季节性流感病毒H1N1和H3N2在BALB/c小鼠中的传代适应与分子机理研究

Study on the Adaptation and Molecular Mechanism of Seasonal Influenza Virus H1N1 and H3N2 in BALB/c Mouse Pneumonitis

目的:建立季节性流感病毒H1N1和H3N2的BALB/c小鼠致死性动物模型,并探索其鼠肺适应的分子机理。方法:将季节性流感病毒A/Guangdong/51/2008(H1N1)(简称为H1N1-GDwt)和A/Anhui/137/2008(H3N2)(简称为H3N2-AHwt)分别滴鼻感染BALB/c小鼠,于病毒增殖高峰期制备肺组织悬液,连续传40代,并对野生型与鼠肺适应株在小鼠中的致死性与全基因组序列进行比较。结果:H3N2-AHwt感染BALB/c小鼠后各代次鼠肺悬液均未检测到病毒;而H1N1-GDwt感染小鼠后第4 d肺部病毒滴度达到高峰,病毒滴度随传代次数的增加而呈现"波浪型"升高,鼠肺适应株对BALB/c小鼠的致死性与致病性明显强于野生型病毒,全基因组序列分析发现鼠肺适应株在血凝素(HA)、酸性聚合酶(PA)、核蛋白(NP)及非结构蛋白(NS)中共发生了10个氨基酸突变。结论: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鼠肺传代而提高,HA、PA、NP及NS蛋白的突变与其鼠肺适应密切相关。

Objective： To establish mouse-lethal model of seasonal influenza virus and investigate the potentialmolecular mechanism of adaptation.Methods： BALB/c mice were intranasally infected with seasonal influenza vi-rus of A/Guangdong/51/2008（H1N1）（H1N1-GDwt） and A/Anhui/137/2008（H3N2）（H3N2-AHwt）,and lungs were dis-sected out at the peak of virus replication to generate the first passage of mouse adapted virus（P1）,which wassubjected serial mouse lung-to-lung passages（from P1 to P40）.Pathogenicity and genetic sequences of wide-typeand mouse-adapted virus were compared.Results： For H3N2-AHwt,virus titer was under detection at any passag-es.In contrast,for H1N1- GDwt,virus replication ability increased as ＂wave＂ by lung- to- lung passage,and thepathogenicity also improved as the passages increased.Sequence analysis indicated that the increased virulence ofmouse-adapted H1N1-GDMA40 was attributed to 10 mutations in HA,PA,NP and NS proteins.Conclusion： H3N2-AHwt was hardly to replicate in mice while H1N1-GDwt could effectively replicate in BALB/c mice and mutationsin HA,PA,NP and NS proteins contributed to the enhanced virulence.