摘要
大鳞大麻哈鱼胚胎细胞(Chinook salmon embryo cells,CHSE-214)和虹鳟性腺细胞(Rainbow trout gonad cells,RTG-2)是传染性胰脏坏死病毒(Infectious pancreatic necrosis virus,IPNV)检测的国家标准所推荐使用的鲑鳟鱼源细胞系。本研究选用24株IPNV毒株,以1000倍半数组织培养物死亡剂量(50%tissue culture infective dose,TCID_(50))分别接种于CHSE-214和RTG-2细胞,以细胞病变情况比较这两种细胞对IPNV的敏感性。将IPNV以10 TCID_(50)和100 TCID_(50)接种在筛选出的最敏感细胞系上,每天观察细胞的病变及变化,收获病毒培养物。利用实时荧光定量PCR(real-time quantitative PCR,RT-q PCR)及病毒滴度测定法分析IPNV在敏感细胞上的增殖状况。结果显示,接毒后7 d时,全部毒株均能在CHSE-214细胞上增殖,并导致细胞病变,而仅有8株IPNV能在RTG-2细胞上增殖,导致细胞病变,表明CHSE-214细胞对IPNV的敏感性高于RTG-2细胞。利用RT-q PCR和TCID_(50)法分析IPNV在CHSE-214上的增殖情况发现,随着接毒时间的增加,病毒量整体呈现先升高后降低的趋势,接毒后3 d时达到最大值。该增殖趋势均出现在不同接毒剂量组,除接毒后1 d时,其余各时间点10 TCID_(50)剂量组的病毒量均显著高于100 TCID_(50)剂量组(P<0.05)。本研究结果表明,CHSE-214细胞比RTG-2更适合用于IPNV的体外分离培养,且在多数细胞出现典型细胞病变而尚未完全脱落时病毒含量最高,此时适合收集病毒培养物进行病毒检测。本研究可为IPNV的检测提供科学指导。
Chinook salmon embryo(CHSE-214)cell line and rainbow trout gonad(RTG-2)cell line are salmonids cell lines that are recommended for detection of infectious pancreatic necrosis virus(IPNV)in Chinese national detection standard.Total of 24 IPNV isolates were selected and inoculated to CHSE-214 and RTG-2 at a high dosage of 1,000-fold 50%tissue culture infective dose(TCID_(50)),and cytopathic effect was observed each day to detect the sensitivity of the cell lines to IPNV to compare the sensitivity of the two cell lines to IPNV.Then IPNV was inoculated to the optimal sensitive cell lines at the dosage of 10 TCID_(50) and 100 TCID_(50),respectively,and the occurrence and change in cytopathic effect were monitored under a microscope every day.The virus cultured was harvested per day,and the propagation of IPNV was analyzed by using real-time quantitative PCR(RT-qPCR)and virus titer measurement.The sensitivity analysis revealed that within 7 days post inoculation,all the IPNV isolates multiplied and induced cytopathic effect on CHSE-214 cells,while only total 8 of them multiplied and induced cytopathic effect on RTG-2 cells indicating that CHSE-214 cells showed more sensitive to IPNV than RTG-2 cells did.RT-qPCR and TCID_(50) assay showed that the total amount of IPNV increased at first and then decreased with the increase in inoculation time,with the maximum on 3 days post inoculation.There was significantly higher virus content in 10 TCID_(50) dose group than that in 100 TCID_(50) dose group at each time points(P<0.05),excluding the 1 day post inoculation,though the proliferation trend in different dose groups.The RTG-2 cells were not shown to be suitable for the isolation and culture of IPNV,while CHSE-214 cells were more appropriate for the isolation and culture of IPNV in vitro.Moreover,it was the best time to collect virus culture for further detection when most cells showed typical cytopathic effect and have not yet shed completely,with the maximal virus content.The findings provided scientific guidance of the detection of IPNV.
作者
赵文闻
徐黎明
陈桂花
段凯越
赵景壮
任广明
邵轶智
张颖
卢彤岩
ZHAO Wenwen;XU Liming;CHEN Guihua;DUAN Kaiyue;ZHAO Jingzhuang;REN Guangming;SHAO Yizhi;ZHANG Ying;LU Tongyan(Dalian Ocean University,Dalian 116023,China;Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province,Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Harbin 150070,China;Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)
出处
《水产学杂志》
CAS
北大核心
2022年第1期1-8,共8页
Chinese Journal of Fisheries
基金
国家重点研发计划(2019YFD0900103)现代农业产业技术CARS-46
中央公益性事业单位基本科研业务费专项资金(HSY2019M和2020GH05)
国家自然科学基金(31802345)
中国博士后科学基金(2018M630893和2019T12027)
关键词
虹鳟
传染性胰脏坏死病毒
大鳞大麻哈鱼胚胎细胞
虹鳟性腺细胞
病毒载量
rainbow trout
infectious pancreatic necrosis virus(IPNV)
chinook salmon embryo cells(CHSE-214)
rainbow trout gonad cells(RTG-2)
viral load
作者简介
赵文闻(1996-)女,硕士研究生,研究方向为鱼类病毒学.E-mail:785147072@qq.com;通信作者:卢彤岩(1967-),女,研究员,从事鱼类病毒学研究.E-mail:lutongyan@hrfri.ac.cn
