Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (17): 3379-3388.doi: 10.3864/j.issn.0578-1752.2018.17.012

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Amino Acid Substitutions of E627V in Polymerase Basic Protein 2 Gene Increases the Pathogenicity of the H7N9 Influenza Virus in Mice

YIN Xin, MA ShuJie, LI Mei, DENG GuoHua, HOU YuJie, CUI PengFei, SHI JianZhong, CHEN HuaLan   

  1. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/State Key Laboratory of Veterinary Biotechnology, Harbin 150001
  • Received:2018-04-08 Online:2018-09-01 Published:2018-09-01

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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

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