Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis


Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma of cattle caused by infection with bovine leukemia virus (BLV). It is defined by clonal and neoplastic expansion of BLV-infected B cells. Currently, multiple examinations are able to comprehensively diagnose this condition. Inverse polymerase chain reaction (PCR) is a useful method to determine retrovirus integration sites. Here, we established a simplified inverse PCR method, involving the evaluation of clonality and similarity of BLV integration sites, to clinically diagnose EBL, and we also assessed its reliability. We found that the novel BLV inverse PCR could detect clonal expansion of infected cells even if they constituted only 5% of the total number of cells, while not amplifying any fragments from BLV-uninfected cells, thus confirming its sufficient sensitivity and specificity for use in EBL diagnosis. Furthermore, 50 clinical cases of bovine leukemia were analyzed using BLV inverse PCR and other PCR-based methods, wherein our method most efficiently determined virus-dependent bovine leukemia, including unidentified clinical cases observed in a previous report. Following further clinical investigations to enhance its reliability, the proposed BLV inverse PCR method has the potential to be applied to EBL diagnosis.

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  1. 1.

    OIE World Organization for Animal Health (2019) Manual of diagnostic tests and vaccines for terrestrial animals 2019, chapter 3.4.9. Enzootic Bovine Leukosis.

  2. 2.

    Gutiérrez G, Rodríguez SM, de Brogniez A, Gillet N, Golime R, Burny A, Jaworski JP, Alvarez I, Vagnoni L, Trono K, Willems L (2014) Vaccination against δ-retroviruses: the bovine leukemia virus paradigm. Viruses 6(6):2416–2427.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    WAHIS Interface (2020) OIE disease distribution maps. Accessed 5 Jun 2020

  4. 4.

    Rhodes JK, Pelzer KD, Johnson YJ (2003) Economic implications of bovine leukemia virus infection in mid-Atlantic dairy herds. J Am Vet Med Assoc 223(3):346–352.

    Article  PubMed  Google Scholar 

  5. 5.

    Somura Y, Sugiyama E, Fujikawa H, Murakami K (2014) Comparison of the copy numbers of bovine leukemia virus in the lymph nodes of cattle with enzootic bovine leukosis and cattle with latent infection. Arch Virol 159(10):2693–2697.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Nishimori A, Konnai S, Okagawa T, Maekawa N, Goto S, Ikebuchi R, Nakahara A, Chiba Y, Ikeda M, Murata S, Ohashi K (2017) Identification of an atypical enzootic bovine leukosis in Japan by using a novel classification of bovine leukemia based on immunophenotypic analysis. Clin Vaccine Immunol 24(9):e00067-e117.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Frie MC, Coussens PM (2015) Bovine leukemia virus: a major silent threat to proper immune responses in cattle. Vet Immunol Immunopathol 163(3–4):103–114.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Asahina M, Kimura K, Murakami K, Ajito T, Wu D, Goryo M, Aida Y, Davis WC, Okada K (1995) Phenotypic analysis of neoplastic cells from calf, thymic, and intermediate forms of bovine leukosis. Vet Pathol 32(6):683–691.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Grünberg W, Eisenberg SWF (2013) Atypical form of sporadic bovine leukosis (SBL) in the Netherlands. Vet Rec 173(16):398.

    Article  PubMed  Google Scholar 

  10. 10.

    Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120(3):621–623

    CAS  Article  Google Scholar 

  11. 11.

    Murakami H, Yamada T, Suzuki M, Nakahara Y, Suzuki K, Sentsui H (2011) Bovine leukemia virus integration site selection in cattle that develop leukemia. Virus Res 156(1–2):107–112.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Miyasaka T, Oguma K, Sentsui H (2015) Distribution and characteristics of bovine leukemia virus integration sites in the host genome at three different clinical stages of infection. Arch Virol 160(1):39–46.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Itohara S, Sekikawa K, Mizuno Y, Kono Y, Nakajima H (1987) Establishment of bovine leukemia virus-producing and -nonproducing B-lymphoid cell lines and their proviral genomes. Leuk Res 11(5):407–414.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Lindsey NJ, Chow TL (1969) Preservation of primary bovine embryonic kidney cells with dimethyl sulfoxide. Appl Microbiol 17(3):484–485

    CAS  Article  Google Scholar 

  15. 15.

