人甲型流感病毒鼠肺适应的分子机理研究

Influenza is a highly contagious and acute respiratory disease caused by influenza virus. Influenza pandemic and pandemic caused by influenza A virus has become a focus in the worldwide public health area. Mouse model plays an important role in the area of influenza disease control and prevention. However, mouse is not a susceptible animal model for influenza virus, and mice are not naturally infected with human or other strains of influenza virus. In addition, although some influenza virus strains can be experimentally adapted for mouse by serial lung-to-lung passages, the molecular mechanism by which the human influenza viruses were able to sufficiently adapt to mice is still poorly understood. Therefore, exploring the molecular basis for adaptation of human influenza A virus in mice to acquire a lethal pneumonia animal model will provide a powerful tool for influenza epidemic and pandemic prevention and control. For this aim, we used the China's 2008-2009 seasonal influenza A virus of H1N1 and H3N2 as study models, and mouse-adapted variants of these viruses were successfully derived through serial lung-lung passages in Balb/C mice. In the present study, full length genomic segments of the influenza virus variants pre- and post-mouse-adapted are to be amplified and sequenced, the nucleotide and amino acid sequences are to be aligned to identify the mutants that may influence the host and virulence determinants. Consequently, the influenza re-assortment or mutation virus variants with mutant segments or amino acids are to be constructed by influenza reverse genetic technology, and the characterization and effect of a single genomic segment or amino acid on virus replication in vitro are to be determined by the cell-culture models. Finally, these recombinant influenza virus variants are to be used to infection Balb/C mice, virulence and replication curve are to be studied, and the effects of the genomic segments and amino acids on adaptation and virulence are compared and assessed in vivo. Results of this research will provide important foundation for the adaptation of human influenza A virus in mice to acquire a lethal pneumonia animal model.

甲型流感病毒引起的流感流行与大流行已成为全世界公共卫生关注的焦点。小鼠模型在流感防控的各个领域发挥了重要作用，但是，小鼠并不是流感病毒的易感动物，流感病毒在小鼠肺部适应的分子基础仍不清楚。因此，建立流感病毒的小鼠致死性肺炎动物模型并探讨其鼠肺适应的分子机理，将为流感流行与大流行的防控提供有力手段。本研究前期已将我国2008-2009年流行的H1N1和H3N2等流感病毒在Balb/C小鼠中进行了肺-肺传代，并获得了多株能引起小鼠致死性肺炎的流感病毒鼠肺适应株。在此基础上，本研究拟将鼠肺适应前后及不同适应代次的流感病毒进行全基因组序列测定和比对，分析可能引起其鼠肺适应的关键氨基酸；然后，通过反向遗传学技术构建含有相应基因片段或突变位点的重组流感病毒重配株与突变株；最后，通过对重组病毒在小鼠中的生长与致病性研究，验证影响其鼠肺适应的关键因素，为建立人甲型流感病毒的小鼠致死性肺炎动物模型提供参考。

建立季节性流感病毒的小鼠致死性肺炎动物模型并探讨其鼠肺适应的分子机理，将为流感病毒宿主范围与致病性的研究提供重要参考。然而，目前针对致病性较弱的季节性流感病毒的鼠肺适应研究仍非常有限。为此，本研究对我国2008-2009年度流行的H1N1和H3N2等季节性流感病毒及相应的疫苗株病毒在BALB/c小鼠中的传代与适应特点进行了研究。. 季节性流感病毒H1N1和H3N2重配疫苗株（A/Brisbane/59/2007(H1N1)-like virus和A/Brisbane/10/2007(H3N2)-like virus）在小鼠中传代适应的研究结果表明，H3N2疫苗株难以在BALB/c小鼠中有效复制与适应，而H1N1疫苗株能够在BALB/c小鼠中有效复制，其毒力可以通过连续的鼠肺传代而提高；全基因组序列测定结果表明，HA 蛋白142位氨基酸由N突变为D（HA-N142D）与H1N1疫苗株致病性的增加密切相关；对野生型（H1N1wt）与突变型（H1N1mu）病毒在细胞和小鼠中的复制动力学与致病性研究进一步验证了HA(N142D)突变是引起A/Brisbane/59/2007 (H1N1)-like virus鼠肺适应的关键氨基酸位点。. 季节性流感病毒H1N1和H3N2流行株病毒（A/Guangdong/51/2008(H1N1) (简称H1N1-GDwt)和A/Anhui/137/2008(H3N2) (简称H3N2-AHwt)）在小鼠中传代适应的研究结果表明，H3N2-AHwt难以在BALB/c小鼠中有效复制与适应，而H1N1-GDwt能够在BALB/c小鼠中有效复制，其毒力可以通过连续的鼠肺传代而提高；全基因组序列测定结果表明，HA、PA、NP及NS蛋白中10个氨基酸的突变与其鼠肺适应密切相关；对野生型（GDwt）与突变型（GDmu）病毒在细胞与小鼠中的复制动力学与致病性的研究进一步验证了上述10个氨基酸突变是引起A/Guangdong/51/2008(H1N1)鼠肺适应的关键氨基酸位点。. 综上所述，本研究通过对人甲型流感病毒H1N1和H3N2疫苗株及流行株在小鼠中的连续传代与鼠肺适应的分子机理研究，建立了季节性流感病毒H1N1小鼠致死性动物模型，为流感病毒宿主范围与致病性的研究提供了重要参考。