A sensitive luminescence syncytium induction assay (LuSIA) based on a reporter plasmid containing a mutation in the glucocorticoid response element in the long terminal repeat U3 region of bovine leukemia virus

Virology Journal

Hiroshi Ishizaki¹  Yuma Hachiya²  Hiroshi Sentsui²  Yasunobu Matsumoto³  Liushiqi Borjigin⁴  Lanlan Bai⁵  Hirotaka Sato⁵  Sonoko Watanuki⁶  Yoko Aida⁶ 

[1] 0000 0000 9191 6962, grid.419600.a, Grazing Animal Unit, Division of Grassland Farming, Institute of Livestock and Grassland Science, NARO, 768 Senbonmatsu, 329-2793, Nasushiobara, Tochigi, Japan;0000 0001 2149 8846, grid.260969.2, Department of Veterinary Medicine, Nihon University, Kameino 1866, 252-0880, Fujisawa, Kanagawa, Japan;0000 0001 2151 536X, grid.26999.3d, Laboratory of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan;Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, 3510198, Saitama, Japan;Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, 3510198, Saitama, Japan;0000000094465255, grid.7597.c, Virus Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, 3510198, Saitama, Japan;Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, 3510198, Saitama, Japan;0000000094465255, grid.7597.c, Virus Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, 3510198, Saitama, Japan;0000 0001 2151 536X, grid.26999.3d, Laboratory of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan;

关键词: Bovine leukemia virus;    Infectivity;    Visualization;    LuSIA (luminescence syncytium induction assay);    Long terminal repeat U3 region;    Tax-dependent reporter;    Glucocorticoid response element;   

DOI  :  10.1186/s12985-019-1172-2

来源: publisher

【 摘 要 】

BackgroundBovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR).MethodsWe changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3GREM-EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells.ResultsWe successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3GREM-EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows.ConclusionThe new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.

【 授权许可】

CC BY