鳜鱼传染性脾肾坏死病和弹状病毒病二联灭活疫苗毒种及种子批的研究  被引量:2

Virus seed and seed batches of mandarin fish ISKNV and SCRV bivalent inactivated vaccine

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作  者:罗霞[1] 付小哲[1] 林强[1] 刘礼辉[1] 牛银杰 梁红茹[1] 李宁求[1] LUO Xia;FU Xiaozhe;LIN Qiang;LIU Lihui;NIU Yinjie;LIANG Hongru;LI Ningqiu(Key Laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Pearl River Fishery Research Institute,Chinese Academy of Fishery Sciences,Guangzhou,Guangdong 510380,China)

机构地区:[1]中国水产科学研究院珠江水产研究所,农业农村部渔用药物创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380

出  处:《西北农林科技大学学报(自然科学版)》2022年第1期1-9,共9页Journal of Northwest A&F University(Natural Science Edition)

基  金:国家重点研发计划资助项目(2019YFD0900103);中国水产科学研究院基本科研业务费项目(2020XT0402);广东省农业产业技术体系创新团队项目(2019KJ141)。

摘  要:【目的】建立鳜鱼传染性脾肾坏死病毒(ISKNV)和弹状病毒(SCRV)二联灭活疫苗毒种库及种子批。【方法】以ISKNV-QY0910株和SCRV-GM1503株为原始毒种,扩繁10代(F1~F10)后检测各代病毒对鳜鱼脑组织细胞系(CPB)的致病性及其滴度,并检测其对鳜鱼的毒力。PCR扩增ISKNV的主衣壳蛋白(MCP)基因、RT-PCR法扩增SCRV G蛋白基因序列,检测ISKNV和SCRV毒种的特异性。对F1、F5和F10代ISKNV、SCRV毒种进行细菌、霉菌、支原体及鳜鱼蛙病毒(RANA)和神经坏死病毒(NNV)检测,检验毒种的纯粹性。分别将F1、F5和F10代ISKNV、SCRV病毒液用甲醛灭活后制备灭活疫苗,免疫鳜鱼21 d后攻毒,连续观察14 d,待鱼体稳定后统计死亡率并计算免疫保护率(RPS)。将ISKNV和SCRV毒种湿毒保存于-80℃冰箱,每6个月取3支检测病毒滴度,以确定毒种的保存期。【结果】各代次毒株感染CPB细胞后产生的CPE形态及病变速度基本相同,SCRV滴度为10^(8.58)~10^(8.875) TCID_(50)/mL,ISKNV滴度为10^(7.38)~10^(7.625) TCID_(50)/mL。F1、F5和F10代ISKNV按10^(4) TCID_(50)/mL、SCRV按10^(6.5) TCID_(50)/mL对鳜鱼攻毒(每尾0.1 mL),致死率均超过90%。ISKNV和SCRV各代次毒种可分别扩增出1300 bp的MCP基因和1500 bp的G基因,特异性良好。ISKNV、SCRV毒种无细菌、霉菌、支原体及鳜鱼蛙病毒和神经坏死病毒污染。各代次病毒灭活疫苗对鳜鱼安全有效,免疫保护率ISKNV可达92%以上,SCRV可达84%以上;湿毒-80℃保存,保存期为36个月。【结论】10代以内ISKNV和SCRV的生物学特性稳定,可用于鳜鱼ISKNV和SCRV二联灭活疫苗的制备与检验。【Objective】This study established virus seed bank and seed batches of the mandarin fish ISKNV and SCRV bivalent inactivated vaccine.【Method】ISKNV-QY0910 strain and SCRV-GM1503 strain were used as the original virus seed.After propagation from F1 to F10 for ten passages,viral pathogenicity to CPB cells,viral titer and virulence against mandarin fish were analyzed.To determine specificity of the viruses,MCP gene for ISKNV and G gene for SCRV were amplified by PCR and RT-PCR,respectively.Purity of the virus seed was determined by detecting bacteria,fungi,mycoplasma,RANA and NNV.Inactivated vaccine was prepared with ISKNV and SCRV at passages 1,5 and 10 using formaldehyde inactivation.Twenty-one days after immunization with the vaccine,mandarin fish were challenged with ISKNV and SCRV and observed for 14 days.Meanwhile,relative percentage survival(RPS)was calculated to analyze the efficacy based on mortality of each group.For storage life study,three tubes of virus seeds stored at-80℃were detected for variation viral titer every six months.【Result】CPE morphology and lesion speed in CPB cells were similar among different passage viruses.CPB cells were highly susceptible to different passage viruses,yielding a viral titer of 10^(8.58)-10^(8.875) TCID_(50)/mL for SCRV and 10^(7.38)-10^(7.625) TCID_(50)/mL for ISKNV.Mortalities were more than 90%when mandarin fish were challenged with ISKNV at a dose of 10^(4) TCID_(50)/mL or SCRV at a dose of 10^(6.5) TCID_(50)/mL.The SCRV G gene with 1500 bp in length and ISKNV MCP gene with 1300 bp in length of different passages were sequenced,and they shared high identity with the homology of 100%to the reference sequences.Purity test and PCR detection demonstrated that there were no bacteria,fungi,mycoplasma,RANA,and NNV contamination.Vaccines prepared with different passage viruses were safe to mandarin fish and provided high RPS of more than 92%against ISKNV and more than 84%against SCRV.The storage life of the humid virus at-80℃was more than 36 months.【Conclusi

关 键 词:鳜鱼病毒病 传染性脾肾坏死病毒(ISKNV) 弹状病毒(SCRV) 毒种库 种子批 二联灭活疫苗 

分 类 号:S941.41[农业科学—水产养殖]

 

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