H9N2 AIV灭活疫苗免疫SPF鸡攻毒后肺组织免疫相关基因表达变化研究

Study on the changes in the expression of immune related genes in the lung of SPF chickens immunized with H9N2 AIV inactivated vaccine and followed by viral infection

[目的]本试验旨在探究H9N2亚型禽流感病毒攻击对H9灭活疫苗免疫SPF鸡呼吸系统的影响。[方法]选用36只3周龄SPF鸡,随机分为对照组(Con)、攻毒组(Flu)、免疫后攻毒组(Vac+Flu)。以每只0.4 mL剂量免疫接种H9灭活疫苗,3周后以10~6 EID_(50)的病毒量进行攻毒,采集拭子、血清、气管、肺组织等样品,通过血凝抑制(HI)试验、RT-PCR、荧光定量PCR(qPCR)等方法检测血清中HI抗体滴度、肺脏流感病毒M基因拷贝数以及免疫相关基因CD4、CD8、GATA3、T-bet、IL-4、IL-13、TNF-α、IFN-γ、Perforin、Granzyme、FasL、Fas等的表达量;并利用HE染色、免疫组织化学的方法观察气管、肺脏的病理变化及病毒特异性抗原NP蛋白的分布特征。[结果]HI试验结果显示,SPF鸡疫苗免疫后可产生较高的H9N2 AIV抗体效价。免疫保护试验和RT-PCR结果显示,SPF鸡疫苗免疫后攻毒仍能检测到机体排毒和流感病毒M基因在肺脏组织中的表达。HE染色和免疫组化结果显示,接种H9N2 AIV灭活疫苗后能够明显减轻SPF鸡气管和肺脏的病理损伤,降低气管、肺脏中的病毒载量。荧光定量PCR结果显示,接种H9N2 AIV灭活疫苗后能够提高SPF鸡肺脏中Th1、Th2细胞因子分泌水平,促进穿孔素/颗粒酶途径相关基因表达进而增强肺脏中细胞毒性反应。[结论]H9N2疫苗免疫鸡感染H9病毒后虽然仍存在排毒现象,但其抗病毒免疫系统活跃、其主要器官病毒载量降低,建议按照流感疫苗免疫程序进行免疫接种,提高对流感的防控水平。

[Objectives]The aim of this experiment was to explore the effect of H9N2 subtype avian influenza virus attack on the respiratory system of SPF chicken immunized with H9 inactivated vaccine.[Methods]36 SPF chickens were selected and randomly divided into the control group(Con),the challenge group(Flu)and the immunization followed challenge group(Vac+Flu).Chickens were immunized with H9 inactivated vaccine at a dose of 0.4 mL per chicken,and after 3 weeks,they were challenged at a dose of 10~6 EID_(50) of virus.The swabs,serum,trachea,lung and other samples were collected.Hemagglutination inhibition(HI)antibody titer,the genomes copies of influenza virus M gene and the expression levels of immune related genes CD4,CD8,GATA3,T-bet,IL-4,IL-13,TNF-α,IFN-γ,Perforin,Granzyme,FasL,Fas were separately detected by HI assay,RT-PCR and quantitative PCR(qPCR).And the pathological changes and distribution of virus specific antigen NP protein of trachea and lung were individually observed by hematoxylin-eosin staining and immunohistochemistry.[Results]The results of HI assay showed that SPF chicken could produce high H9N2 AIV antibody titer after immunization.The results of immune protection test and RT-PCR showed that the virus shedding and the expression of influenza virus M gene in lung tissue could still be detected after SPF chickens were immunized with vaccine and followed by viral infection.The result of hematoxylin-eosin staining and immunohistochemistry showed that inoculating the H9N2 AIV inactivated vaccine significantly alleviated the pathological damage of trachea and lung and significantly reduced the viral load of trachea and lung.Quantitative PCR showed that inoculating the H9N2 AIV inactivated vaccine significantly up-regulated the secretion of Th1 and Th2 cytokines and promoted the expression of perforin/granzyme pathway related genes to enhance cytotoxicity in the lung.[Conclusions]Although chickens inoculated with H9N2 vaccine were infected with H9 virus,there was still a phenomenon of virus shedding.Their antiviral immune system was active and the viral load of main organs was reduced.It is recommended to carry out vaccination according to the influenza vaccine immunization procedure to improve the level of influenza prevention.