Archives of Virology

, Volume 160, Issue 5, pp 1325–1332 | Cite as

BLV-CoCoMo-qPCR-2: improvements to the BLV-CoCoMo-qPCR assay for bovine leukemia virus by reducing primer degeneracy and constructing an optimal standard curve

  • Shin-nosuke Takeshima
  • Yuri Kitamura-Muramatsu
  • Yuan Yuan
  • Meripet Polat
  • Susumu Saito
  • Yoko Aida
Brief Report


Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. Because BLV infection can remain clinically silent, the proviral load is an important index for estimating disease progression. CoCoMo-qPCR, an assay developed to estimate BLV proviral load, allows the highly sensitive detection of BLV originating in different countries. Here, we developed a modified version of the CoCoMo-qPCR assay, the “BLV-CoCoMo-qPCR-2” assay, which uses optimized degenerate primers. We also constructed a new plasmid standard. Finally, we used both assays to examine DNA samples from BLV-infected cattle and compared the results.


Bovine leukemia virus Quantitative PCR BLV-CoCoMo-qPCR Cattle CoCoMo algorithm Viral load 


  1. 1.
    Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59:284–291PubMedCentralPubMedGoogle Scholar
  2. 2.
    Aida Y, Murakami H, Takahashi M, Takeshima SN (2013) Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus. Front Microbiol 4:328CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Buehring GC, Shen HM, Jensen HM, Choi KY, Sun D, Nuovo G (2014) Bovine leukemia virus DNA in human breast tissue. Emerg Infect Dis 20:772–782CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Hugerth LW, Wefer HA, Lundin S, Jacobsson HE, Lindberg M, Rodin S, Engstrand L, Andersson AF (2014) DegePrime: a program for degenerate primer design for broad taxonomic-range PCR for microbial ecology studies. Appl Environ Microbiol 80:5116–5123CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Inabe K, Ikuta K, Aida Y (1998) Transmission and propagation in cell culture of virus produced by cells transfected with an infectious molecular clone of bovine leukemia virus. Virology 245:53–64CrossRefPubMedGoogle Scholar
  6. 6.
    Jimba M, Takeshima SN, Matoba K, Endoh D, Aida Y (2010) BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm. Retrovirology 7:91CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Jimba M, Takeshima SN, Murakami H, Kohara J, Kobayashi N, Matsuhashi T, Ohmori T, Nunoya T, Aida Y (2012) BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status. BMC Vet Res 8:167CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Kobayashi N, Konishi H, Sabe H, Shigesada K, Noma T, Honjo T, Hatanaka M (1984) Genomic structure of HTLV (human T-cell leukemia virus): detection of defective genome and its amplification in MT-2 cells. EMBO J 3:1339–1343PubMedCentralPubMedGoogle Scholar
  9. 9.
    Konishi H, Kobayashi N, Hatanaka M (1984) Defective human T-cell leukemia virus in adult T-cell leukemia patients. Mol Biol Med 2:273–283PubMedGoogle Scholar
  10. 10.
    Korber B, Okayama A, Donnelly R, Tachibana N, Essex M (1991) Polymerase chain reaction analysis of defective human T-cell leukemia virus type I proviral genomes in leukemic cells of patients with adult T-cell leukemia. J Virol 65:5471–5476PubMedCentralPubMedGoogle Scholar
  11. 11.
    Lee J, Kim Y, Kang CS, Cho DH, Shin DH, Yum YN, Oh JH, Kim SH, Hwang MS, Lim CJ, Yang KH, Han K (2005) Investigation of the bovine leukemia virus proviral DNA in human leukemias and lung cancers in Korea. J Korean Med Sci 20:603–606CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Miyasaka T, Takeshima SN, Jimba M, Matsumoto Y, Kobayashi N, Matsuhashi T, Sentsui H, Aida Y (2013) Identification of bovine leukocyte antigen class II haplotypes associated with variations in bovine leukemia virus proviral load in Japanese Black cattle. Tissue Antigens 81:72–82CrossRefPubMedGoogle Scholar
  13. 13.
    Ohshima K, Kikuchi M, Masuda Y, Kobari S, Sumiyoshi Y, Eguchi F, Mohtai H, Yoshida T, Takeshita M, Kimura N (1991) Defective provirus form of human T-cell leukemia virus type I in adult T-cell leukemia/lymphoma: clinicopathological features. Cancer Res 51:4639–4642PubMedGoogle Scholar
  14. 14.
    Ott SL, Johnson R, Wells SJ (2003) Association between bovine-leukosis virus seroprevalence and herd-level productivity on US dairy farms. Prev Vet Med 61:249–262CrossRefPubMedGoogle Scholar
  15. 15.
    Panei CJ, Takeshima SN, Omori T, Nunoya T, Davis WC, Ishizaki H, Matoba K, Aida Y (2013) 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 Vet Res 9:95CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Tajima S, Ikawa Y, Aida Y (1998) Complete bovine leukemia virus (BLV) provirus is conserved in BLV-infected cattle throughout the course of B-cell lymphosarcoma development. J Virol 72:7569–7576PubMedCentralPubMedGoogle Scholar
  17. 17.
    Tajima S, Tsukamoto M, Aida Y (2003) 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 77:4423–4430CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Tang L, Wang X, Ru B, Sun H, Huang J, Gao H (2014) MDC-analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites. Biotechniques 56:301–310PubMedGoogle Scholar
  19. 19.
    Zhao TM, Robinson MA, Bowers FS, Kindt TJ (1995) Characterization of an infectious molecular clone of human T-cell leukemia virus type I. J Virol 69:2024–2030PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Shin-nosuke Takeshima
    • 1
    • 2
  • Yuri Kitamura-Muramatsu
    • 3
  • Yuan Yuan
    • 3
  • Meripet Polat
    • 1
    • 2
  • Susumu Saito
    • 3
  • Yoko Aida
    • 1
    • 2
  1. 1.Viral Infectious Diseases UnitRIKENWakoJapan
  2. 2.Laboratory of Viral Infectious Diseases, Department of Medical Genome Sciences, Graduate School of Frontier ScienceThe University of TokyoWakoJapan
  3. 3.Riken Yokohama InstituteRIKEN GENESIS CO., LTD.YokohamaJapan

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