两株J亚型禽白血病病毒分离鉴定及全基因序列分析

doi:10.11843/j.issn.0366-6964.2025.12.035

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (12): 6326-6338.doi: 10.11843/j.issn.0366-6964.2025.12.035

两株J亚型禽白血病病毒分离鉴定及全基因序列分析

夏琦琦¹(), 桂亚萍¹(), 刘健¹, 杨显超¹, 陈琦¹, 沈莉萍¹, 冯桂丹¹, 向蒙¹, 徐平¹, 唐聪圣¹, 卫龙兴², 洪云超², 石慧花², 王建¹^,*(), 赵洪进¹^,*()

1. 上海市动物疫病预防控制中心, 上海 201103
2. 上海市奉贤区生态养殖服务中心, 上海 201400

收稿日期:2025-04-03 出版日期:2025-12-23 发布日期:2025-12-24
通讯作者: 王建,赵洪进 E-mail:1766854941@qq.com;1679163002@qq.com;jianwhlj@163.com;zhaohongjin945@163.com
作者简介:夏琦琦(1996-)，女，黑龙江哈尔滨人，兽医师，博士，主要从事动物疫病防控的研究，E-mail: 1766854941@qq.com, Tel: 010-62696318
桂亚萍(1995-)，女，安徽池州人，兽医师，硕士，主要从事动物病毒学研究，E-mail: 1679163002@qq.com
第一联系人：
夏琦琦和桂亚萍为同等贡献作者
基金资助:
2023上海东方英才领军项目

Isolation, Identification, and Whole Gene Sequence Analysis of Two J Subtype Avian Leukemia Virus

XIA Qiqi¹(), GUI Yaping¹(), LIU Jian¹, YANG Xianchao¹, CHEN Qi¹, SHEN Liping¹, FENG Guidan¹, XIANG Meng¹, XU Ping¹, TANG Congsheng¹, WEI Longxing², HONG Yunchao², SHI Huihua², WANG Jian¹^,*(), ZHAO Hongjin¹^,*()

1. Shanghai Animal Disease Prevention and Control Center, Shanghai 201103, China
2. Shanghai Fengxian District Ecological Breeding Service Center, Shanghai 201400, China

Received:2025-04-03 Online:2025-12-23 Published:2025-12-24
Contact: WANG Jian, ZHAO Hongjin E-mail:1766854941@qq.com;1679163002@qq.com;jianwhlj@163.com;zhaohongjin945@163.com

摘要/Abstract

摘要：

为了探究禽白血病病毒(ALV)的进化趋势和分子特征，本研究从上海市某鸡场采集了1 600份血液样品，接种鸡成纤维细胞系(DF-1)分离病毒，通过PCR扩增gp85基因序列进行亚群分型，进一步通过间接免疫荧光试验(IFA)和多步生长曲线对其生物学特性进行鉴定，并将分离株和ALV各亚群毒株的编码区和非编码区进行序列比对和分子构象分析。结果显示，通过PCR和IFA鉴定本研究共分离到了6株ALV-J亚群毒株，其中ALV-J-345株和ALV-J-480株序列存在明显差异，且ALV-J-480株在DF-1细胞上比ALV-J-345株具有更强的复制能力，进一步比对两株分离株和ALV-J其他毒株的序列发现，两株分离株的gp85和gp37蛋白之间共存在46个差异氨基酸位点，且ALV-J-480株和其他ALV-J毒株的相似性更低，除此之外ALV-J-480株pol基因在第866个氨基酸位置提前产生一个终止密码子，使其缩短了8个氨基酸，在非编码区位置上ALV-J-345株在3'UTR的E(XSR)元件区缺失了7个核苷酸，而ALV-J-480株在5'UTR的第358~376位置处多插入了19 nt的核苷酸，这些位置上的序列差异，可能是导致ALV-J-480株具有更强复制能力的原因，这使得其在鸡群中有成为新的流行毒株的竞争优势。因此本研究提供了关于ALV-J分子特征的最新数据，这将有助于揭示ALV-J的进化趋势，并为制定相关的防控措施提供参考。

关键词: 禽白血病病毒, 分离鉴定, gp85, 序列分析, 基因变异

Abstract:

