H9N2亚型禽流感病毒聚合酶基因适应性变异对跨种间传播的影响及其作用机制

H9N2 avian influenza virus (AIV) is the gene donor for multi-subtype viruses infecting animal and humans. Therefore, it is crucial to explore the molecular basis and host mechanism for H9N2 AIV gene donor on the inter-species transmission of AIV. Our previous study demonstrated that the PB2-mediated tissue damage contribute to the increased virulence of a H9N2 AIV strain in mice; PA L672F contribute to aerosol-transmission of H9N2 AIV in chicken. However, it is unclear which viral and host factors contribute to inter-species caused by the genes. In this study, to further understand the pathogenesis of the H9N2 AIV polymerase genes (PB2, PB1 and PA) as donor, we will identify crucial residues in PB2, PB1 and PA proteins that influence pathogenicity of recombinant viruses, and the host proteins that interact with the proteins and explore the potential biological significance of the interaction. The following experiments will be performed. First, we will construct the recombinant and mutant H9N2 viruses. Second, we will characterize the replication level and transmission of recombinant and mutant AIVs in mammal cell, chicken and mice. Third, we will construct recombinant H7N9 viruses introducing the PB2, PB1 or PA gene with mutation influence AIV pathogenicity. Finally, cellular proteins interacting with PB2, PB1 or PA will be identified by LC-MS/MS and analyzed by bioinformatics software, and the interaction of the cellular protein and polymerase proteins will be confirmed by GST-pull down, co-immunoprecipitation and confocal microscopy assays. It will be carried out to identify the molecular mechanism and biological significance of crucial mutation in polymerase on the inter-species transmission. This study may accelerate our understanding of the inter-species of the influenza virus.

H9N2亚型禽流感病毒（AIV）与多个亚型流感病毒重组，使其获得跨种间传播的能力，急需了解其潜在的分子机制。前期研究发现禽源H9N2 PB2或PA单个位点变异介导的对小鼠致病性增强的机制，但潜在的跨越种间限制的机制还不清楚。本研究旨在挖掘聚合酶（PB2、PB1和PA）基因适应性变异影响跨种间传播的分子基础及宿主机制从而探索H9N2基因供体影响跨种间传播的分子机制。研究内容：（1）构建聚合酶基因及其突变基因真核表达质粒，并在不同基因型H9N2骨架上互换重组：rH9N2，权重变异位点对聚合酶活性的影响；（2）测定rH9N2对不同宿主致病力差异，确定跨种间传播的关键分子标记；（3）关键位点相关基因引入H7N9重组：rH7N9，验证其对致病性的影响；（4）质谱技术等筛选与rH7N9互作的宿主蛋白，用IPA等软件进行分析；共聚焦技术等对互作验证，进一步分析适应性变异位点影响AIV跨种间传播的机制。

1. S（G57）基因型H9N2 病毒PB2和M基因对其内部基因盒稳定性的影响机制研究.S（G57）基因型H9N2自2010年以后持续占据优势流行地位，与早期优势流行的H基因型相比，其PB2和M基因来源于G1-like，含有G1-like 来源PB2和M 基因比F/98-like PB2和M的内部基因盒更稳定，病毒复制水平也上升。S基因型H9N2病毒频繁为H7N9、H5N6和H10N3等新型重组病毒提供整套内部基因G1-like PB2增加了新型重组病毒H7N9和H5Nx的聚合酶活性和核聚集能力。2021年出现的新型H10N3无需提前适应即能在小鼠体内复制，甚至引起小鼠的死亡；PB2发生E627V的突变，感染小鼠鼻腔中的病毒滴度高，并可通过直接接触和气溶胶途径在豚鼠中传播。.2. S基因型H9N2 病毒适应性变异位点影响致病性的机制研究.H9N2病毒HA的受体结合位点通常发生Q226L和G228S突变，大多数毒株PB2基因627E，只有个别毒株PB2具有E627K突变。含有HA（Q226L，G228S）和PB2（E627K）突变的H9N2毒株对小鼠中的致病性仍然较低，这表明这些突变的存在不足以改变该菌株在哺乳动物中的毒力。HA受体结合位点198N在自然毒株中盛行，毒株PB2即使发生627K的突变，毒株仍然保持对哺乳动物的低致病性，当发生N198T的突变后，联合PB2 627K可以明显增强H9N2病毒对小鼠的致病性。.3. H9N2 亚型 AIV 对小鼠致病性的宿主因素分析.决定病毒对宿主致病力强弱的因素包括两个方面：一个是病毒本身的特性， 另一个是病毒感染后宿主所作出的应答。小鼠感染模型中肺脏转录组和蛋白质组联合测序分析，发现 HA 蛋白222-224 缺失使H9N2 毒株诱导更多的宿主基因和蛋白差异表达。缺失毒株诱导的差异基因/蛋白参与的免疫相关生物过程条目显著多于rSDKD1-WT 毒株；野生型毒株诱导的少数差异基因/蛋白仅参与 RIG-I-like 受体信号通路，而缺失株诱导的差异基因/蛋白参与了几乎所有重要的免疫相关信号通路。缺失株毒株可使大部分天然免疫相关蛋白/基因表达水平上调，个别下调。 综上所述 HA 蛋白222-224 缺失 突变使 H9N2 毒株诱导了更广泛以及更强烈的免疫反应，异常的免疫应答导致了严重的肺脏病理变化，因此增强了对小鼠的致病性。