H9N2亚型禽流感病毒气源性传染及跨种传播分子机制研究

The low pathogenic H9N2 AI（avian influenza） prevail in our country's poultry flocks. It has been leading not only to enormous economic losses to the poultry industry, but also infected human repeatly. The amino acids in NA protein region of H9N2 AIVs which owed different transmissibility were mutated and viruses were rescued using influenza reverse genetic system of eight plasmids. The transmissibility in chickens of every virus was studied in aerosol transmission and infection model in order to indicate these mutations affecting virus airborne transmissibility. Bioinformatics technology was used to simulate the three-dimensional structure of the mutant NA proteins before and after obtaining, in-depth analysis of the relationship between NA protein three-dimensional structure and the aerosol transmission. To track and assess the risk of the virus after the adaption in the mammalian, H9N2 AIV was serially passaged in guinea pigs and the whole gene sequences of passage virus were analyzed to reveal the influence of passage on virus gene mutation. The structural differences of HA and PB2 proteins before and after passage of the were simulated to understand the impact of mutant protein structure to the cross-species transmission. Genes of parent virus were mutated using site-directed mutagenesis according to the results of passage study to induce the same characteristics as passage virus. Transmissibility of mutated virus was studied in guinea pigs to ensure which were the important amino acids affecting H9N2 AIV transmission and to access its risk of infection in human.

H9N2禽流感在我国家禽群普遍存在，不仅对养禽业造成了严重危害，而且常感染人。本研究采用8质粒反向遗传操作系统，对气源性传染能力不同的H9N2 AIV（Avian Influenza Virus）毒株NA蛋白氨基酸位点突变，拯救病毒株。将SPF鸡带入气溶胶发生、传播和感染模型，验证AIV突变株的气源性传染力，阐明氨基酸位点的突变对其传染的影响。应用生物信息学技术，模拟AIV突变前后NA蛋白三维结构，分析其与气溶胶传播的关系。为评估AIV在哺乳动物体适应后的危害，将AIV在豚鼠体内传代后，对其全基因序列分析，揭示其适应后的变异，展示传代前后模拟的HA和PB2蛋白结构差异，揭示变异株蛋白结构对其跨种传播的影响。据此结果，反向遗传操作对原代毒株基因定点突变，使人工突变毒株与传代株具有相同特性，再带入豚鼠模型予以验证，确定H9N2 AIV在哺乳动物间传播的重要的基因位点，评估其在人际间的传染风险。

H9N2禽流感在我国家禽群普遍存在，不仅对养禽业造成了严重危害，而且常感染人，对公共卫生形成威胁。本研究采用8质粒反向遗传操作系统，对气源性传染能力不同的H9N2 AIV（Avian Influenza Virus）毒株NA蛋白氨基酸位点突变，拯救病毒株，并验证其气溶胶发生、传播和感染过程及机制。验证AIV突变株的气源性传染力，阐明氨基酸位点的突变对其传染的影响；应用生物信息学技术，模拟AIV突变前后NA蛋白三维结构，分析其与气溶胶传播的关系，揭示了跨种传染的机制；为评估AIV在哺乳动物体适应后的危害，将AIV在豚鼠体内传代后，对其全基因序列分析，揭示其适应后的变异，展示传代前后模拟的HA和PB2蛋白结构差异，阐明变异株蛋白结构对其跨种传播的影响。.H9N2 AIV SD01株能够感染豚鼠，通过直接接触的方式感染接触组豚鼠，检测到了病毒气溶胶，但气溶胶被动感染组未发生感染。进一步研究证实，其豚鼠产生的气溶胶的量不足以导致传染。揭示了该亚型禽流感病毒危害性增强，获得了跨种传染的能力，能够引起哺乳动物和人的感染；具备了形成气溶胶的能力，传播扩散的风险增大。.在此基础上，反向遗传操作对原代毒株基因定点突变，使人工突变毒株与传代株具有相同特性，再带入豚鼠模型予以验证，确定H9N2 AIV在哺乳动物间传播的重要的基因位点，评估其在人际间的传染风险。结果表明，将SD01病毒NA的四个位点（D368E, S370L, E313K 和 G381D)）进行突变，发现突变毒株在鸡呼吸道的复制能力降低，病毒NA的活性下降，同时病毒在鸡之间气源性传播的能力降低。.将SD01病毒的 PA片段替换H1N1的PA后，重组毒株rSD01-PA仍能对家禽感染致病，且通过直接接触感染接触组家禽。然而，rSD01-PA失去了在家禽间气溶胶传播的能力；rSD01-PA仍能感染豚鼠，并通过直接接触方式感染接触组豚鼠。但气溶胶感染失败，说明PA片段的替换没有影响毒株跨种传染和在哺乳动物间气源性传播的能力，或影响较小。本研究对评估该亚型流感对哺乳动物及人危害风险及大范围流行的趋势，采取合适的防控措施，减少畜牧业的损失和公共卫生风险具有重要指导意义。