Sato Hirotaka, Watanuki Sonoko, Murakami Hironobu, Sato Reiichiro, Ishizaki Hiroshi, Aida Yoko
Virus Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Arch Virol. 2018 Jun;163(6):1519-1530. doi: 10.1007/s00705-018-3744-7. Epub 2018 Feb 17.
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 (R = 0.8942).
牛白血病病毒（BLV）可引发地方流行性牛白血病，且与人类T细胞白血病病毒密切相关。由于BLV感染大多通过细胞间传播发生，BLV的传染性通常通过将感染BLV的细胞与报告细胞共培养来测定，这些报告细胞在感染BLV后会形成多核巨细胞。然而，这种方法耗时且需要技巧。为了可视化BLV的传染性，我们开发了一种名为发光多核巨细胞诱导试验（LuSIA）的新检测方法，该方法基于一种名为CC81 - BLU3G的新报告细胞系。CC81 - BLU3G用pBLU3 - EGFP稳定转染，pBLU3 - EGFP包含与增强型绿色荧光蛋白（EGFP）基因相连的BLV长末端重复序列U3区域。CC81 - BLU3G在特异性响应BLV Tax表达时表达EGFP，并且在转染感染性BLV质粒或与感染BLV的细胞共培养时形成发荧光的多核巨细胞。与传统检测方法相比，LuSIA更具特异性，能够检测到前病毒载量低的牛样本。通过肉眼和自动扫描很容易检测到发荧光的多核巨细胞，并且LuSIA计数与感染牛的前病毒载量密切相关（R = 0.8942）。
