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

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。

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

Abstract:

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.

Key words:GIFT Oreochromis niloticus;E-11 cell;TiLV;HEF protein;polyclonal antibody;immunohistochemistry