Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice

Aobaixue Zhou; Jiahao Zhang; Huanan Li; Qiang Xu; Yiqun Chen; Bo Li; Wanying Liu; Guanming Su; Xingxing Ren; Guangjie Lao; Baozheng Luo; Ming Liao; Wenbao Qi

doi:10.1016/j.virs.2022.01.001

February 2022

Aobaixue Zhou, Jiahao Zhang, Huanan Li, Qiang Xu, Yiqun Chen, Bo Li, Wanying Liu, Guanming Su, Xingxing Ren, Guangjie Lao, Baozheng Luo, Ming Liao and Wenbao Qi. Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice[J]. Virologica Sinica, 2022, 37(1): 38-47. doi: 10.1016/j.virs.2022.01.001

Citation: Aobaixue Zhou, Jiahao Zhang, Huanan Li, Qiang Xu, Yiqun Chen, Bo Li, Wanying Liu, Guanming Su, Xingxing Ren, Guangjie Lao, Baozheng Luo, Ming Liao, Wenbao Qi. Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice .VIROLOGICA SINICA, 2022, 37(1) : 38-47. http://dx.doi.org/10.1016/j.virs.2022.01.001

高致病性H7N9禽流感病毒的裂解位点处碱性氨基酸和非碱性氨基酸的联合插入促进鸡和小鼠的致病性

周傲白雪 ^a,b,d ,
张家豪 ^a,b,e,f,g,h ,
李华楠 ^a,b,e,f,g,h ,
徐强 ^a,b,e,f ,
陈意群 ^a,b,e,f ,
李波 ^a,b,e,f ,
刘婉莹 ^a,b,e,f ,
苏冠铭 ^a,b,e,f ,
任行星 ^a,b,e,f ,
劳光杰 ^a,b,e,f ,
罗宝正 ^d ,
廖明 ^{a,b,c,e,f,g,h,,} ,
亓文宝 ^{a,b,c,e,f,g,h,,}

国家禽流感专业实验室（广州），华南农业大学

通讯作者： 廖明, mliao@scau.edu.cn; 亓文宝, qiwenbao@scau.edu.cn
收稿日期： 2021-04-02
录用日期： 2021-07-22

摘要

自2016年中旬以来，低致病性H7N9流感病毒在我国演变为高致病性H7N9流感病毒，对家禽产业和公共卫生构成了极大的威胁。在高致病性H7N9流感病毒流行的早期，其裂解位点主要插入“KRTA”基序。随着H7N9流感病毒的传播，禽源和人源高致病性H7N9流感病毒的裂解位点更具有多态性，表现出碱性和非碱性氨基酸插入和氨基酸的替换，但这些插入和替换的潜在功能仍不清楚。在这里，我们通过反向遗传技术拯救出6株携带PEIPKGR/G、PEVPKGR/G、PEVPKRKRTAR/G、PEVPKGKRTAR/G、PEVPKGKRIAR/G和PEVPKRKRR/G基序的H7N9流感病毒。我们的研究结果表明，PEVPKRKRTAR/G基序的氨基酸插入在我国流行的高致病性H7N9病毒中占主导地位。有趣的是，删除H7N9病毒裂解位点的苏氨酸和丙氨酸后，H7N9病毒显著降低了热稳定性并且降低小鼠致病性。含有PEVPKRKRTAR/G基序的病毒在鸡上能够增强致病性，但是与其他高致病性H7N9病毒相比，其在鸡上的传播能力有所降低。以上结论解释了为何含有PEVPKRKRTAR/G基序的H7N9病毒在我国能够持续流行。与此相反，携带异亮氨酸和丙氨酸的H7N9病毒可降低鸡的传播率和小鼠的毒力。值得注意的是，H7N9病毒的HA蛋白的I335V氨基酸突变能够显著增强鸡的致病性和传播能力，表明H7N9的碱性氨基酸和非碱性氨基酸的联合插入以及裂解位点处I335V的替换促进了鸡和小鼠的复制能力和致病性。随着H7N9病毒的不断进化，其对家禽养殖业和人类健康的威胁越来越大，因此需要加强H7N9病毒的全面监测和预防。
- H7N9
- , 高致病性
- , 裂解位点
- , 碱性氨基酸和非碱性氨基酸
- , 致病性

Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice

Aobaixue Zhou ^a,b,d ,
Jiahao Zhang ^a,b,e,f,g,h ,
Huanan Li ^a,b,e,f,g,h ,
Qiang Xu ^a,b,e,f ,
Yiqun Chen ^a,b,e,f ,
Bo Li ^a,b,e,f ,
Wanying Liu ^a,b,e,f ,
Guanming Su ^a,b,e,f ,
Xingxing Ren ^a,b,e,f ,
Guangjie Lao ^a,b,e,f ,
Baozheng Luo ^d ,
Ming Liao ^{a,b,c,e,f,g,h,,} ,
Wenbao Qi ^{a,b,c,e,f,g,h,,}

a College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China

Corresponding author: Ming Liao, mliao@scau.edu.cn Wenbao Qi, qiwenbao@scau.edu.cn
Received Date: 02 April 2021
Accepted Date: 22 July 2021

Abstract

Since mid-2016, the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic (HP) phenotype in China, raising many concerns about public health and poultry industry. The insertion of a “KRTA” motif at hemagglutinin cleavage site (HACS) occurred in the early stage of HP H7N9 variants. During the co-circulation, the HACS of HP-H7N9 variants were more polymorphic in birds and humans. Although HP-H7N9 variants, unlike the H5 subtype virus, exhibited the insertions of basic and non-basic amino acids, the underlying function of those insertions and substitutions remains unclear. The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China. Then, we generated six H7N9 viruses bearing the PEIPKGR/G, PEVPKGR/G, PEVPKRKRTAR/G, PEVPKGKRTAR/G, PEVPKGKRIAR/G, and PEVPKRKRR/G motifs. Interestingly, after the deletion of threonine and alanine (TA) at HACS, the H7N9 viruses manifested decreased thermostability and virulence in mice, and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence. By contrast, the insertion of non-basic amino acid isoleucine and alanine (IA) decreased the transmissibility in chickens and virulence in mice. Remarkably, the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens, suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice. The ongoing evolution of H7N9 increasingly threatens public health and poultry industry, so, its comprehensive surveillance and prevention of H7N9 viruses should be pursued.
- H7N9
- , Highly pathogenic
- , Cleavage site
- , Basic and non-basic amino acids
- , Pathogenicity

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10.1016j.virs.2022.01.001-ESM.docx