G57基因型H9N2亚型禽流感病毒反向遗传系统的建立与验证

doi:10.11843/j.issn.0366-6964.2025.04.030

Abstract

Abstract:

To establish a reverse genetic manipulation platform for the currently prevalent H9N2 subtype avian influenza virus (AIV), we isolated the A/Chicken/Guangdong/M/2021 (H9N2) strain (designated as M) from a diseased chicken farm in Guangdong. The full genome sequence was determined to identify the viral genotype and analyze differences from circulating strains. Plasmids for the eight genes were constructed using RT-PCR and seamless cloning, followed by co-transfection into 293T cells to rescue the virus, named rM. We assessed viral stability and growth characteristics, and conducted animal experiments to verify the consistency of rM with the original M strain in terms of transmission efficiency and pathogenicity. The M strain, classified as the G57 genotype, shares consistent biological properties with rM, including acid and heat stability, pathogenicity, and transmissibility. The reverse genetic system of AIV subtype H9N2 of the G57 genotype was successfully established, and the biological properties of M and rM were comprehensively verified to remain consistent from both in vivo and in vitro experiments.

Key words: reverse genetics system, H9N2, avian influenza virus, G57 genotype

CLC Number:

S852.659.5

SHI Yuting, HAN Xinyu, ZHONG Muhui, LIN Yaozhong, LIU Tengfei, LI Yan, YIN Huifang, JIA Weixin. Establishment and Validation of the Reverse Genetics System for G57 Genotype H9N2 Subgenotype Avian Influenza Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1825-1833.

Figures/Tables 5

Table 1

Fig. 1

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Table 2

References 21

1	YANG J , YAN J , ZHANG C , et al. Genetic, biological and epidemiological study on a cluster of H9N2 avian influenza virus infections among chickens, a pet cat, and humans at a backyard farm in Guangxi, China[J]. Emerg Microbes Infect, 2023, 12 (1): 2143282. doi: 10.1080/22221751.2022.2143282
2	ZHANG K , ZHANG Z , YU Z , et al. Domestic cats and dogs are susceptible to H9N2 avian influenza virus[J]. Virus Res, 2013, 175 (1): 52- 57. doi: 10.1016/j.virusres.2013.04.004
3	LY H . Highly pathogenic avian influenza H5N1 virus infections of dairy cattle and livestock handlers in the United States of America[J]. Virulence, 2024, 15 (1): 2343931. doi: 10.1080/21505594.2024.2343931
4	ZHOU P , ZHU W , GU H , et al. Avian influenza H9N2 seroprevalence among swine farm residents in China[J]. J Med Virol, 2014, 86 (4): 597- 600. doi: 10.1002/jmv.23869
5	LI S , ZHOU Y , SONG W , et al. Avian influenza virus H9N2 seroprevalence and risk factors for infection in occupational poultry-exposed workers in Tai'an of China[J]. J Med Virol, 2016, 88 (8): 1453- 1456. doi: 10.1002/jmv.24483
6	LI X , TIAN B , JIANFANG Z , et al. A comprehensive retrospective study of the seroprevalence of H9N2 avian influenza viruses in occupationally exposed populations in China[J]. PLoS One, 2017, 12 (6): e0178328. doi: 10.1371/journal.pone.0178328
7	裴瑞青, 张如胜, 叶文, 等. 湖南省首例人感染H3N8亚型禽流感病例的病毒分离及分子进化分析[J]. 中国人兽共患病学报, 2023, 39 (04): 364- 375.
	PEI R QG , ZHANG R S , YE W , et al. Viral isolation and molecular evolution analysis of the first human case of H3N8 subtype avian influenza infection in Hunan Province[J]. Chinese Journal of Zoonoses, 2023, 39 (04): 364- 375.
8	BI Y , LI J , SHI W . The time is now: a call to contain H9N2 avian influenza viruses[J]. Lancet Microbe, 2022, 3 (11): e804- e805. doi: 10.1016/S2666-5247(22)00232-4
9	LI C , WANG S , BING G , et al. Genetic evolution of influenza H9N2 viruses isolated from various hosts in China from 1994 to 2013[J]. Emerg Microbes Infect, 2017, 6 (1): 1- 11.
10	WANG X , LIU K , GUO Y , et al. Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China[J]. Emerg Microbes Infect, 2023, 12 (2): 2249558. doi: 10.1080/22221751.2023.2249558
11	KATOH K , ROZEWICKI J , YAMADA K D . Mafft online service: multiple sequence alignment, interactive sequence choice and visualization[J]. Brief Bioinform, 2019, 20 (4): 1160- 1166. doi: 10.1093/bib/bbx108
12	KALYAANAMOORTHY S , MINH B Q , WONG T , et al. Modelfinder: fast model selection for accurate phylogenetic estimates[J]. Nat Methods, 2017, 14 (6): 587- 589. doi: 10.1038/nmeth.4285
13	LETUNIC I , BORK P . Interactive tree of life (itol) v5: an online tool for phylogenetic tree display and annotation[J]. Nucleic Acids Res, 2021, 49 (W1): W293- W296. doi: 10.1093/nar/gkab301
14	LIN S , CHEN J , LI K , et al. Evolutionary dynamics and comparative pathogenicity of clade 2.3. 4.4 b H5 subtype avian influenza viruses, China, 2021-2022[J]. Virol Sin, 2024, 39 (3): 358- 368. doi: 10.1016/j.virs.2024.04.004
15	DING S , ZHOU J , XIONG J , et al. Continued evolution of H10N3 influenza virus with adaptive mutations poses an increased threat to mammals[J]. Virol Sin, 2024, 39 (4): 546- 555. doi: 10.1016/j.virs.2024.06.005
16	李梦娇, 徐凯, 杨彬, 等. H9N2亚型禽流感病毒A/Chicken/Shandong/LY1/2017株反向遗传操作系统的建立[J]. 中国动物传染病学报, 2020, 28 (3): 49- 54.
	LI M J , XU K , YANG B , et al. Establishment of Reverse Genetics System of H9n2 Avian Influenza Virus A/Chicken/Shandong/LY1/2017[J]. Chinese Journal of Animal Infectious Diseases, 2020, 28 (03): 49- 54.
17	JIN X , ZHA Y , HU J , et al. New molecular evolutionary characteristics of H9N2 avian influenza virus in Guangdong Province, China[J]. Infect Genet Evol, 2020, 77, 104064. doi: 10.1016/j.meegid.2019.104064
18	孙华鹏, 崔新鑫, 潘亮奇, 等. 中国H9N2亚型禽流感病毒的流行现状[J]. 畜牧兽医学报, 2021, 52 (5): 1218- 1229. doi: 10.11843/j.issn.0366-6964.2021.05.008
	SUN HUAPENG , CUI XINXIN , PAN LIANGQI , et al. The epidemiology of H9N2 avian influenza virus in China[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52 (5): 1218- 1229. doi: 10.11843/j.issn.0366-6964.2021.05.008
19	陈子轩, 张楠, 胡群, 等. 我国H9N2亚型禽流感病毒血凝素蛋白145和153位抗原位点变异分析[J]. 畜牧兽医学报, 2022, 53 (4): 1165- 1172. doi: 10.11843/j.issn.0366-6964.2022.04.016
	CHEN Z X , ZHANG N , HU Q , et al. Mutation analysis of antigen sites 145 and 153 on hemagglutinin of H9N2 avian influenza virus in China[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (4): 1165- 1172. doi: 10.11843/j.issn.0366-6964.2022.04.016
20	ZHANG D , GAO F , JAKOVLI? I , et al. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies[J]. Mol Ecol Resour, 2020, 20 (1): 348- 355. doi: 10.1111/1755-0998.13096
21	HOFFMANN E , NEUMANN G , KAWAOKA Y , et al. A DNA transfection system for generation of influenza A virus from eight plasmids[J]. Proc Natl Acad Sci U S A, 2000, 97 (11): 6108- 6113. doi: 10.1073/pnas.100133697

