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

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Abstract

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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Acknowledgements

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 (www.editage.com) for English language editing.

Funding

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

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Authors and Affiliations

  1. Bovine Viral Disease Unit, Division of Viral Disease and Epidemiology, National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki, 305-0856, Japan

    Asami Nishimori, Kiyohiko Andoh, Yuichi Matsuura, Asuka Kumagai & Shinichi Hatama

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  1. Asami Nishimori
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  2. Kiyohiko Andoh
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  3. Yuichi Matsuura
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  4. Asuka Kumagai
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Contributions

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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The authors have no conflicts of interest to disclose.

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Handling Editor: Zhongjie Shi.

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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). https://doi.org/10.1007/s00705-020-04945-4

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  • DOI: https://doi.org/10.1007/s00705-020-04945-4

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