-infected cows at the subclinical stage of EBL as examined by cell sorting and BLV-CoCoMo-qPCR.ResultsPhenotypic characterization of five BLV-infected but clinically normal cattle with a proviral load of > 100 copies per 1 × 105 cells identified a high percentage of CD5+ IgM+ cells (but not CD5- IgM+ B cells, CD4+ T cells, or CD8+T cells). These lymphocyte subpopulations were purified from three out of five cattle by cell sorting or using magnetic beads, and the BLV proviral load was estimated using BLV-CoCoMo-qPCR. The CD5+ IgM+ B cell population in all animals harbored a higher BLV proviral load than the other cell populations. The copy number of proviruses infecting CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells (per 1 ml of blood) was 1/34 to 1/4, 1/22 to 1/3, and 1/31 to 1/3, respectively, compared with that in CD5+ IgM+ B cells. Moreover, the BLV provirus remained integrated into the genomic DNA of CD5+ IgM+ B cells, CD5- IgM+ B cells, CD4+ T cells, and CD8+ T cells, even in BLV-infected cattle with a proviral load of <100 copies per 105 cells.ConclusionsThe results of the recent study showed that, although CD5+ IgM+ B cells were the main cell type targeted in BLV-infected but clinically normal cattle, CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells were infected to a greater extent than previously thought.

Major Histocompatibility Complex-Associated Resistance to Infectious Diseases: The Case of Bovine Leukemia Virus Infection

“…Although evidence of infection of other peripheral blood cell subpopulations has been reported [76], these results have not been confirmed by others. Soon after infection of a cell, the viral RNA is copied into DNA by the virus-encoded reverse transcriptase.…”

Genetic diversity of bovine leukemia virus worldwide

“…As shown in Figure 1 Panei et al 2013). Among these, the Tax protein of BLV which is a transcriptional activator of viral replication and a key contributor to oncogenic potential, immortalizes some infected cells, probably CD5 + IgM + B cells (Aida et al 1993), and induces polyclonal proliferation.…”

Seroprevalencia de infección por el virus de leucosis bovina durante 2015 en rodeos de cría de la Zona Deprimida del Río Salado, provincia de Buenos Aires, Argentina

“…La transmisión natural del virus se produce principalmente a través de las células infectadas con el provirus integrado (Mammerickx et al, 1987), detectándose altas copias de estos provirus en diferentes subpoblaciones de linfocitos (Panei et al, 2013). Por lo tanto, la sangre es una fuente importante de infección, principalmente en la práctica veterinaria (Hopkins & DiGiacomo 1997;Kohara, 2006;Mammerickx et al, 1987), ya que solo cinco microlitros de sangre periférica inoculados por diferentes vías en forma experimental fueron suficientes para infectar a un bovino (González et al, 2001).…”