为了研发用于预防传染性造血器官坏死病(infectious hematopoietic necrosis，IHN)灭活疫苗，实验以不同感染复数(multiplicity of infection，MOI)在胖头鱥上皮细胞(Epithelioma papulosum cyprinid，EPC)上对传染性造血器官坏死病毒(infectious hematopoietic necrosis virus，IHNV)进行连续传代培养，通过测定各代病毒滴度，结合病毒收获时间确定最佳增殖方案；采用不同浓度的β-丙内酯(β-propanolactone，BPL)在24 °C下灭活，经安全性实验验证后确定最佳灭活条件。以不同剂量腹腔注射免疫虹鳟，以磷酸盐缓冲溶液(PBS)为阴性对照组，通过检测攻毒后相对免疫保护率(relative percent survival，RPS)、免疫相关因子表达量及血清中和抗体效价来分析该疫苗的保护效果。结果显示，最佳增殖方案为以MOI为0.000 1接种，15 °C培养3 d；最佳灭活条件为以终浓度3.0 mmol/L的BPL将IHNV在24 °C下灭活24 h；以最佳免疫剂量10 μL/尾腹腔注射免疫虹鳟，免疫后7、21、45和60 d的相对免疫保护率分别为91.37%、84.28%、84.15%和47.5%，免疫后60 d时RPS显著低于其他时间点免疫组；免疫相关基因的实时荧光定量PCR(realtime quantitative PCR，RT-qPCR)结果显示,与阴性对照组相比，Mx-1和IFN-γ表达量在免疫后7、15和30 d脾脏和头肾中均显著上调，在第7天时达到最大值(5倍)；CD4和IgM表达量在免疫后15 d脾脏和头肾中均显著上调；在免疫后第30、45和60天虹鳟血清IHNV中和抗体效价依次为67.25、43.40和29.78，呈下降趋势且各组间差异显著。研究表明，该灭活疫苗可诱导虹鳟产生特异性免疫和非特异性免疫反应，对虹鳟具有显著的免疫保护作用。本研究为IHNV灭活疫苗研发提供参考。

Infectious hematopoietic necrosis disease (IHN) is an acute infectious viral disease that can cause sudden death of salmon. Vaccine immunization is the most effective way to prevent and control the disease. At present, there is no commercial vaccine to prevent the disease in China. The objective of the present study was to prepare an inactivated vaccine against IHN and evaluate its protective immunity in Oncorhynchus mykiss. In this study, infectious hematopoietic necrosis viruses (IHNV) were successively cultured on Epithelioma papulosum cyprinid (EPC) cells with different multiplicity of infection (MOI). The optimal proliferation pattern of IHNV on EPC cells was determined by measuring the titer of IHNV in each passage combined with the virus harvest time. IHNV was inactivated by β-propanolactone (BPL) at different final concentrations at 24 °C, and the inactivity was then verified in vitro and in vivo to determine the optimal inactivation condition. Inactivated IHNV prepared with the optimal inactivation protocol was intraperitoneally injected into O. mykiss [(10±2) g)] with different doses, and the protective effect of the inactivated vaccine was analyzed by detecting relative percent survival (RPS) after challenge, expression levels of immune-related factors and serum neutralizing antibody titers at different time post vaccination. It was shown that different proliferation patterns had some effects on the proliferation of IHNV on EPC. We chose MOI of 0.0001 as the best inoculation dose on EPC cells, and the virus was harvested on the 3rd day post inoculation at 15 °C. The in vivo and in vitro safety tests showed that the best inactivation condition was to inactivate IHNV at 24 °C for 24 h with the final concentration of 3.0 mmol/L BPL. 10 μL per fish was chosen as the optimal immunization dose, and more O. mykiss were immunized. The RPS was 91.37%, 84.28%, 84.15% and 47.5% at 7, 21, 45 and 60 d post immunization (d.p.i), respectively, and significant difference was observed on RPS between 60 d.p.i and other time points. Compared with the negative group, the expression levels of Mx-1 and IFN-γ were significantly up-regulated in spleen and head-kidney at 7, 15 and 30 d.p.i, and reached the maximum at 7 d.p.i (5 folds). The expressions of CD4 and IgM genes were significantly up-regulated in spleen and head-kidney at 15 d.p.i. In the detection of neutralizing antibody titer, the average neutralizing antibody titer in O. mykiss serum was 67.25, 43.40 and 29.78 at 30, 45 and 60 d.p.i respectively, with a decreasing trend and significant differences among different groups. The results indicated that the IHN-BPL inactivated vaccine developed in this study could induce specific and non-specific immune response in O. mykiss, and could provide significant immunoprotection, which will provide references for the development of inactivated vaccines against IHN.

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国家自然科学基金(31802345)；国家重点研发计划(2019YFE0115500)；中央公益性事业单位基本科研业务费专项(HSY2019M，2020GH05)；中国博士后科学基金(2018M630893，2019T12027)