H9N2 avian influenza virus has spread worldwide, and vaccination with an inactivated virus is currently the major prevention method in China. To further understand the effect of the selection pressure from antibodies on the evolution of H9N2 avian influenza virus, F/98 (A/Chicken/Shanghai/F/98), which is the vaccine representative of H9N2 avian influenza virus in East China, was used for serial passaging for 20 generations in chickens with and without vaccination. After plaque purification from trachea and lung tissues, 390 quasispecies were obtained. The second-generation quasispecies under the selection pressure of vaccine antibodies had undergone 100% antigen variation, while after passaging to the fifth generation, only 30–40% of the quasispecies displayed antigen variation when there was no selection pressure of vaccine antibodies, implying that the selection pressure of vaccine antibodies promotes the antigen variation of F/98. We found for the first time that there were three mutation hotspots in the HA genes of the quasispecies under the selection pressure of vaccine antibodies, which were K131R, A168T, and N201D. Moreover, under the selection pressure of vaccine antibodies, 10 amino acids (67–76) of the NA protein of all quasispecies were deleted, and PB2 of the quasispecies had undergone a high-frequency R355K mutation. However, without selection pressure of vaccine antibodies, NP had undergone two high-frequency mutations, namely, V186I and L466I, and a high-frequency mutation of L77I appeared in the NS gene. This result shows that the vaccine antibody selection pressure could control and regulate gene variation of the F/98 virus. Compared to that of the parental virus F/98, the EID₅₀ of the twentieth passaged virus under the selection pressure of vaccine antibodies did not change, while the EID₅₀ of the twentieth passaged virus without selection pressure of vaccine antibodies was significantly enhanced by 794 times. Furthermore, the twentieth passaged virus with selection pressure from vaccine antibodies lost its lethal ability in embryonated chicken eggs, whereas the EID₅₀ of the twentieth passaged virus without selection pressure of vaccine antibodies increased to 6.3 times that of the F/98 strain. All the above results show that the selection pressure of vaccine antibodies promotes the antigen variation of H9N2 avian influenza virus and plays a role in regulating and controlling gene mutation of H9N2 avian influenza virus.

H9N2禽流感病毒已在​​全球范围内传播，灭活病毒疫苗接种是目前我国主要的预防方法。为了进一步了解抗体选择压力对H9N2禽流感病毒进化的影响，对华东地区H9N2禽流感病毒的代表疫苗F/98（A/Chicken/Shanghai/F/98）进行了研究。用于在接种疫苗和未接种疫苗的鸡中连续传代 20 代。从气管和肺组织中纯化噬菌斑后，获得了 390 个准种。疫苗抗体选择压力下的第二代准种发生了100%的抗原变异，而传至第五代后，在没有疫苗抗体选择压力的情况下，只有30%~40%的准种出现了抗原变异，这意味着疫苗抗体的选择压力促进了F/98的抗原变异。我们首次发现在疫苗抗体选择压力下准种的HA基因存在3个突变热点，分别是K131R、A168T和N201D。而且，在疫苗抗体的选择压力下，所有准种的NA蛋白有10个氨基酸（67-76）被删除，准种的PB2发生了高频的R355K突变。然而，在没有疫苗抗体选择压力的情况下，NP发生了两次高频突变，即V186I和L466I，并且NS基因中出现了L77I高频突变。这一结果表明疫苗抗体选择压力可以控制和调节F/98病毒的基因变异。与亲本病毒F/98相比，在疫苗抗体选择压力下的第20代病毒的EID ₅₀没有变化，而在没有疫苗抗体选择压力的情况下第20代病毒的EID ₅₀显着增强794次。此外，具有疫苗抗体选择压力的第20代病毒失去了对含胚鸡蛋的致死能力，而没有疫苗抗体选择压力的第20代病毒的EID ₅₀增加至F/98毒株的6.3倍。以上结果表明，疫苗抗体的选择压力促进了H9N2禽流感病毒的抗原变异，对H9N2禽流感病毒的基因突变起到调控作用。