PA-X 基因C末端氨基酸的缺失增强H5N6亚型流感病毒致病性的机制研究

We have found that after four years continued epidemics and spread, the pathogenicity of H5N6 subtype influenza viruses is significantly enhanced. What is the mechanism led to the enhanced pathogenicity of H5N6 influenza viruses? We sequenced all these H5N6 influenza viruses in our hands and found that these contemporary isolates had C-terminal twenty amino acids deletion in the PA-X gene after four years gradually variation. Furthermore, we found that the replication ability and infectivity of those mutant viruses in ex vivo human lung tissues were attenuated by using reverse genetics to repair the C-terminal twenty amino acids deletion in the PA-X gene. Based on these pre-experimental results, we propose a new mechanism closely related to the enhanced pathogenicity of H5N6 influenza viruses: Deletion of C-terminal amino acids of PA-X gene might lead to the enhanced pathogenicity of H5N6 influenza viruses. In this study, we would use reverse genetics to artificial construct series of H5N6 PA-X gene deletion and extension variants and use these variants to infect cell lines and ferrets to evaluate biological characteristics in vitro and pathogenicity and transmissibility in vivo. This study would help us to clarify new mechanism involving enhancement of pathogenicity and transmissibility H5N6 influenza viruses and provide molecular basis for exploring the cross-species of H5N6 influenza viruses.

我们已经发现：经过4年的流行和传播，H5N6亚型流感病毒的致病性明显增强。那么，导致H5N6病毒致病性增强的机制是什么？对H5N6病毒进行全基因测序和比对后发现，经过4年的逐渐变异，H5N6病毒在PA-X基因C末端发生了20个氨基酸的缺失。近一步实验发现：当用反向遗传学技术人为补齐H5N6病毒PA-X基因C末端的20个氨基酸缺失后，病毒对离体培养的人肺组织的复制能力和感染性反而减弱。为此，我们提出了导致H5N6病毒致病性增强的新机制，即：H5N6病毒PA-X基因C末端氨基酸的缺失可导致病毒的致病性明显增强。为此，本研究拟通过反向遗传学技术，人工构建系列H5N6 PA-X基因缺失和延长毒株，并用这些毒株进行体外细胞生物学及动物的致病性及传播性研究，阐明导致H5N6病毒致病性明显增强的新机制，此研究结果将为探索H5N6病毒的跨种属传播提供分子学基础。

近年来，H5N6亚型流感病毒在我国家禽中广泛流行传播并时常感染人，对公众健康构成直接威胁。陆地小种群（鹌鹑、鹧鸪等）宿主作为流感病毒“基因混合器”在流感病毒生态系统中的作用也日益受到关注。为了了解和掌握H5N6亚型流感病毒在陆地小种群中的流行情况、病毒进化和重组规律及病毒的生物学特性，我们开展了此项研究。.通过近2年的流行病学调查，我们发现H5N6亚型流感病毒在华南地区陆地小种群宿主鹌鹑或鹧鸪中有少量流行，且病毒主要在宿主的支气管进行复制，增加了病毒通过宿主呼吸道感染人的可能性。对病毒的遗传进化分析发现，陆地小种群宿主中的H5N6亚型流感病毒是水禽鹅中的H5N6病毒通过跨种属传播进入鹌鹑且经历了较长的进化时间形成的。病毒的受体结合实验提示病毒的受体结合特性以结合α-2，3 唾液酸为主，仍保持禽流感病毒的受体结合特性。用小鼠进行的病毒致病性和传播性研究中发现，该鹌鹑来源的H5N6亚型流感病毒对小鼠的感染性和致病性较弱，引发炎症反应的部位主要在支气管和肺部，且炎症反应相对轻微。.因此，本项研究将为探索和预防H5N6亚型流感病毒的跨种属传播提供更多的科学依据。