H9N2不同进化谱系PB2和M基因对H5亚型重组禽流感病毒致病力的作用及机制研究

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.

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。

H5与H9亚型禽流感病毒在我国长期的共同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及其相关机制，开展了该项目研究。通过对我国H9N2禽流感病毒的流行动态分析，明确了S基因型（H9N2/S）为近年来国内鸡群中流行的优势基因型，其重要的分子进化特征是在前期流行的H基因型（H9N2/H）骨架上将PB2和M基因由F/98-like替换为G1-like进化谱系。以同时含有G1-like与F/98-like PB2和M基因的10质粒转染体系进行重组H5N6病毒的竞争性拯救，表明G1-like PB2相较于G1-like M或F/98-like PB2基因均更具竞争优势且PB2基因的作用更占主导；进而通过在体外测定真核表达的PB2 Cap-binding结构域（PB2cap）与m7GTP的帽结合能力表明G1-like PB2cap显著强于F/98-like PB2cap。通过将不同流行分支H5病毒的HA和NA基因与来自优势流行H9N2/S病毒的PB2、PB1、PA、NP、M和NS基因进行定向的“2+6”基因重配构建重组H5/S病毒，表明H9N2/S病毒内部基因盒的引入显著致弱重组H5/S病毒对SPF鸡和BLAB/c小鼠的致病力。进一步地，在重组H5/S病毒骨架上分别单替换或同时替换入F/98-like PB2、M基因构建重组H5/PB2、H5/M、H5/H病毒，表明无论是在哺乳类细胞MDCK上还是哺乳类动物模型BALB/c小鼠体内，携带G1-like PB2、M基因的重组H5病毒相较于携带F/98-like PB2、M基因的重组H5病毒均具有显著更强的毒力和复制能力，且G1-like PB2基因的作用更为关键。并且，基于G1-like与F/98-like PB2基因vRNA包装信号序列的差异，通过构建含有嵌合型PB2基因的重组H9N2病毒，表明G1-like PB2基因3’、5’端包装信号可明显提高F/98-like PB2基因的RNA合成和蛋白表达水平，以及重组H9N2病毒在MDCK和CEF细胞上的复制能力。该研究为深入探讨H9N2/S病毒作为H7N9、H5N6等新型重组流感病毒内部基因供体的生存优势及相关机制奠定了重要的理论基础。