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

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (4): 1825-1833.doi: 10.11843/j.issn.0366-6964.2025.04.030

G57基因型H9N2亚型禽流感病毒反向遗传系统的建立与验证

史玉婷¹(), 韩心雨¹, 钟沐卉¹, 林耀忠¹, 柳腾飞¹, 李焰², 尹会方², 贾伟新¹^,*()

1. 华南农业大学兽医学院, 国家禽流感专业实验室(广州), 人兽共患病防控制剂国家地方联合工程实验室, 农业农村部人畜共患病重点实验室, 农业农村部兽用疫苗创制重点实验室, 广东省动物源性人兽共患病预防与控制重点实验室, 广东省人兽共患病防控制剂工程实验室, 广州 510640
2. 龙岩学院生命科学学院, 龙岩 364012

收稿日期:2024-05-22 出版日期:2025-04-23 发布日期:2025-04-28
通讯作者: 贾伟新 E-mail:shiyuting_01@stu.scau.edu.cn;jiaweixin@scau.edu.cn
作者简介:史玉婷(1999-), 女, 回族, 湖北武汉人, 兽医硕士生, 主要从事禽流感病毒相关研究, E-mail: shiyuting_01@stu.scau.edu.cn
基金资助:
广东省科技计划项目(2024B1212070013；2022B1111010004)；广东省现代农业产业技术体系创新团队建设项目(粤农农计[2024]147号)；国家肉鸡产业技术体系(CARS-41)；国家动物疫病监测与流行病学调查计划(2021-2025年)(农牧发[2021]11号)；龙岩市科技局产学研联合创新项目(龙岩)(2023LYF18001)

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

SHI Yuting¹(), HAN Xinyu¹, ZHONG Muhui¹, LIN Yaozhong¹, LIU Tengfei¹, LI Yan², YIN Huifang², JIA Weixin¹^,*()

1. College of Veterinary Medicine, South China Agricultural University, National Avian Influenza Para-Reference Laboratory (Guangzhou), National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Key Laboratory of Zoonoses, Key Laboratory of Animal Vaccine Development of Ministry of Agriculture, Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangdong Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510640, China
2. School of Life Sciences, Longyan University, Longyan 364012, China

Received:2024-05-22 Online:2025-04-23 Published:2025-04-28
Contact: JIA Weixin E-mail:shiyuting_01@stu.scau.edu.cn;jiaweixin@scau.edu.cn

摘要/Abstract

摘要：

旨在构建当前流行毒株H9N2亚型禽流感病毒(avian influenza virus, AIV)的反向遗传操作平台。毒株A/Chicken/Guangdong/M/2021(H9N2)(简称为M)分离自广东某养鸡场病料。鉴定并分析其全基因组序列，通过RT-PCR以及无缝克隆技术完成质粒构建，共转染293T细胞成功拯救毒株命名为rM。测定病毒的稳定性和生长曲线；进行动物试验，从传播效率、致病性角度验证克隆毒株与原毒的一致性。结果显示：M毒株为G57基因型，为近年的流行毒株。M毒株与rM毒株在酸稳定性、热稳定性、致病性以及传播性等方面保持一致的生物学特性。综上，成功建立了G57基因型H9N2亚型AIV的反向遗传系统，且从体内和体外试验两个方面全面验证M与rM的生物学特性保持一致。

关键词: 反向遗传, H9N2, 禽流感病毒, G57基因型

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

中图分类号:

S852.659.5

史玉婷, 韩心雨, 钟沐卉, 林耀忠, 柳腾飞, 李焰, 尹会方, 贾伟新. G57基因型H9N2亚型禽流感病毒反向遗传系统的建立与验证[J]. 畜牧兽医学报, 2025, 56(4): 1825-1833.

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.

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参考文献 21

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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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G57基因型H9N2亚型禽流感病毒反向遗传系统的建立与验证

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

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