| 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 |
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| 引用本文： | 杨西西,池洪树,郑在予,刘晓东,罗潘潘,许斌福,陈秀霞,龚晖.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[J].水产学报,2023,47(9):099412-099412. |
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| 作者姓名： | 杨西西 池洪树 郑在予 刘晓东 罗潘潘 许斌福 陈秀霞 龚晖 |
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| 作者单位： | 福建省农业科学院生物技术研究所，福建 福州 350003;福建农林大学动物科学学院，福建 福州 350002;中山大学生命科学学院，广东 广州 510275 |
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| 基金项目： | 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) |
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| 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(large yellow croaker iridovirus, LYCIV) LYCIV-Zhoushan (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。
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| 关 键 词： | 大黄鱼 肿大细胞病毒 细胞培养 主要衣壳蛋白 |
| 收稿时间： | 2021/8/18 0:00:00 |
| 修稿时间： | 2021/10/26 0:00:00 |
Cell culture and major capsid protein (mcp) gene analysis of Megalocytivirus isolates from large yellow croaker (Larimichthys crocea) |
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| YANG Xixi,CHI Hongshu,ZHENG Zaiyu,LIU Xiaodong,LUO Panpan,XU Binfu,CHEN Xiuxi,GONG Hui.Cell culture and major capsid protein (mcp) gene analysis of Megalocytivirus isolates from large yellow croaker (Larimichthys crocea)[J].Journal of Fisheries of China,2023,47(9):099412-099412. |
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| Authors: | YANG Xixi CHI Hongshu ZHENG Zaiyu LIU Xiaodong LUO Panpan XU Binfu CHEN Xiuxi GONG Hui |
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| Institution: | 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 |
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| Abstract: | 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 highly homologous to LYCIV-Zhoushan (GenBank: MW139932. 1), while the homological identity of mcp gene between FD201807 and lateolabrax maculatus iridovirus (LMIV, GenBank: MH577517. 1) was up to 99. 93%. 12 of the total 15 Fujian L. crocea Megalocytivirus isolates were found clustered with SA201808, and the other 3 were clustered with FD201807. In this study, L. crocea Megalocytivirus were isolated through MFF-1 cell culture, indicating the differences among L. crocea Megalocytivirus strains, which could benefit for better understanding of this virus group. |
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| Keywords: | Larimichthys crocea Megalocytivirus virus culture major capsid protein |
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