文章摘要
罗非鱼湖病毒对尼罗罗非鱼(Oreochromis niloticus)和E-11细胞的感染研究
The Infection of Tilapia Lake Virus in Nile Tilapia (Oreochromis niloticus) and E-11 Cell
投稿时间:2019-01-30  修订日期:2019-05-05
DOI:
中文关键词: 尼罗罗非鱼  E-11 细胞  罗湖病毒  HEF蛋白  多克隆抗体  免疫组化
英文关键词: Nile Tilapia  E-11 cell  TiLV  HEF protein  Polyclonal antibody  Immunochemistry
基金项目:现代农业产业技术体系专项资金(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资金
作者单位E-mail
林蠡 华中农业大学 linli@mail.hzau.edu.cn 
李嘉波 华中农业大学  
秦真东 仲恺农业工程学院  
赵丽娟 仲恺农业工程学院  
刘志刚 中国水产科学研究院珠江水产研究所  
可小丽 中国水产科学研究院珠江水产研究所  
吴灶和 仲恺农业工程学院  
刘小玲 华中农业大学  
卢迈新 中国水产科学研究院珠江水产研究所  
摘要点击次数: 329
全文下载次数: 0
中文摘要:
      2009年以来,罗非鱼湖病毒(Tilapia lake virus ,TiLV)在以色列、厄瓜多尔、埃及、泰国和印度等多个国家养殖的罗非鱼中流行和暴发,对罗非鱼养殖业造成了严重威胁。为研究罗非鱼湖病毒在罗非鱼体内和敏感细胞E-11中的感染特性,本实验使用的罗非鱼湖病毒(TiLV)由德国弗里德里希洛弗勒研究所Sven Bergmann 博士赠送。首先从人工感染罗非鱼湖病毒的罗非鱼脾脏获得罗非鱼湖病毒第四片段基因组,cDNA 全长1250 bp,开放读码框长度为1065 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(Hemagglutinin-esterase-fusion HEF protein,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。通过人工感染实验,发现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。HE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可以通过感染罗非鱼幼鱼的肝、脾、头肾、体肾、鳃和脑等组织而引起疾病。研究结果可为防控罗非鱼湖病毒感染提供参考。
英文摘要:
      Since 2009, Tilapia lake virus (TiLV) has been endemic and outbreaks in the cultured tilapia in many countries such as Israel, Ecuador, Egypt, Thailand and India, posing a serious threat to the tilapia aquaculture industry. In order to study the infection characteristics of TiLV in the cultured tilapia species and susceptible cells, Nile Tilapia (Oreochromis niloticus,GIFT strain) and E-11 cells were chosen as models. TiLV for present study was kindly gifted by Dr. Sven Bergmann from Institute of Infectology, Friedrich Loffler Institut (FLI). First of all, the whole nucleotide sequences of the fourth genome segment of TiLV from the experimental infected tilapia were determined. The length of the cDNA of the fourth genome segment was 1250 bp containing an open reading frame of 1065 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 1:51200, and the serum could specifically recognize the HEF protein from the spleen of TiLV infected tilapia. Through artificial infection experiments, it was found that TiLV infected juvenile tilapia 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 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 tissue of tilapia, and will shed a new light on the prevention and control of Tilapia Lake virus infection in tilapia.
HTML     下载PDF阅读器
关闭

手机扫一扫看