Development of a luminescence syncytium induction assay (LuSIA) for easily detecting and quantitatively measuring bovine leukemia virus infection
Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T cell leukemia virus. Since BLV infection mostly occurs via cell-to-cell transmission, BLV infectivity is generally measured by culturing BLV-infected cells with reporter cells that form syncytia upon BLV infection. However, this method is time-consuming and requires skill. To visualize the infectivity of BLV, we developed a new assay called the luminescence syncytium induction assay (LuSIA) that is based on a new reporter cell line designated CC81-BLU3G. CC81-BLU3G is stably transfected with pBLU3-EGFP, which contains the BLV long terminal repeat U3 region linked to the enhanced-green fluorescence protein (EGFP) gene. CC81-BLU3G expresses the EGFP in response to BLV Tax expression specifically, and forms fluorescing syncytia when transfected with an infectious BLV plasmid or when cultured with BLV-infected cells. Compared to the conventional assay, LuSIA was more specific and detected cattle samples with low proviral loads. The fluorescing syncytia was easily detected by eye and automated scanning and LuSIA counts correlated strongly with the proviral load of infected cattle (R2 = 0.8942).
The authors thank Ms. Yoshiko Sakuma for providing technical support and Dr. Meripet Polat for constructing the pME18neo/BLV Tax-FLAG plasmid. We also thank all members of the virus infectious disease unit of RIKEN, Grazing Animal Unit, Institute of Livestock and Grassland Science and Nasu Operation Unit, Technical Support Center, NARO for providing advice and samples. We are grateful to the Support Unit, Bio-material Analysis, RIKEN BSI Research Resources Center for helping with the sequence analysis.
This study was supported by Grants-in-Aid for Scientific Research (A) and Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) [Grant Nos. JP16H02590 and JP17K18356], and by grants from the Project of the NARO Bio-oriented Technology Research Advancement Institution (the special scheme regarding regional development strategies) [Grant No. 16817983].
Compliance with ethical standards
Conflict of interest
The authors have no conflicts of interests to declare.
All animal experiments were conducted in accordance with the Guidelines for Laboratory Animal Welfare and Animal Experiment Control that were set out by the Institute of Livestock and Grassland Science, NARO (permit numbers: 1711B082) and by the School of Veterinary Medicine of Azabu University (permit numbers: 161121-2).
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