    Shimizu M, Satou K (1987) Frequency of persistent infection of cattle with bovine viral diarrhea-mucosal disease virus in epidemic areas. Nihon Juigaku Zasshi 6:1045–1051.

    Article  Google Scholar 

  16. 16.

    Fechner H, Blankenstein P, Looman AC, Elwert J, Geue L, Albrecht C, Kurg A, Beier D, Marquardt O, Ebner D (1997) Provirus variants of the bovine leukemia virus and their relation to the serological status of naturally infected cattle. Virology 237(2):261–269.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Rosewick N, Durkin K, Artesi M, Marçais A, Hahaut V, Griebel P, Arsic N, Avettand-Fenoel V, Burny A, Charlier C, Hermine O, Georges M, van den Broeke A (2017) Cis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis. Nat Commun 8:15264.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Gillet NA, Malani N, Melamed A, Gormley N, Carter R, Bentley D, Berry C, Bushman FD, Taylor GP, Bangham CRM (2011) The host genomic environment of the provirus determines the abundance of HTLV-1-infected T-cell clones. Blood 117(11):3113–3122.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Artesi M, Hahaut V, Ashrafi F, Marçais A, Hermine O, Griebel P, Arsic N, Van der Meer F, Burny A, Bron D, Charlier C, Georges M, van den Broeke A, Durkin K (2019) Pooled CRISPR inverse PCR sequencing (PCIP-seq): simultaneous sequencing of retroviral insertion points and the associated provirus in thousands of cells with long reads. bioRxiv.

  20. 20.

    Okayama A, Stuver S, Matsuoka M, Ishizaki J, Tanaka G, Kubuki Y, Mueller N, Hsieh C, Tachibana N, Tsubouchi H (2004) Role of HTLV-1 proviral DNA load and clonality in the development of adult T-cell leukemia/lymphoma in asymptomatic carriers. Int J Cancer 110(4):621–625.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Kobayashi S, Yamamoto T, Hayama Y, Murai K, Tsutsui T (2016) Descriptive epidemiology of bovine leukemia in Japan. J Vet Epidemiol 20(1):17–18.

    Article  Google Scholar 

  22. 22.

    Kondo S, Kotani T, Tsumori S, Narahara S, Aratake Y, Kobayashi M, Takahashi M, Inoue S, Ohtaki S (1995) Identification of biclonal (duplex) leukaemic cells expressing either CD4+/CD8- or CD4-/CD8+ from a patient with adult T-cell leukaemia/lymphoma. Br J Haematol 89(3):669–671.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Kato N, Sugawara H, Aoyagi S, Mayuzumi M (2001) Lymphoma-type adult T-cell leukaemia-lymphoma with a bulky cutaneous tumour showing multiple human T-lymphotropic virus-1 DNA integration. Br J Dermatol 144(6):1244–1248.

    CAS  Article  PubMed  Google Scholar 

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We are grateful to the staff at Ibaraki Kenpoku Livestock Hygiene Service Center, Ibaraki Kensei Meat Inspection Office, Himeji Meat Hygiene Inspection Center, Hyogo Meat Hygiene Inspection Center, Awaji Meat Inspection Office, Nishiharima Meat Inspection Office, and Tajima Meat Inspection Office for kindly providing clinical EBL samples. Expert technical advice was provided by Yuto Suda (National Institute of Animal Health, NARO) and Tomohiro Okagawa (Faculty of Veterinary Medicine, Hokkaido University). We would like to thank Editage ( for English language editing.


This work was supported by a subsidy from the National Agriculture and Food Research Organization.

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The study conception was determined by AN. All authors contributed to the design of the study. Material preparation was performed by AN, KA, and SH. Data collection and analysis were performed by AN and KA. The first draft of the manuscript was written by AN. All authors commented on previous versions of the manuscript and have read and approved the final manuscript.

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Correspondence to Asami Nishimori.

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Nishimori, A., Andoh, K., Matsuura, Y. et al. Establishment of a simplified inverse polymerase chain reaction method for diagnosis of enzootic bovine leukosis. Arch Virol 166, 841–851 (2021).

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