PB2蛋白E627V突变可增强H7N9病毒对小鼠的致病力

doi:10.3864/j.issn.0578-1752.2018.17.012

摘要/Abstract

摘要： 【背景】低致病性H7N9病毒自2013年在我国首次出现以来，研究发现其对家禽均呈现低致病力，对哺乳动物模型小鼠也不表现任何的致病力。但是，2015年在湖南分离的1株低致病性H7N9病毒（简称HuN/S40726），却对哺乳动物小鼠表现为高致病力。分析、推测导致该病毒对哺乳动物致病力改变的原因可能在于该病毒PB2蛋白E627V的改变。【目的】为了揭示该病毒致病力的变化原因以及对哺乳动物致病性增强的机制，提供人类H7N9病毒感染、危害增强风险预警，展开该研究。【方法】选取2013年低致病性H7N9病毒代表株（简称SH/S1053）和上述湖南HuN/S40726病毒，开展了哺乳动物致病力对比试验以及可能导致病毒发生致病力变化的相关基因位点对比分析。然后以HuN/S40726病毒为模式毒株，利用反向遗传学技术，成功建立了病毒的反向遗传操作系统；利用基因点突变技术定点突变了HuN/S40726病毒PB2蛋白的627位氨基酸，救获了重组病毒rHuN/S40726、rHuN/S40726-PB2/627E和rHuN/S40726-PB2/627K。通过小鼠感染模型评估了以上3株重组突变病毒对哺乳动物的致病力，分析了PB2蛋白627位氨基酸的突变对小鼠致病力的差异。然后通过构建HuN/S40726病毒及其突变病毒的聚合酶复合表达质粒系统，以SH/S1053病毒为背景毒株构建聚合酶复合表达质粒系统作为对照，使用双荧光素酶法检测了PB2蛋白627位氨基酸的不同突变体在293T细胞中的聚合酶活性，进一步分析PB2蛋白627位氨基酸影响病毒毒力的内在机制。【结果】通过哺乳动物致病力对比试验以及可能导致病毒发生致病力变化的相关基因位点对比分析，推测导致HuN/S40726病毒对哺乳动物致病力改变的原因可能在于该病毒PB2蛋白E627V的改变。重组救获病毒和突变株对小鼠致病性试验结果显示，PB2蛋白E627V的改变显著的增强了HuN/S40726病毒对小鼠的致病力，使得病毒MLD₅₀由≥6.5 log₁₀EID₅₀变化为3.5 log₁₀EID₅₀，病毒毒力增强了至少1 000倍以上。聚合酶活性试验结果表明，无论是在33℃还是37℃下，PB2蛋白E627V的改变，显著提高了HuN/S40726病毒在哺乳动物细胞中的聚合酶活性，与病毒对小鼠的致病性增强呈正相关性。【结论】PB2蛋白627位氨基酸（V）决定了HuN/S40726病毒对小鼠的高致病力。PB2蛋白E627V的改变能够显著增强HuN/S40726病毒在哺乳动物细胞中的聚合酶活性，是引起HuN/S40726病毒对哺乳动物致病力的重要因素。

关键词: H7N9, PB2, E627V, 致病力, 聚合酶活性

Abstract: 【Background】 The low pathogenic H7N9 virus (see note 1.1) that emerged in 2013 in China showed low virulence to poultry and were not virulent to the mammalian model-mice. However, a low-pathogenic H7N9 virus isolated in Hunan in 2015 (HuN/S40726 in abbreviation) showed high virulence in mammalian mice. The reason of the change in the virulence of the virus in mammals might be the alteration of the PB2 E627V mutation of the virus. 【Objective】 in order to reveal the causes of the viral virulence change and the mechanism of the enhancement of the pathogenicity in mammals, and to provide human H7N9 virus infection and early warning of the increased risks, we conducted this study. 【Method】We conducted comparative experiments on mammalian virulence and related gene loci that could lead to changes in viral pathogenicity with the low pathogenic H7N9 virus strain (SH/S1053 in abbreviation) and HuN/S40726 virus strain. We had successfully established a reverse genetic operating system of HuN/S40726 virus and rescued related viruses of rHuN/S40726, rHuN/S40726-PB2/627E and rHuN/S40726-PB2/627K. The virulences of the above three mutants against mammals were evaluated by using a mouse infection model, and then the differences in the pathogenicity of the related viruses in mice were analyzed. The polymerase complex expression plasmid system of HuN/S40726 and its mutant plasmids were constructed, whereas the SH/S1053 polymerase complex expression plasmid system were constructed as a background control. The dual-luciferase assay system was used to detect the polymerase activity of different mutants of amino acid 627 of PB2 protein in 293T cells, and the intrinsic mechanism of the virulence of amino acid at position 627 of PB2 protein was further analyzed. 【Result】 By comparative analysis of mammalian virulence and related gene loci that might cause changes in viral pathogenicity, we speculated that the cause of the change in the virulence of the HuN/S40726 virus in mammals might be in the E627V mutation of the PB2 protein. The results of pathogenicity tests on rescued virus and mutant strains in mice showed that the change of PB2 protein E627V significantly enhanced the virulence of HuN/S40726 virus in mice. The virus MLD₅₀ was changed from ≥ 6.5 log₁₀EID₅₀ to 3.5 log₁₀EID₅₀ and the virulence of the virus was increased by at least 1 000 folds. The results of polymerase activity assay showed that the change of PB2 E627V significantly improved the polymerase activity of HuN/S40726 virus in mammalian cells both at 33℃ or 37℃, and it was associated with the pathogenicity of the virus in mice. 【Conclusion】 The 627 amino acid (V) of PB2 protein determined the high virulence of the HuN/S40726 virus in mice. The alteration of PB2 protein E627V could significantly enhance the polymerase activity of HuN/S40726 virus in mammalian cells, and it was an important factor that caused the HuN/S40726 virus to be highly pathogenic to mammals.

Key words: H7N9, PB2, E627V, virulence, polymerase activity

尹馨，马树杰，李梅，邓国华，侯玉杰，崔鹏飞，施建忠，陈化兰. PB2蛋白E627V突变可增强H7N9病毒对小鼠的致病力[J]. 中国农业科学, 2018, 51(17): 3379-3388.

YIN Xin, MA ShuJie, LI Mei, DENG GuoHua, HOU YuJie, CUI PengFei, SHI JianZhong, CHEN HuaLan. Amino Acid Substitutions of E627V in Polymerase Basic Protein 2 Gene Increases the Pathogenicity of the H7N9 Influenza Virus in Mice[J]. Scientia Agricultura Sinica, 2018, 51(17): 3379-3388.

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