Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China

H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010–2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605–4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.

H9N2亚型禽流感病毒（AIVs）对全球家禽养殖业构成日益严峻的威胁，并具备大流行潜力。自1998年以来，疫苗接种一直是我国防控H9N2亚型禽流感的主要策略，但疫苗效力持续受到基因和/或抗原变异株出现的挑战。本研究分析了我国H9N2亚型禽流感病毒的基因与抗原特性，分析序列涵盖2010-2020年间70株家禽源H9N2分离株的血凝素（hemagglutinin，HA）基因序列、7358条来自在线数据库的HA序列，以及15株早期参考毒株的HA序列。基于HA基因的贝叶斯分析显示，2012年4月出现了一个新命名的进化分支16，该分支自2013年起在我国流行并与进化分支15共同循环传播。进化分支16毒株与进化分支15毒株的交叉反应活性有所降低。抗原制图（Antigenic Cartography）分析显示，代表性毒株可分为3个抗原群，分别命名为Group1、Group2和Group3；其中Group3中的绝大多数毒株（15/17，88.2%）均来自进化分支16，而Group2中的大部分毒株（26/29，89.7%）则来自进化分支15。Group3与Group2之间的平均距离为4.079（95%置信区间：3.605~4.554），表明进化分支16的出现伴随着病毒抗原特性的显著转变。基因分析显示，主要位于抗原位点的11个协同进化氨基酸替换，与进化分支15和16之间的抗原差异相关。本研究结果凸显了H9N2亚型禽流感病毒基因进化的复杂性，并为H9N2亚型禽流感病毒新兴进化分支16的遗传基础与抗原特性研究提供了分析框架。