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

BMC Veterinary Research

Yoko Aida¹  Kazuhiro Matoba⁵  Hiroshi Ishizaki⁵  William C Davis²  Tetsuo Nunoya³  Takashi Omori³  Shin-nosuke Takeshima¹  Carlos Javier Panei⁴ 

[1] Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan;Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, 99164-7040, USA;Nippon Institute for Biological Science, 9-2221-1 Shinmachi Ome, Tokyo, 198-0024, Japan;CONICET and Virology Laboratory, Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina;NARO Institute of Livestock and Grassland Sciences, 768 Senbonmatsu, Nasushiobara, Tochigi, 329-2793, Japan

关键词: Flow cytometry;    Cell sorting;    CD5⁺IgM⁺ B cell;    BLV-CoCoMo-qPCR;    Proviral load;    Bovine leukemia virus (BLV);   

Others  :  1119543
DOI  :  10.1186/1746-6148-9-95

 received in 2013-01-22, accepted in 2013-04-17,  发布年份 2013

【 摘 要 】

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 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.

Results

Phenotypic 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.

Conclusions

The 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.

【 授权许可】

   
2013 Panei et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Burny A, Cleuter Y, Kettmann R, Mammerickx M, Marbaix G, Portetelle D, van den Broeke A, Willems L, Thomas R: Bovine leukaemia: facts and hypotheses derived from the study of an infectious cancer. Vet Microbiol 1988, 17(3):197-218.
• [2]Aida Y, Miyasaka M, Okada K, Onuma M, Kogure S, Suzuki M, Minoprio P, Levy D, Ikawa Y: Further phenotypic characterization of target cells for bovine leukemia virus experimental infection in sheep. Am J Vet Res 1989, 50(11):1946-1951.
• [3]Djilali S, Parodi AL, Levy D, Cockerell GL: Development of leukemia and lymphosarcoma induced by bovine leukemia virus in sheep: a hematopathological study. Leukemia 1987, 1(11):777-781.
• [4]Stott ML, Thurmond MC, Dunn SJ, Osburn BI, Stott JL: Integrated bovine leukosis proviral DNA in T helper and T cytotoxic/suppressor lymphocytes. J Gen Virol 1991, 72(Pt 2):307-315.
• [5]Mirsky ML, Olmstead CA, Da Y, Lewin HA: The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection. J Virol 1996, 70(4):2178-2183.
• [6]Schwartz I, Bensaid A, Polack B, Perrin B, Berthelemy M, Levy D: In vivo leukocyte tropism of bovine leukemia virus in sheep and cattle. J Virol 1994, 68(7):4589-4596.
• [7]Murakami K, Okada K, Ikawa Y, Aida Y: Bovine leukemia virus induces CD5- B cell lymphoma in sheep despite temporarily increasing CD5+ B cells in asymptomatic stage. Virology 1994, 202(1):458-465.
• [8]Takahashi M, Tajima S, Takeshima SN, Konnai S, Yin SA, Okada K, Davis WC, Aida Y: Ex vivo survival of peripheral blood mononuclear cells in sheep induced by bovine leukemia virus (BLV) mainly occurs in CD5- B cells that express BLV. Microbes Infect 2004, 6(6):584-595.
• [9]Takahashi M, Tajima S, Okada K, Davis WC, Aida Y: Involvement of bovine leukemia virus in induction and inhibition of apoptosis. Microbes and infection / Institut Pasteur 2005, 7(1):19-28.
• [10]Kettmann R, Deschamps J, Cleuter Y, Couez D, Burny A, Marbaix G: Leukemogenesis by bovine leukemia virus: proviral DNA integration and lack of RNA expression of viral long terminal repeat and 3' proximate cellular sequences. Proc Natl Acad Sci U S A 1982, 79(8):2465-2469.
• [11]Lagarias DM, Radke K: Transcriptional activation of bovine leukemia virus in blood cells from experimentally infected, asymptomatic sheep with latent infections. J Virol 1989, 63(5):2099-2107.
• [12]Tajima S, Tsukamoto M, Aida Y: Latency of viral expression in vivo is not related to CpG methylation in the U3 region and part of the R region of the long terminal repeat of bovine leukemia virus. J Virol 2003, 77(7):4423-4430.
• [13]Tajima S, Ikawa Y, Aida Y: Complete bovine leukemia virus (BLV) provirus is conserved in BLV-infected cattle throughout the course of B-cell lymphosarcoma development. J Virol 1998, 72(9):7569-7576.
• [14]Jimba M, Takeshima SN, Murakami H, Kohara J, Kobayashi N, Matsuhashi T, Ohmori T, Nunoya T, Aida Y: BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status. BMC Vet Res 2012, 8(1):167. BioMed Central Full Text
• [15]Lew AE, Bock RE, Molloy JB, Minchin CM, Robinson SJ, Steer P: Sensitive and specific detection of proviral bovine leukemia virus by 5' Taq nuclease PCR using a 3' minor groove binder fluorogenic probe. J Virol Methods 2004, 115(2):167-175.
• [16]Monti GE, Frankena K: Survival analysis on aggregate data to assess time to sero-conversion after experimental infection with Bovine Leukemia virus. Prev Vet Med 2005, 68(2–4):241-262.
• [17]Kurdi A, Blankenstein P, Marquardt O, Ebner D: [Serologic and virologic investigations on the presence of BLV infection in a dairy herd in Syria]. Berl Munch Tierarztl Wochenschr 1999, 112(1):18-23.
• [18]Zaghawa A, Beier D, Abd El-Rahim IH, Karim I, El-ballal S, Conraths FJ, Marquardt O: An outbreak of enzootic bovine leukosis in upper Egypt: clinical, laboratory and molecular-epidemiological studies. J Vet Med B Infect Dis Vet Public Health 2002, 49(3):123-129.
• [19]Jimba M, Takeshima SN, Matoba K, Endoh D, Aida Y: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm. Retrovirology 2010, 7:91. BioMed Central Full Text
• [20]Williams DL, Barta O, Amborski GF: Molecular studies of T-lymphocytes from cattle infected with bovine leukemia virus. Vet Immunol Immunopathol 1988, 19(3–4):307-323.
• [21]Wu D, Murakami K, Morooka A, Jin H, Inoshima Y, Sentsui H: In vivo transcription of bovine leukemia virus and bovine immunodeficiency-like virus. Virus Res 2003, 97(2):81-87.
• [22]Miyasaka M, Trnka Z: Sheep as an experimental model for immunology immunological techniques in vitro and in vivo. In Immunological Methods Volume 3, edn edition. Edited by Lefkovits I. 1985, 403-423.
• [23]Mammerickx M, Otte J, Rase F, Braibant E, Portetelle D, Burny A, Dekegel D, Ghysdael J: Large scale serological detection in Belgium of enzootic bovine leukosis. Zentralblatt fur Veterinarmedizin Reihe B Journal of veterinary medicine Series B 1978, 25(5):416-424.
• [24]Tajima S, Takahashi M, Takeshima SN, Konnai S, Yin SA, Watarai S, Tanaka Y, Onuma M, Okada K, Aida Y: A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity, increases expression and propagation of BLV in vitro but not in vivo. J Virol 2003, 77(3):1894-1903.
• [25]Lavanya M, Kinet S, Montel-Hagen A, Mongellaz C, Battini JL, Sitbon M, Taylor N: Cell surface expression of the bovine leukemia virus-binding receptor on B and T lymphocytes is induced by receptor engagement. J Immunol 2008, 181(2):891-898.