囊膜糖蛋白突变在H7N9禽流感病毒受体结合及跨种传播中的作用研究

血凝素(HA)与神经氨酸酶(NA)是流感病毒重要的囊膜糖蛋白，HA对细胞受体的结合与NA对受体的破坏特性之间存在功能匹配，在病毒入侵宿主过程中发挥关键作用。当前，高致病性H7N9禽流感病毒仍在我国部分地区持续存在；且近两年分离株在HA受体结合区和NA第二个唾液酸结合位点附近呈现部分位点规律性的氨基酸突变，并会导致糖基化修饰的改变。为了明确这些位点变异是否有利于H7N9病毒获得更强的结合人型受体的能力而促进其跨种传播，本项目拟开展如下研究：(1)构建系列HA和/或NA点突变H7N9重组病毒；(2)权重各位点变异对受体结合特性的影响，筛选差异性H7N9病毒作为配对模式病毒；(3)测定模式病毒的细胞生长曲线、热/低pH值稳定性、NA活性、红细胞解凝能力等体外特性及在鸡、小鼠、豚鼠体内的感染性；(4)借助转录组测序技术并结合内质网应激通路，探讨HA、NA位点变异引起H7N9病毒特性差异的相关机制。

Hemagglutinin (HA) and neuraminidase (NA) are important envelope glycoproteins of influenza virus. There is a functional match between the properties of binding to cell receptors by HA and the cutting of receptors by NA, which plays a key role in the process of virus invasion to host. Currently, the highly pathogenic H7N9 avian influenza virus still continues to exist in some regions of China, especially that the isolates in the past two years exhibit regular amino acid mutations at some sites near the receptor binding region of HA and the second sialic acid binding site of NA, which could also lead to changes in glycosylation modification. In order to determine whether such site mutations are conducive for the H7N9 virus to acquire stronger binding affinity to human-type receptors and thus promote its interspecies transmission, the following studies are planned in this project: (1) construction of a series of H7N9 reassortants with site mutation(s) in HA and/or NA; (2) weight the effect of those site variations on receptor binding property, and screen the differential H7N9 viruses as paired model viruses; (3) determine the in vitro characteristics including cell growth curve, heat/low pH stability, NA activity, hemagglutination-elution capacity, and the in vivo infectivity in chickens, mice and guinea pigs of the model H7N9 viruses; (4) explore the relevant mechanism of differential characteristics of H7N9 viruses caused by site mutations in HA and/or NA with transcriptome sequencing technology and endoplasmic reticulum stress pathway.