文章摘要
李嘉波,秦真东,赵丽娟,刘志刚,可小丽,吴灶和,刘小玲,卢迈新,林蠡.罗非鱼湖病毒对吉富罗非鱼和E-11细胞的感染[J].水产学报,2020,44(1):142~155
罗非鱼湖病毒对吉富罗非鱼和E-11细胞的感染
Infection of tilapia lake virus in GIFT Oreochromis niloticus and E-11 cell
投稿时间:2019-01-30  修订日期:2019-05-05
DOI:10.11964/jfc.20190111649
中文关键词: 吉富罗非鱼  E-11细胞  罗非鱼湖病毒  HEF蛋白  多克隆抗体  免疫组化
英文关键词: GIFT Oreochromis niloticus  E-11 cell  TiLV  HEF protein  polyclonal antibody  immunohistochemistry
基金项目:现代农业产业技术体系专项(CARS-46);中国-东盟海上合作基金(CAMC-2018F);国家自然科学基金(31872606,31572657,U1701233);广东省海洋与渔业局基金(GDME-2018C006,D21822202,A201512C003,2015-115);广东省教育厅基金(KA170500G,TK222001G,KA18058B3,KA1819604);广东省现代农业产业技术体系创新团队建设专项
作者单位E-mail
李嘉波 华中农业大学水产学院, 湖北 武汉 430070  
秦真东 仲恺农业工程学院动物科技学院, 广东省水环境与水产品安全工程技术研究中心, 广州市水产病害与水禽养殖重点实验室, 广东 广州 510225  
赵丽娟 仲恺农业工程学院动物科技学院, 广东省水环境与水产品安全工程技术研究中心, 广州市水产病害与水禽养殖重点实验室, 广东 广州 510225  
刘志刚 中国水产科学研究院珠江水产研究所, 农业农村部热带亚热带水产资源利用与养殖重点实验室, 广东 广州 510380  
可小丽 中国水产科学研究院珠江水产研究所, 农业农村部热带亚热带水产资源利用与养殖重点实验室, 广东 广州 510380  
吴灶和 仲恺农业工程学院动物科技学院, 广东省水环境与水产品安全工程技术研究中心, 广州市水产病害与水禽养殖重点实验室, 广东 广州 510225  
刘小玲 华中农业大学水产学院, 湖北 武汉 430070  
卢迈新 中国水产科学研究院珠江水产研究所, 农业农村部热带亚热带水产资源利用与养殖重点实验室, 广东 广州 510380 mx-lu@163.com 
林蠡 仲恺农业工程学院动物科技学院, 广东省水环境与水产品安全工程技术研究中心, 广州市水产病害与水禽养殖重点实验室, 广东 广州 510225 linli@zhku.edu.cn 
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中文摘要:
      为研究罗非鱼湖病毒在吉富罗非鱼体内和敏感细胞E-11中的感染特性,实验首先从人工感染罗非鱼湖病毒的吉富罗非鱼脾脏中获得罗非鱼湖病毒第4片段基因组,其cDNA全长1 250 bp,开放读码框长度为1 065 bp,编码354个氨基酸。通过进化树分析,该蛋白是罗非鱼湖病毒血凝素—酯酶融合蛋白(HEF)。随后通过在大肠杆菌大量表达和提纯GST融合HEF蛋白,免疫新西兰大白兔,制备了兔抗TiLV-HEF多克隆抗体。ELISA结果显示获得的抗血清效价高于1∶51 200,并且获得的抗体可以特异性识别病毒的TiLV-HEF蛋白。人工感染实验结果显示,TiLV的感染造成鱼体表面溃疡、全身性出血以及眼晶状体混浊等症状。H.E染色结果显示,肝脏形成合胞体,脾脏中含铁血黄素增加和部分细胞空泡变性。头肾出现淋巴细胞坏死,体肾蛋白质沉淀和肾小球坏死等病理症状。Western blot和免疫组织化学结果显示该病毒在所有组织中均有分布,其中脾脏、头肾和鳃中的病毒丰度高于肝脏、体肾和脑组织。通过细胞间接免疫荧光实验,发现TiLV感染E-11细胞后,HEF蛋白在细胞质中。TiLV可以通过感染吉富罗非鱼幼鱼的肝脏、脾脏、头肾、体肾、鳃和脑等组织而引起疾病。
英文摘要:
      In order to study the infection characteristics of TiLV in the cultured tilapia species and susceptible cells, GIFT Oreochromis niloticus and E-11 cells were chosen as models. For the present study, first of all, the whole nucleotide sequences of the fourth genome segment of TiLV from the experimental infected GIFT O. niloticus were determined. The length of the cDNA of the fourth genome segment was 1 250 bp containing an open reading frame of 1 065 bp, encoding a protein with 354 amino acids. The sequences and phylogenetic tree analysis showed that the fourth genome segment encoded TiLV Hemagglutinin-esterase-fusion (HEF) protein. Subsequently, GST fusion HEF was expressed in Escherichia coli and purified, and it was used to immunize New Zealand white rabbits according to the conventional method to prepare rabbit anti-HEF polyclonal antibody. The results showed that the antiserum titer obtained by ELISA was higher than 1∶51 200, and the serum could specifically recognize the HEF protein from the spleen of TiLV infected GIFT O. niloticus. Through artificial infection experiments, it was found that TiLV infected juvenile GIFT O. niloticus severely and caused surface ulceration, systemic bleeding and ocular lens opacity. Furthermore, hematoxylin and eosin (HE) stain showed the syncytium in liver, hemosiderin and vacuolar degeneration in spleen, necrosis in head kidney lymphocytes, protein precipitation and glomerulus necrosis in trunk kidney. Western blot and immunohistochemistry results showed that the virus was distributed in all the tissues with the higher abundance in the spleen, head kidney and gill than that in the liver, trunk kidney and brain tissues. Through indirect immunohistochemistry assay, it was found that HEF protein was mainly distributed in the cytoplasm in E-11 cells infected with TiLV. Our results demonstrate that TiLV infection could cause disease by targeting liver, spleen, kidney, gill and brain tissues of GIFT O. niloticus.
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