引物 Primer		序列(5′→3′)Sequences
类型 Type	名称 Name	序列(5′→3′)Sequences
扩增引物 Amplification primer	HA-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCAAAAGCAGGGG
	HA-2	$\underline{{\rm{TTTTGGGCCGCCGGGTT}}}$ATTAGTAGAAACAAGGGTGTTTT
	NA-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCAAAAGCAGGAGT
	NA-2	$\underline{{\rm{TTTTGGGCCGCCGGGTT}}}$ATTAGTAGAAACAAGGAGTTTTTT
	PB2-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCGAAAGCAGGTC
	PB2-2	$\underline{{\rm{TTTTGGGCCGCCGGGTTATT}}}$AGTAGAAACAAGGTCGTTT
	PB1-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCGAAAGCAGGCA
	PB1-2	$\underline{{\rm{TTTTGGGCCGCCGGGTT}}}$ATTAGTAGAAACAAGGCATTT
	PA-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCGAAAGCAGGTAC
	PA-2	$\underline{{\rm{TTTTGGGCCGCCGGGTT}}}$ATTAGTAGAAACAAGGTACTT
	NP-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCAAAAGCAGGGTA
	NP-2	$\underline{{\rm{TTTTGGGCCGCCGGGTT}}}$ATTAGTAGAAACAAGGGTATTTTT
	MP-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCAAAAGCAGGTAG
	MP-2	$\underline{{\rm{TTTTGGGCCGCCGGGTT}}}$ATTAGTAGAAACAAGGTAGTTTTT
	NS-1	$\underline{{\rm{ACCTCCGAAGTTGGGGG}}}$GGAGCAAAAGCAGGGTG
	NS-2	$\underline{{\rm{TTTTGGGCCGCCGGGTT}}}$ATTAGTAGAAACAAGGGTGTTTT
通用引物 Universal primer	pHW2000-F	CTAGAGAACCCACTGCTTAC
通用引物 Universal primer	pHW2000-R	TTGGGGACAGGTGTCCGTGT

试验组别 Group		拭子类型 Swab type	测定阳性拭子数/采集拭子总数 Determine the number of positive swabs/total number of collected swabs
试验组别 Group		拭子类型 Swab type	1 dpi	3 dpi	5 dpi①	7 dpi	9 dpi
M	感染组 Infection group	咽拭子	6/6	6/6	3/3	3/3	1/3
M	感染组 Infection group	肛拭子	4/6	6/6	2/3	2/3	1/3
rM		咽拭子	6/6	6/6	3/3	1/3	0/3
		肛拭子	5/6	6/6	3/3	1/3	1/3
M	同居组 Cohabitation group	咽拭子	6/6	6/6	3/3	3/3	2/3
M	同居组 Cohabitation group	肛拭子	4/6	5/6	3/3	2/3	2/3
rM		咽拭子	6/6	6/6	3/3	3/3	3/3
rM		肛拭子	6/6	5/6	3/3	3/3	2/3
PBS	对照组 Control group	咽拭子	0/6	0/6	0/3	0/3	0/3
PBS	对照组 Control group	肛拭子	0/6	0/6	0/3	0/3	0/3

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Establishment and Validation of the Reverse Genetics System for G57 Genotype H9N2 Subgenotype Avian Influenza Virus

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