猪流感H1N1病毒逃避宿主免疫的分子机制研究

The H1N1 influenza A virus is one of the important causative agents of swine influenza. The H1N1 virus not only causes outbreaks in pigs but also poses a significant threat to public health. Our previous analysis of whole genome sequencing and pathological study in mouse identified a number of reassortments and mutations occurred in H1N1 swine influenza virus isolates, leading to dramatic alterations in virulence and pathogenicity of the isolates. This study will be focused on investigation of the antigenicity and immunogenicity of these H1N1 viruses, aiming at determining the molecular mechanism of difference among these strains in terms of antigenicity and immunogenicity. Toward this end, two H1N1 swine influenza A viruses will be selected to generate a number of infectious viruses bearing various fragment(s) for reassortment using reverse genetics. The impact of the inserted fragment(s) on the host immune response will be analyzed and compared to determine the role of the fragment(s) in host immune evasion of the recombinant viruses. Meanwhile, a representative H1N1 swine influenza isolate will be used to generate a series of infectious viruses in which a variety of mutations are introduced in the gene. The level of neutralizing antibody and cytokines including B and T lymphocytes induced by the wild-type and mutant viruses will be compared and investigated. Finally, the functional role of amino acids examined will be addressed. This study would determine the molecular mechanism by which H1N1 swine influenza A virus uses to evade the host immune response and would provide the molecular basis for the development of novel vaccines against H1N1 swine influenza.

H1Nl亚型猪流感病毒不仅引起猪发病和死亡，而且会引发人流感的爆发和流行，具有重要的公共卫生学意义。前期工作中，我们发现很多分离株均发生了基因重组和氨基酸突变，导致毒株的致病力存在明显差异。本研究旨在通过对H1N1亚型猪流感病毒分离株的抗原性和免疫原性进行系统研究，阐明分离株之间在抗原性和免疫原性上存在差异的分子机理；从中选取两株代表性分离株进行片段重排，通过所建立的流感病毒反向遗传操作技术拯救感染性人工重排病毒，然后比较和分析不同片段对宿主免疫反应的影响，进而分析病毒基因片段或基因区域在逃避宿主免疫方面的功能；选取一株H1N1亚型猪流感病毒，对不同片段上的不同氨基酸位点进行突变，拯救突变株病毒，比较突变株与野毒株在诱导中和抗体水平和细胞因子分泌水平上的差异，从而确定分离株基因组上与宿主免疫相关的氨基酸位点或功能区，最终在分子水平上阐明H1N1亚型猪流感病毒逃避宿主免疫的分子机制。

H1Nl亚型猪流感病毒不仅引起猪发病和死亡，也会引发人流感的爆发和流行，如1918 年人类流感爆发，特别是2009年始发于美国和墨西哥的人甲型H1N1 流感疫情，引起全球关注和极大恐慌，具有重要的公共卫生学意义。本项目主要通过病毒的分离、鉴定、序列测定分析、毒株的拯救和突变以及毒株的致病性、细胞因子变化和抗流感病毒防治技术等方面研究取得了一些重要成果：一是本课题分离到14株类禽型H1N1流感病毒，多数毒株是重组病毒，基因片段中2009甲型流感病毒片段呈不断增加的趋势，部分毒株PB2氨基酸701位发生了天冬氨酸到天冬酰胺的突变，这种突变趋向人源变化，为类禽型H1N1流感病毒适应猪群进而感染人敲响了警钟。二是对小鼠致病性研究结果发现H1N1类禽型猪流感病毒攻毒BABL/c小鼠后可引起血浆中抗炎细胞因子IL-10和促炎细胞因子IL-6的含量极显著上调，对BABL/c小鼠的致病力与IL-10 、IL-6的上调成正相关；促炎因子TNF-α则有不同程度的下调，毒株对BABL/c小鼠的致病力与TNF-α成负相关，血浆中TNF-α的含量越低则毒株对BABL/c小鼠的致病力越强；6号毒株接种小鼠后出现典型的流感症状，在1周内全部死亡，该毒株为流感病毒的疫苗、药物、致病性等后续研究提供了平台。三是对H1N1亚型流感病毒A/swine/Guangxi/12/2011毒株成功完成了病毒拯救和NA基因点突变，氨基酸119位点发生E到G（的变化，突变毒株对小鼠的致病性有所增强，而神经氨酸酶活性有一定的下降。四是研发了治疗流感的中草药胶囊制剂；从合成的36种S-色氨酸苯甲酰胺类衍生物和苦参碱衍生物筛选出了抗流感病毒的药物；通过抗体效价的测定、免疫原性分析、血清中和试验以及动物保护试验，筛选出A/SW/6和A/SW/8两个毒株是H1N1流感病毒较好的备选疫苗毒株。项目成果对流感病毒深入研究和防治具有重要意义。