欧亚类禽H1N1猪流感病毒抗原性变异关键氨基酸的鉴定与分析  

Identification of Key Amino Acids in the Antigenic Variation of Eurasian Avian-Like H1N1 Swine Influenza Viruses

作　　者：张乃心 许程志 杨玉莹 张亚萍 万云飞 乔传玲[1] 陈化兰[1] ZHANG NaiXin;XU ChengZhi;YANG YuYing;ZHANG YaPing;WAN YunFei;QIAO ChuanLing;CHEN HuaLan(Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences/State Key Laboratory of Animal Disease Control and Prevention,Harbin 150069)

机构地区：[1]中国农业科学院哈尔滨兽医研究所/动物疫病防控全国重点实验室,哈尔滨150069

出　　处：《中国农业科学》2023年第14期2828-2836,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31872472)。

摘　　要：【背景】流感病毒的抗原性主要是由病毒的表面糖蛋白HA决定的,前期利用欧亚类禽型H1N1猪流感病毒(EA H1N1 SIV)HA蛋白单克隆抗体(mAb)筛选抗原逃逸株发现,HA蛋白190、230和269位(H3编码)氨基酸的改变能够导致病毒发生抗原逃逸,并发现在新近分离的EA H1N1 SIV HA蛋白广泛存在这3个氨基酸的变异。【目的】进一步探索哪些氨基酸对病毒的抗原性具有关键作用,为流感的防控提供科学依据。【方法】选取一株病毒A/swine/Liaoning/SY72/2018(H1N1)SY72为模式病毒,建立该病毒的反向遗传系统(RGS),又以SY72为骨架,拯救含有HA基因190、230和269单点突变重组病毒;利用rSY72病毒制备灭活疫苗,分别免疫SPF鸡和非免疫猪制备其相应的血清,通过中和试验和血凝抑制(HI)试验分析病毒的抗原性,确定影响病毒抗原性的关键氨基酸位点;继而评估HA蛋白不同位点氨基酸的改变对病毒复制特性及受体结合特性等的影响。【结果】分析SY72病毒的HA氨基酸序列发现,该病毒HA蛋白分别含有190D、230M和269R。利用反向遗传技术,分别拯救了病毒rSY72及单点突变病毒rSY72HA/D190N、rSY72HA/M230I和rSY72HA/R269M;中和试验结果表明,与rSY72相比较,3株突变病毒均能够与两株mAbs发生一定反应;HI试验结果显示,与rSY72相比较,突变病毒rSY72HA/D190N与rSY72免疫鸡血清和猪血清的反应减弱,HI抗体效价分别出现了4倍和8倍的差异,而病毒rSY72HA/M230I和rSY72HA/R269M与两种血清的反应差异不明显,表明HA蛋白190位氨基酸改变对病毒的抗原性影响最为明显;病毒蚀斑测定及复制动力学研究结果发现D190N的氨基酸替换导致病毒rSY72HA/D190N在MDCK细胞上形成的蚀斑减小,而R269M的氨基酸替换导致病毒rSY72HA/R269M在MDCK细胞上的复制能力下降;3个位点的氨基酸替换均未影响病毒的受体结合特性。【结论】HA蛋白190位氨基酸对EA H1N1 SIV抗原性具有决定作用,269位�【Background】The antigenicity of influenza virus is mainly determined by the hemagglutinin(HA),a surface glycoprotein of the virus.Our previous study indicated that amino acid changes at positions 190,230 and 269(H3 numbering)of HA protein resulted in antigenic escape by using the monoclonal antibody(mAb)against the HA protein of Eurasian avian-like H1N1 swine influenza virus(EA H1N1 SIV).These three amino acid substitutions widely existed in the HA protein of the recently isolated EA H1N1 SIVs.【Objective】This study aimed to explore which amino acids played a key role in the antigenicity of the virus,and further provide scientific basis for the control of influenza.【Method】In this study,A/swine/Liaoning/SY72/2018(H1N1)(SY72)was selected as the model virus,and its reverse genetic system(RGS)was established.Then,using SY72 virus as backbone,three reassortant viruses were rescued by introducing the respective single amino acid mutation at position 190,230,and 269 into HA protein.Antisera were raised by inoculating the rSY72-inactivated vaccine into specific-pathogen-free(SPF)chickens and non-immunized pigs.The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition(HI)titers with mAbs and polyclonal sera raised against the rSY72 virus.Then their effects on viral replication capacities and receptor binding properties were further evaluated.【Result】Sequence analysis showed that the HA of SY72 virus carried 190D,230M,and 269R,respectively.The viruses,including rSY72,rSY72HA/D190N,rSY72HA/M230I,and rSY72HA/R269M,were rescued by RGS.The results of neutralization test showed that all the three mutant viruses could react with two mAbs,to some extent,compared with the rSY72 virus.The HI results indicated that the HI antibody titers of the rSY72HA/D190N reacted with the rSY72-immunized chicken and pig sera were 4-and 8-fold lower than those of the rSY72 virus with the respective sera.However,the rSY72HA/M230I and rSY72HA/R269M

关 键 词：猪流感病毒 HA蛋白 抗原性 

分 类 号：S852.65[农业科学—基础兽医学]