Tang Xiaoqian, Qin Yinghui, Sheng Xiuzhen, Xing Jing, Zhan Wenbin
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
Dis Aquat Organ. 2018 Jun 4;128(3):203-213. doi: 10.3354/dao03222.
Hirame novirhabdovirus (HIRRV) causes severe disease in fish cultures, resulting in great economic loss in Asia and Europe. In this study, the matrix protein (M) of HIRRV was recombinantly expressed as the immunogen to produce monoclonal antibodies (MAbs) using hybridoma cell fusion technology, and 3 MAbs were produced and characterized by indirect ELISA, Western blotting and immunofluorescence assay (IFA). Western blotting and mass spectrometric analysis showed that the MAbs could specifically react with the nature M protein of HIRRV. The MAbs were employed to detect virions in HIRRV-infected epithelioma papulosum cyprini (EPC) cells and flounder Paralichthys olivaceus by IFA and immunohistochemistry (IHC). In the virus-infected EPC cells, the virions were mainly located in the cytoplasm, whereas in flounder, HIRRV was present in all 10 tested tissues, and the positive signals in spleen, head-kidney and heart were higher than in other tissues, consistent with the results obtained by RT-PCR. Moreover, strong positive signals were observed in the endothelial cells of blood vessels, but only the leukocytes were infected by HIRRV in the whole blood cells. These results indicate that the high susceptibility to HIRRV of leukocytes and endothelial cells may facilitate the spread of HIRRV and finally cause systemic infection in flounder. This study provides a foundation for further studies on rapid diagnosis of HIRRV and its infection mechanisms.
牙鲆弹状病毒（HIRRV）在鱼类养殖中引发严重疾病，给亚洲和欧洲造成巨大经济损失。在本研究中，利用杂交瘤细胞融合技术，将HIRRV的基质蛋白（M）重组表达为免疫原以制备单克隆抗体（MAb），共产生了3株MAb，并通过间接ELISA、Western印迹和免疫荧光分析（IFA）对其进行了鉴定。Western印迹和质谱分析表明，这些MAb能与HIRRV的天然M蛋白特异性反应。通过IFA和免疫组织化学（IHC），利用这些MAb检测了感染HIRRV的鲤上皮瘤细胞（EPC）和牙鲆中的病毒粒子。在病毒感染的EPC细胞中，病毒粒子主要位于细胞质中，而在牙鲆中，HIRRV存在于所有10个检测组织中，脾脏、头肾和心脏中的阳性信号高于其他组织，这与RT-PCR结果一致。此外，在血管内皮细胞中观察到强阳性信号，但在全血细胞中只有白细胞被HIRRV感染。这些结果表明，白细胞和内皮细胞对HIRRV的高易感性可能促进HIRRV的传播，并最终导致牙鲆全身感染。本研究为进一步研究HIRRV的快速诊断及其感染机制奠定了基础。
