我国H9N2亚型禽流感病毒血凝素蛋白145和153位抗原位点变异分析

H9N2亚型禽流感病毒(AIV)血凝素蛋白(HA)易发生抗原漂移，但识别我国H9N2亚型AIV流行株抗原差异性的关键抗原位点还不清楚。选取两株血凝抑制(HI)效价高的H9N2亚型AIV单克隆抗体2E4与2D6对A/Chicken/Shanghai/F/1998(H9N2)毒株施加抗体压力制备单抗逃逸突变株，鉴定抗原位点；利用单抗对2017—2019年H9N2亚型AIV代表株进行HI抗原谱分析，确定抗原位点与基因分型关系；分析我国H9N2亚型AIV相应抗原位点的变异规律。结果显示：单抗2E4与2D6分别识别HA头部的145位和153位抗原位点；2017—2019年华东地区H9N2亚型AIV分离株根据HA基因遗传进化树可分为Group 1和Group 2两大分支，Group 1代表株与两株单抗反应性好，而Group 2代表株与两株单抗反应性差，序列分析发现已发生S145D (16/16)和D153G (15/16)的突变；通过对我国1996—2021年禽源H9N2亚型AIV HA蛋白位点自然突变率分析发现，含HA蛋白145D和153G的毒株占比分别上升至68%和66%，成为优势流行株。单抗2E4可用于当前H9N2亚型AIV的抗原分型，抗原位点145位是鉴别Group 1和Group 2抗原群差异的分子靶标之一。

Mutation Analysis of Antigen Sites 145 and 153 on Hemagglutinin of H9N2 Avian Influenza Virus in China

Antigenic drift on hemagglutinin (HA) protein of H9N2 subtype avian influenza virus (AIV) happens frequently, however, the key antigenic sites for identification of antigenic changes of H9N2 subtype AIV in China remain unclear. Two monoclonal antibodies(mAbs)2E4 and 2D6 with high hemagglutination inhibition (HI) titers against H9N2 subtype AIV were used, and antigenic sites were identified by selection of mAbs escape mutants with H9N2 AIV strain A/Chicken/Shanghai/F/1998. The antigenic profile of representative strains of H9N2 subtype AIV in 2017-2019 was investigated by HI assay using mAbs 2E4 or 2D6. The relationship between antigenic sites and genotype and variation characteristic of antigenic sites for H9N2 AIV in China were also analyzed. Two antigen sites 145 and 153 on the globe of HA protein of H9N2 AIV were identified by mAbs 2E4 or 2D6. H9N2 AIVs isolated in Eastern China in 2017-2019 were divided into two major branches:Group 1 and Group 2, according to the genetic distance of the HA gene. The representative viruses in Group 1 had good reactoin with two mAbs. In contrast, the viruses in Group 2 had poor reaction with two mAbs. Sequence analysis showed that S145D (16/16) and D153G (15/16) mutation happened in the viruses of Group 2. Natural mutation rate analysis showed that the rates of H9N2 subtype AIV strains isolated in China in 1996-2021 with 145D or 153G have risen to 68% or 66%, respectively, which become prevailing strains. The mAb 2E4 may be a tool for antigenic typing of the prevailing H9N2 subtype AIV, and antigenic site 145 may be one of molecular target to identify the difference between Group 1 and Group 2.