Key Laboratory of Fishery Drug Development of Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, PR China.
Key Laboratory of Fishery Drug Development of Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, PR China; College of Veterinary, South China Agricultural Universtiy, Guangzhou, PR China.
Microb Pathog. 2018 Jan;114:68-74. doi: 10.1016/j.micpath.2017.11.042. Epub 2017 Nov 24.
Grass carp reovirus (GCRV) caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus, in southeast Asian. It was first isolated in 1983 in China, and clade analysis of the different GCRV isolates indicates there are at least three different genotypes I, II, and III. In recent years, GCRV genotype II has been determined as a dominant virus type which cause severe obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. TCID is one of standard method to quantity infectious virus particles. In the present study, an indirect immunofluorescence assay (IFA) was developed using antibody against a protein encoded by segment 10 of GCRV genotype II. Moreover, the specific assay to differentitate GCRV of different genotypes and a sensitive assay for determination of GCRV genotype II were developed respectively. The results showed the IFA only can recognize genotype II virus at the lowest initial concentration of 550 genomic copies/ml. Furthermore, comparison of results obtained from qPCR and the TCID assay combined IFA was conducted. The results indicated that TCID of GCRV isolates JX0901 and HZ08 differs with 2 log steps reduction in the numbers of viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is sensitive, specific, and the TCID combined with IFA will be a standardizable technique for the quantitation and detection of infectious GCRV in cell culture without cytolysis.
草鱼呼肠孤病毒 (GCRV) 引起了严重的出血性疾病，导致中国东南地区的草鱼幼鱼和鱼种大量死亡。该病毒于 1983 年首次在中国分离得到，不同 GCRV 分离株的系统发育分析表明，至少存在三种不同的基因型 I、II 和 III。近年来，GCRV 基因型 II 已确定为主要病毒类型，其在鱼类中引起严重的明显临床症状，但对目前可用的细胞培养物没有细胞病变效应。半数组织感染剂量 (TCID) 是定量感染性病毒颗粒的标准方法之一。在本研究中，开发了一种使用针对 GCRV 基因型 II 第 10 节编码蛋白的抗体的间接免疫荧光测定法 (IFA)。此外，还分别开发了用于区分不同基因型 GCRV 的特异性检测方法和用于检测 GCRV 基因型 II 的敏感检测方法。结果表明，IFA 仅能在 550 个基因组拷贝/ml 的最低初始浓度下识别基因型 II 病毒。此外，还比较了 qPCR 和结合 IFA 的 TCID 测定法获得的结果。结果表明，与 qPCR 检测到的基因组拷贝数相比，JX0901 和 HZ08 分离株的 TCID 减少了 2 个对数级。所开发的免疫荧光测定法具有敏感性、特异性，并且 TCID 结合 IFA 将成为一种可标准化的技术，可用于在无细胞溶解的情况下定量和检测细胞培养物中的传染性 GCRV。
