Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China, 5 Yushan Road, Qingdao, 266003, PR China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No.1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao, 266071, China.
Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China, 5 Yushan Road, Qingdao, 266003, PR China.
Microb Pathog. 2021 May;154:104868. doi: 10.1016/j.micpath.2021.104868. Epub 2021 Mar 23.
Hirame rhabdovirus (HIRRV) is one of the most important viruses of fish, posing a great threat to the fish industry in Asia and Europe. The glycoprotein (G) of HIRRV is known to play important roles in virus attachment and entry, making it an ideal target for both diagnosis and therapy. In this study, a truncated G of HIRRV was expressed as a fusion protein in Escherichia coli. Using the recombinant G protein (rG), monoclonal antibodies (mAbs) were prepared by the hybridoma technology. Subsequently, positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA) and further characterized by Western blot and immunofluorescence assay (IFA). ELISA results showed that two mAbs (3E5 and 4D10) could react with the rG, as well as the purified HIRRV. Western blot analysis showed that the mAbs belong to the IgG isotype and could recognize a 60 kDa viral protein, which is consistent with the molecular weight of G protein and determined to be the G protein of HIRRV by mass spectrometry. The virions in HIRRV-infected EPC could also be recognized by two mAbs in IFA. Moreover, neutralization assay showed that mAb 4D10 could significantly inhibit the proliferation of HIRRV and delay the development of cytopathic effect in viral-infected EPC cells, and in vivo neutralization assay also showed that mAb 4D10 could significantly reduce the mortality of HIRRV-infected flounder, indicating that mAb 4D10 can partially neutralize the HIRRV infection. Western blot analysis showed that mAb 4D10 could specifically bind the C-terminal domain of HIRRV-G protein. These results demonstrated that the produced mAbs could specifically recognize the G protein of HIRRV and displayed virus-neutralizing activity in vitro and in vivo, which could serve as effective detection probes and potential neutralizing antibodies for HIRRV.
虹彩病毒（HIRRV）是鱼类最重要的病毒之一，对亚洲和欧洲的鱼类产业构成了巨大威胁。虹彩病毒的糖蛋白（G）已知在病毒附着和进入中发挥重要作用，使其成为诊断和治疗的理想目标。在这项研究中，虹彩病毒的截短 G 作为融合蛋白在大肠杆菌中表达。使用重组 G 蛋白（rG），通过杂交瘤技术制备单克隆抗体（mAbs）。随后，通过间接酶联免疫吸附试验（ELISA）筛选阳性克隆，并通过 Western blot 和免疫荧光试验（IFA）进一步表征。ELISA 结果表明，两种 mAbs（3E5 和 4D10）可与 rG 以及纯化的 HIRRV 反应。Western blot 分析表明，mAbs 属于 IgG 同种型，可识别 60 kDa 的病毒蛋白，与 G 蛋白的分子量一致，并用质谱法确定为 HIRRV 的 G 蛋白。IFA 中两种 mAbs 也可识别 HIRRV 感染的 EPC 中的病毒粒子。此外，中和试验表明，mAb 4D10 可显著抑制 HIRRV 的增殖并延迟病毒感染的 EPC 细胞中细胞病变效应的发展，体内中和试验也表明，mAb 4D10 可显著降低 HIRRV 感染牙鲆的死亡率，表明 mAb 4D10 可部分中和 HIRRV 感染。Western blot 分析表明，mAb 4D10 可特异性结合 HIRRV-G 蛋白的 C 末端结构域。这些结果表明，所产生的 mAbs 可特异性识别 HIRRV 的 G 蛋白，并在体外和体内显示出病毒中和活性，可作为 HIRRV 的有效检测探针和潜在的中和抗体。
