李呈军一直从事流感病毒研究，尤其是在流感病毒变异及其机制研究方面取得重要进展。以第一作者或共同通讯作者在PNAS、JVI、TIB等杂志上发表SCI论文9篇，累计影响因子超过40，被SCI论文他引200多次。这些成果深化了对流感病毒的科学认知，在禽流感的风险预警和有效防控中发挥了重要作用。流感病毒通过基因突变和重配两种方式持续进化，不断产生出威胁动物和人类健康的新病毒。2013年我国出现人感染H7N9流感疫情，对人的致死率达到30%。H7N9病毒具有较高致死率的深层次原因是什么，病毒下一步在致病性和抗原性上将会如何变异是科学界亟需解决的重要命题。本项目拟开展H7N9流感病毒感染后对人产生较高致死率的分子机制研究，探索H7N9流感病毒演化为高致病性病毒的可能性，揭示H7N9病毒抗原变异的分子基础，为H7N9流感病毒的风险评估和防控策略的制定实施提供重要依据。

Chengjun Li has been working on influenza virus for more than 13 years, and has made important accomplishments especially in the field of influenza virus variations and the underlying mechanisms. He was the first author or co-corresponding author for 9 research articles published in peer reviewed journals such as the Proceedings of the National Academy of Sciences of the United States of America, Trends in Biotechnology and Journal of Virology in recent years, which were cited more than 200 times by other researchers. These accomplishments have deepened the scientific understanding of influenza viruses and played an important role in the risk warning, and prevention and control of avian influenza. Influenza viruses continually evolve through mutation and genetic reassortment in nature, resulting in the continuous emergence of new viruses that pose great threats for the health of animals and humans. In February and March, 2013, a previously unidentified novel A (H7N9) influenza virus caused human infections in China, leading to a lethality rate of approximately 30%. So far, it is still unknown what the underlying reason is for the considerable lethality rate of H7N9 virus and how H7N9 virus will further evolve with respect to its pathogenicity and antigenicity. This project will study the molecular mechanism responsible for the considerable lethality, the possibility of evolving into highly pathogenic virus, and the molecular basis of antigenic drift for H7N9 influenza virus. The implementation of this project will help to understand the risk potential of H7N9 influenza virus and provide a theoretical basis for the development and implementation of prevention and control strategies.

自2013年我国首次发生人感染H7N9流感疫情以来，截至目前已造成1500多人感染，死亡率接近40%，对公共卫生安全构成重大威胁。H7N9流感病毒家禽分离株PB2蛋白均为627E，但在感染人后70%以上毒株获得PB2蛋白E627K突变从而导致致病力增强。因此，本项目围绕H7N9流感病毒感染人后获得PB2蛋白E627K突变的分子机制进行了一系列研究。通过将2013年分离的H7N9禽流感病毒及早期分离的H9N2禽流感病毒在小鼠体内传代，我们发现A/pigeon/Shanghai/S1421/2013(S1421, H7N9)仅在小鼠体内传代一次其 PB2蛋白即发生E627K突变，而 A/chicken/Henan/5/1998(HeN5, H9N2)在小鼠体内传代四次其 PB2蛋白依然未发生突变。为此我们选择S1421和HeN5两株病毒作为模式病毒，在S1421背景下，通过反向遗传操作系统拯救一系列单基因替换病毒。小鼠实验结果发现包含HeN5-PA基因的重组病毒S1421/HeN5-PA在小鼠上连续传代两次，其PB2蛋白仍未发生任何突变，即PA蛋白在H7N9禽流感病毒PB2蛋白获得627K突变过程中发挥重要作用。我们将两株病毒16种不同RNP组合利用双荧光素酶报告系统进行聚合酶活性检测，发现HeN5-PA蛋白的存在可以显著提高RNP的聚合酶活性。随后我们构建了一系列PA嵌合突变体及单点/多点突变体，小鼠实验证明在S1421背景下，PA蛋白上三个氨基酸142R/147V/182L（PAmut）协同作用对于H7N9病毒感染小鼠后保持PB2 蛋白的稳定发挥关键作用。进一步研究发现HeN5-PA蛋白 N端及PAmut可以显著提高H7N9流感病毒RNP的聚合酶活性。HeN5-PA蛋白与S1421-PA蛋白相比，核酸内切酶活性减弱，而且HeN5PA及PAmut的存在可以显著提高H7N9流感病毒感染细胞后病毒基因组的复制及转录水平。此外，HeN5-PA 可以影响病毒RNP复合体蛋白同宿主因子的结合，以及影响RNP复合体对宿主因子的依赖程度。本项目发现了驱动PB2蛋白产生致病力增强突变E627K的内在病毒因素和宿主因素，深化了我们对禽流感病毒感染哺乳动物和人类宿主后产生适应性变异而导致致病力增强机制的认知，为人类新发流感的预警和防控提供了参考依据。