Gambaryan A S, Lomakina N F, Boravleva E Y, Mochalova L V, Sadykova G K, Prilipov A G, Matrosovich T Y, Matrosovich M N
Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products, Russian Academy of Sciences, Moscow, 108819 Russia.
Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, 123098 Russia.
Mol Biol (Mosk). 2018 Jul-Aug;52(4):644-658. doi: 10.1134/S0026898418040055.
To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.
为研究甲型H1N1流感大流行病毒的致病因素，通过在鸡胚、小鼠肺脏和MDCK细胞培养物中传代，获得了A/汉堡/5/2009（H1N1）pdm09毒株的多个突变变体。在小鼠体内对A/汉堡/5/2009进行17次肺传代后，衍生变体的最小致死剂量与亲代病毒相比降低了五个数量级。该变体与原始病毒在以下病毒蛋白中的九个氨基酸残基上存在差异：血凝素（HA）、神经氨酸酶（NA）和聚合酶复合物的组分。对中间变体进行额外传代和克隆，使得能够获得仅相差一个氨基酸替换的毒株对。对这些变体的复制活性、受体特异性和毒力进行比较分析，揭示了病毒对小鼠致病性增加的两种机制。因此，（1）HA中的替换（Asp225Gly或Gln226Arg）以及降低HA电荷的补偿性突变（Lys123Asn、Lys157Asn、Gly158Glu、Asn159Asp或Lys212Met）改变了病毒受体结合特异性，并恢复了HA和NA之间的功能平衡；（2）PA蛋白中的Phe35Leu替换增加了病毒聚合酶活性。
