| 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108.58~108.875 TCID50/mL，ISKNV滴度为107.38~107.625 TCID50/mL。F1、F5和F10代ISKNV按104 TCID50/mL、SCRV按106.5 TCID50/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1 300 bp的MCP基因和1 500 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。 |
| 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 |
| DOI： |
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| 基金项目: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） |
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| Virus seed and seed batches of mandarin fish ISKNV and SCRV bivalent inactivated vaccine |
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LUO Xia,FU Xiaozhe,LIN Qiang,et al
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| Abstract: |
| 【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 108.58-108.875 TCID50/mL for SCRV and 107.38－107.625 TCID50/mL for ISKNV.Mortalities were more than 90% when mandarin fish were challenged with ISKNV at a dose of 104 TCID50/mL or SCRV at a dose of 106.5TCID50/mL.The SCRV G gene with 1 500 bp in length and ISKNV MCP gene with 1 300 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.【Conclusion】Biological characteristics of the virus seed bacthes within 10 passages were stable and could be used for preparation and virulence test for mandarin fish ISKNV and SCRV bivalent inactivated vaccine. |
| Key words: mandarin fish virus disease infectious spleen and kidney necrosis virus（ISKNV） Siniperca chuatsi rhabdoviruses（SCRV） virus seed bank seed batches bivalent inactivated vaccine |
