甲型流感病毒X31(H3N2)小鼠适应后毒力增强与HA及PB2突变相关

Increased Virulence of Influenza A Virus X31 (H3N2) after Adaptation in Mice is Related to the Mutations on HA and PB2 Protein

流感小鼠适应毒株及感染模型是研究流感病毒致病机理、评价抗流感病毒药物和疫苗效果的重要工具。为建立甲型流感病毒X31(H3N2)的鼠肺适应毒株,并探讨其在小鼠适应过程中毒力增强的分子机制,本研究将X31在BALB/c小鼠肺组织中连续传代,通过观察病毒滴度测定、感染小鼠症状变化、体重改变和致死力等指标,发现X31经过鼠肺连续传代10次以后,病毒毒力逐渐增强,与原代病毒相比,鼠肺中病毒滴度提高10倍,半数致死量(50%lethal dose,LD50)提高大于1 000倍。确定获得高致病力的鼠肺适应毒株(Mouse adapted X31,X31MA);对X31MA毒株全基因组测序发现,血凝素(Hemagglutinin,HA)基因出现3个有义突变(P221H、V309I、K411T),聚合酶碱性蛋白2(Basic protein 2,PB2)基因出现1个有义突变(M483T)。结果表明,通过在小鼠肺组织中连续传代X31(H3N2)致病力增强,其HA和PB2蛋白中四个适应性突变位点可能与流感病毒X31MA毒力增加相关。本研究为流感疫苗评价及致病机制研究提供了重要技术支持。

Mouse-adapted strains and infection models of influenza virus are important tools for studying the pathogenesis and evaluating the effects of anti-influenza drugs and vaccines. To establish a mouse lung-adapted strain of influenza A virus X31(H3N2) and explore the molecular mechanism of its virulence enhancement during mouse adaptation,primary X31 stock(X31P0) was serially passaged in lungs of BALB/c mice.Observation results of changes in virus titer,symptoms of infected mice,weight and lethality revealed that the virulence of the virus gradually increased during serial passage of X31 in mice lungs for 10 generations.Compared with the original virus(X31P0),the virus titer in the lungs of the mouse increased by 10-fold and the LD50 increased by >1000-fold. Therefore,a highly-pathogenic mouse lung-adapted strain was obtained and named X31MA. Sequencing of the whole genome of X31MA revealed three sense mutations(P221H,V309I,K411T) on HA protein and one sense mutation on PB2 protein(M483T). These results suggested that the virulence of influenza virus X31(H3 N2) was enhanced by serial passages in mouse lung,and four mutation sites on HA and PB2 proteins might be associated with increased virulence. X31MA provides important technical support for influenza vaccine evaluation and pathogenesis research.