当前，国内H5与H9亚型禽流感病毒的共流行，使两者极易发生自然重组，且自然界已出现内部基因完全来源于H9N2的新型重组H5病毒。为评估此类重组H5病毒的生物危害，明确何种H9N2的内部基因组合对于重组H5病毒具有更强的生存力，拟分别构建内部基因由PB2和M基因分属不同进化谱系的S和H基因型H9N2提供的重组H5病毒，开展如下研究：(1)将含2份不同PB2和M基因的10质粒共转染细胞进行病毒拯救，体外评价不同重组H5病毒的竞争优势；(2)测定细胞复制能力和体内感染能力的差异，明确不同PB2和M基因对重组H5病毒致病力的影响；(3)分别互换不同PB2和M节段的非编码区以构建嵌合型重组H5病毒，经细胞和动物感染实验，探讨流感病毒RNA非编码区影响重组H5病毒致病力的相关机制。本项目将为H9N2病毒频繁作为H7N9、H0N8等新型重组流感病毒内部基因供体的机制研究和人类新发流感的预警提供重要参考。

Currently, the endemicity of both H5 and H9 subtype avian influenza viruses in China has made natural reassortment between each other more easily. Urgently, novel reassorted H5 viruses harboring the whole cassette of internal genes from circulating H9N2 have already emerged naturally. In order to evaluate the biohazard of those H5 variants and to make sure which kind of H9N2 internal combination confers the optimal viral adaptability, we plan to construct H5 reassortants containing internal genes all from S or H genotype H9N2 with PB2 and M genes affiliating to different lineages for subsequent studies: (1) estimate the competitive advantages of distinct H5 reassortants in vitro, by virus rescue from cotransfection of 10 plasmids expressing a set of eight individual influenza A virus genes plus additional PB2 and M genes from different lineage; (2) determine the effect of distinct H9N2 lineages of PB2 and M genes on the pathogenic phenotype of the ressorted H5 viruses, by comparing viral infectivity on poultry and mammalian cells in vitro and on animals in vivo, respectively; (3) exchange the viral RNA (vRNA) non-coding regions between different lineages of PB2 and M segments to construct chimeric H5 reassortants accordingly, and explore the related mechanism of non-coding vRNA affecting the pathogenicity of H5 reassortants through infection experiments executed on cells and animals, respectively. The results of this project would provide important suggestions for the mechanism study that H9N2 continually donating all the internal genes to novel influenza reassortants such as H7N9 and H10N8, and the early warning of emerging human influenza.