欧亚类禽H1N1猪流感病毒抗原性变异关键氨基酸的鉴定与分析

doi:10.3864/j.issn.0578-1752.2023.14.016

摘要/Abstract

摘要：

【背景】流感病毒的抗原性主要是由病毒的表面糖蛋白HA决定的，前期利用欧亚类禽型H1N1猪流感病毒（EA H1N1 SIV）HA蛋白单克隆抗体（mAb）筛选抗原逃逸株发现，HA蛋白190、230和269位（H3编码）氨基酸的改变能够导致病毒发生抗原逃逸，并发现在新近分离的EA H1N1 SIV HA蛋白广泛存在这3个氨基酸的变异。【目的】进一步探索哪些氨基酸对病毒的抗原性具有关键作用，为流感的防控提供科学依据。【方法】选取一株病毒A/swine/Liaoning/SY72/2018（H1N1） SY72为模式病毒，建立该病毒的反向遗传系统（RGS），又以SY72为骨架，拯救含有HA基因190、230和269单点突变重组病毒；利用rSY72病毒制备灭活疫苗，分别免疫SPF鸡和非免疫猪制备其相应的血清，通过中和试验和血凝抑制（HI）试验分析病毒的抗原性，确定影响病毒抗原性的关键氨基酸位点；继而评估HA蛋白不同位点氨基酸的改变对病毒复制特性及受体结合特性等的影响。【结果】分析SY72病毒的HA氨基酸序列发现，该病毒HA蛋白分别含有190D、230M和269R。利用反向遗传技术，分别拯救了病毒rSY72及单点突变病毒rSY72HA/D190N、rSY72HA/M230I和rSY72HA/R269M；中和试验结果表明，与rSY72相比较，3株突变病毒均能够与两株mAbs发生一定反应；HI试验结果显示，与rSY72相比较，突变病毒rSY72HA/D190N与rSY72免疫鸡血清和猪血清的反应减弱，HI抗体效价分别出现了4倍和8倍的差异，而病毒rSY72HA/M230I和rSY72HA/R269M与两种血清的反应差异不明显，表明HA蛋白190位氨基酸改变对病毒的抗原性影响最为明显；病毒蚀斑测定及复制动力学研究结果发现D190N 的氨基酸替换导致病毒rSY72HA/D190N在MDCK细胞上形成的蚀斑减小，而R269M的氨基酸替换导致病毒rSY72HA/R269M在MDCK细胞上的复制能力下降；3个位点的氨基酸替换均未影响病毒的受体结合特性。【结论】HA蛋白190位氨基酸对EA H1N1 SIV抗原性具有决定作用，269位氨基酸突变使得病毒在MDCK细胞上的复制能力降低。这提示在后续开展的病原学监测中要密切关注这些氨基酸的变异情况，提高对动物流感的预警预报。

关键词: 猪流感病毒, HA蛋白

Abstract:

【Background】 The antigenicity of influenza virus is mainly determined by the hemagglutinin (HA), a surface glycoprotein of the virus. Our previous study indicated that amino acid changes at positions 190, 230 and 269 (H3 numbering) of HA protein resulted in antigenic escape by using the monoclonal antibody (mAb) against the HA protein of Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV). These three amino acid substitutions widely existed in the HA protein of the recently isolated EA H1N1 SIVs. 【Objective】This study aimed to explore which amino acids played a key role in the antigenicity of the virus, and further provide scientific basis for the control of influenza. 【Method】 In this study, A/swine /Liaoning /SY72/2018 (H1N1) (SY72) was selected as the model virus, and its reverse genetic system (RGS) was established. Then, using SY72 virus as backbone, three reassortant viruses were rescued by introducing the respective single amino acid mutation at position 190, 230, and 269 into HA protein. Antisera were raised by inoculating the rSY72-inactivated vaccine into specific-pathogen-free (SPF) chickens and non-immunized pigs. The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the rSY72 virus. Then their effects on viral replication capacities and receptor binding properties were further evaluated. 【Result】 Sequence analysis showed that the HA of SY72 virus carried 190D, 230M, and 269R, respectively. The viruses, including rSY72, rSY72HA/D190N, rSY72HA/M230I, and rSY72HA/R269M, were rescued by RGS. The results of neutralization test showed that all the three mutant viruses could react with two mAbs, to some extent, compared with the rSY72 virus. The HI results indicated that the HI antibody titers of the rSY72HA/D190N reacted with the rSY72-immunized chicken and pig sera were 4- and 8-fold lower than those of the rSY72 virus with the respective sera. However, the rSY72HA/M230I and rSY72HA/R269M virus reacted well with these two sera. The results indicated that residue 190 in the HA had the important effects on the viral antigenicity. Results of the viral plaque assay and growth curve experiments demonstrated that plaque sizes generated by the rSY72HA/D190N virus were smaller than those by the rSY72 virus in MDCK cells. The replication ability of the rSY72HA/R269M virus was significantly decreased in MDCK cells, compared with that of the rSY72 virus. Furthermore, the three amino acid mutations had no impact on the viral receptor-binding preference. 【Conclusion】 The amino acid at position 190 of HA protein played an important role in determining the antigenicity of EA H1N1 SIV. The mutation of amino acid at position 269 reduced the viral replication ability in MDCK cells. These results suggested that more attentions should be paid for monitoring these residue changes in the influenza surveillance, so as to improve early warning of influenza in animals.

Key words: swine influenza virus, HA protein

张乃心, 许程志, 杨玉莹, 张亚萍, 万云飞, 乔传玲, 陈化兰. 欧亚类禽H1N1猪流感病毒抗原性变异关键氨基酸的鉴定与分析[J]. 中国农业科学, 2023, 56(14): 2828-2836.

ZHANG NaiXin, XU ChengZhi, YANG YuYing, ZHANG YaPing, WAN YunFei, QIAO ChuanLing, CHEN HuaLan. Identification of Key Amino Acids in the Antigenic Variation of Eurasian Avian-Like H1N1 Swine Influenza Viruses[J]. Scientia Agricultura Sinica, 2023, 56(14): 2828-2836.

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病毒 Virus	上游引物Forward primer（5' - 3'）	下游引物Reverse primer（5' - 3'）
rSY72HA/D190N	ACCGACAGTAACCAACAAACTCTCTACCAG	TTTGTTGGTTACTGTCGGTCGGAGGGTGAT
rSY72HA/M230I	AGGCAGAATAAATTATTACTGGACACTGTT	GTAATAATTTATTCTGCCTGCCTGTTCTCT
rSY72HA/R269M	GAATTATGATGTCGGATGCTCAGGTTCA	GCATCCGACATCATAATTCCAGAACTAGAG

病毒 Virus	氨基酸差异位点 Amino acid difference site
病毒 Virus	4	7	95	132	144	152	190	211	230	269	290	304	347	406	376	522
SY72	V	T	D	A	T	I	D	K	M	R	G	K	V	G	V	I
HeN11	A	A	N	T	A	L	N	Q	I	M	S	E	I	R	E	V

病毒 Virus	mAbs中和抗体效价 Neutralization antibody titer of mAbs		血清HI抗体效价 HI antibody titers of sera
病毒 Virus	2B6	4C7	鸡血清（rSY72） Chicken sera (rSY72)	猪血清（rSY72） Pig sera (rSY72)
HeN11	640	640	NT *	NT
rSY72	<10	<10	512	2560
rSY72HA/D190N	320	80	128	320
rSY72HA/M230I	20	20	256	2560
rSY72HA/R269M	40	160	512	2560