大黄鱼肿大细胞病毒不同毒株的细胞培养及主要衣壳蛋白基因比较  

Cell culture and major capsid protein(mcp)gene analysis of Megalocytivirus isolates from large yellow croaker(Larimichthys crocea)

作　　者：杨西西 池洪树[1] 郑在予[1] 刘晓东[1] 罗潘潘 许斌福[1] 陈秀霞[1] 龚晖[1] YANG Xixi;CHI Hongshu;ZHENG Zaiyu;LIU Xiaodong;LUO Panpan;XU Binfu;CHEN Xiuxia;GONG Hui(Institute of Biotechnology,Fujian Academy of Agricultural Sciences,Fuzhou 350003,China;College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China;College of Life Sciences,Sun Yat-sen University,Guangzhou 510275,China)

机构地区：[1]福建省农业科学院生物技术研究所,福建福州350003 [2]福建农林大学动物科学学院,福建福州350002 [3]中山大学生命科学学院,广东广州510275

出　　处：《水产学报》2023年第9期145-154,共10页Journal of Fisheries of China

基　　金：国家重点研发计划(2019YFD0900102);福建省重大专项专题(2020NZ08003);福建省公益类科研院所基本科研专项(2020R1027006,2020R1027002,2020R1027004)。

摘　　要：为建立大黄鱼肿大细胞病毒的培养方法,明确其分类地位,用肿大细胞病毒检测呈阳性的大黄鱼幼鱼病料(FD201807和SA201808)肾组织匀浆液感染鳜仔鱼细胞系(mandarin fish fry cell line-1,MFF-1)并连续传代,从病料组织匀浆液和细胞冻融液中提取病毒DNA,克隆病毒主要衣壳蛋白基因(mcp),测序后与NCBI GenBank中的虹彩病毒科肿大细胞病毒属病毒mcp以及2018—2020年所检出的15株大黄鱼肿大细胞病毒mcp进行比对分析。结果显示,病毒传至第4代才可引起MFF-1细胞病变,细胞病变的主要特征为细胞脱壁、变圆、折光度增强;感染时间越长脱壁细胞越多,同时培养液中的颗粒增加;透射电镜下可见感染细胞的细胞质散在大小为130~150 nm的六边形病毒粒子和空壳。感染细胞的病变周期随传代代次的增加而缩短,第15代次的FD201807株感染细胞80%细胞病变的时间为3 d,第15代次的SA201808株感染细胞80%细胞病变的时间为7~8 d。mcp序列比对和聚类分析发现,SA201808株与FD201807株的mcp序列存在21个碱基差异,二者的mcp序列分别与大黄鱼虹彩病毒(largeyellowcroakeriridovirus,LYCIV)LYCIVZhoushan(GenBank:MW139932.1)和花鲈虹彩病毒(Lateolabrax maculatus iridovirus,LMIV)(GenBank:MH577517.1)相近。15株从大黄鱼病料检出的肿大细胞病毒中,12株的mcp序列与SA201808株聚类;3株与FD201807聚类。本研究利用MFF-1细胞系分离培养了大黄鱼肿大细胞病毒,揭示了大黄鱼肿大细胞病毒存在差异,为更好地了解大黄鱼肿大细胞病毒提供了数据参考。In the last decades,the marine cage-cultured large yellow croaker(Larimichthys crocea)in Fujian province suffered from Megalocytivirus frequently.The Megalocytivirus isolated from L.crocea have not found any sensitive cell-culture model yet.The missing of culture methods has set back the studies on Megalocytivirus from L.crocea.The Siniperca chuatsi cell line mandarin fish fry cell line-1(MFF-1)derived from S.chuasti fry,which has been proved to be highly sensitive to multiple ISKNV-like and RSIV-like Megalocytivirus members,may also be a promising culture system for L.crocea Megalocytivirus.To establish methods for cell culture and classification of Megalocytivirus strains from L.crocea,and to make a comparison of their major capsid protein(mcp)genes,which will benefit for the studies of invasion and prevention of these unclassified Megalocytivirus,kidney tissue homogenates of Megalocytivirus-positive L.crocea juveniles(FD201807 and SA201808)were inoculated to the MFF-1 cell line and subcultured continuously.From the tissue homogenates and freeze-thawed infected cells,the virus genome was extracted.The virus mcp was then cloned and sequenced,and compared with the NCBI GenBank records of Megalocytivirus,and a 2018–2020 Fujian collection of 15 Megalocytivirus isolates from L.crocea as well.The results showed both two Megalocytivirus isolates caused typical cytopathic effects(CPE)on MFF-1 cells after 3 passages,of which the key features included cell rounding and shrinking,increased cell diopter,continuous cell detachment and particulates secretion with time.Hexagonal viral particles and empty capsids with a size of 130-150 nm were observed in the cytoplasm of infected MFF-1 cells under a transmission electron microscope(TEM).With the processing of virus subculture,the CPE interval of FD201807 shortened from 10 d to 3-5 d,while which of SA201808 remained 7-8 d.mcp gene revealed a 21-bases difference between SA201808 and FD201807.Phylogenetic and clustering analysis indicated that the mcp gene of SA201808 was high

关 键 词：大黄鱼 肿大细胞病毒 细胞培养 主要衣壳蛋白 

分 类 号：Q785[生物学—分子生物学] S942.2[农业科学—水产养殖]