To investigate the evolutionary trends and molecular characteristics of Avian leukosis virus (ALV), this study collected 1 600 blood samples from a poultry farm in Shanghai. Viruses were isolated by inoculating chicken fibroblast cells (DF-1), followed by subgroup identification through PCR amplification of the gp85 gene. Biological characterization was performed using indirect immunofluorescence assay (IFA) and multi-step growth curves. Coding and non-coding regions of the isolated strains were compared with representative ALV subgroups for sequence alignment and molecular conformation analysis. The results showed that six ALV-J strains were successfully isolated and identified via PCR and IFA. Notably, strains ALV-J-345 and ALV-J-480 exhibited significant sequence divergence, with strain ALV-J-480 demonstrating stronger replication efficiency in DF-1 cells than strain ALV-J-345. Further comparison with other ALV-J strains revealed 46 distinct amino acid variations in the gp85 and gp37 protein between the two isolates, and strain ALV-J-480 displayed lower homology to other ALV-J strains. Additionally, a premature termination codon at position 866 in the pol gene of strain ALV-J-480 truncated the protein by eight amino acids. In non-coding regions, strain ALV-J-345 harbored a 7-nucleotide deletion in the E(XSR) element region of the 3'UTR, whereas strain ALV-J-480 carried a 19 nt nucleotide insertion at positions 358-376 in the 5'UTR. These sequence variations likely contributed to the enhanced replication capacity of strain ALV-J-480, endowing it with a competitive advantage to emerge as a potential epidemic strain in poultry populations. Therefore, this study provides updated molecular insights into ALV-J, which will aid in elucidating its evolutionary dynamics and inform the development of targeted control strategies.

Key words: avian leukosis virus, isolation and identification, gp85, sequence analysis, genetic variation

中图分类号:

S852.659.3

夏琦琦, 桂亚萍, 刘健, 杨显超, 陈琦, 沈莉萍, 冯桂丹, 向蒙, 徐平, 唐聪圣, 卫龙兴, 洪云超, 石慧花, 王建, 赵洪进. 两株J亚型禽白血病病毒分离鉴定及全基因序列分析[J]. 畜牧兽医学报, 2025, 56(12): 6326-6338.

XIA Qiqi, GUI Yaping, LIU Jian, YANG Xianchao, CHEN Qi, SHEN Liping, FENG Guidan, XIANG Meng, XU Ping, TANG Congsheng, WEI Longxing, HONG Yunchao, SHI Huihua, WANG Jian, ZHAO Hongjin. Isolation, Identification, and Whole Gene Sequence Analysis of Two J Subtype Avian Leukemia Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6326-6338.

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	产物长度/bp Product length
ALV-F	CCGGAGAAGACACCCTTGCT
ALV-A-R	TTGCCACAGCGGTACTGCTG	710
ALV-B-R	AGAAAGGATTGYTCCCTGGG	576
ALV-J-R	GAGTTGCGGCCTGGACCAAT	425
ALV-K-R	CACCGAACCAAAGGTAACAC	534

亚群 Subgroup	毒株 Strain	GenBank登录号 GenBank No.	年份 Year	分离地区 Region of isolation
A	A46	DQ412726.1	2006	美国
	SDAU09C1	HM452339.1	2009	中国
	MQNCSU	DQ365814.1	2016	美国
	JS-A1201	KC282889.1	2012	中国
	RAV-1	MF926337.1	1980	美国
	WB11031a	JX570801.1	2011	中国
B	GifN 001	MK757486.1	2015	日本
	GX10LS01	KM058055.1	2010	中国
	SDLYB1901	MT648687.1	2019	中国
	Schmidt-Ruppin B	AF052428.1	1998	美国
	JS-B1204	KC282899.1	2012	中国
	RAV-2	M14902.1	1986	美国
	WB11149b	JX570799.1	2011	中国
C	Prague C	J02342.1	1977	美国
D	Schmidt-Ruppin D	D10652	1992	日本
E	RAV0	MF817822.1	1980	美国
	SDO5O1	EF467236.1	2007	中国
	ev-1	AY013303.1	2000	美国
	AF229	MF817820	2010	美国
	ALVE-B11	KC610517.1	2013	加拿大
J	HLJ13SH01	KM376510.1	2013	中国
	SVR807	HM776937.1	2008	俄罗斯
	AHaq02	KF534753.1	2012	中国
	PK19FA01	MN956379.1	2018	中国
	HPRS103	Z46390.1	1994	英国
	ADOL-7501	AY027920.1	2001	美国
	PDRC-59831	KP284572.1	2007	美国
	GX14NN01	MN735292.1	2014	中国
	NX0101	DQ115805.1	2005	中国
K	GD14LZ	KU605774.1	2014	中国
	TW-3593	HM582658.1	2008	中国台湾
	KmN 115	OP620428.1	2022	日本
	JS13-DX5	KF999961.1	2013	中国
	SDAUAK-13	KY773923.1	2016	中国
	JS11C1	KF746200.1	2012	中国

引物名称 Primer Name	引物序列(5′→3′) Primer Sequence
gp85-qPCR-F	TTGCAACAACCAGGAAACGT
gp85-qPCR-R	GGTGAGGTCGCTGACTGTA
qGAPDH-F	CCCCCATGTTTGTGATGGGT
qGAPDH-R	GCACGATGCATTGCTGACAA

毒株 Strain	差异氨基酸位点 Differential amino acid sites
毒株 Strain	193	217	236	288	435	750	755	789	848
ALV-J-345	G	I	A	T	V	K	S	V	T
ALV-J-480	E	V	T	A	M	R	N	I	I

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两株J亚型禽白血病病毒分离鉴定及全基因序列分析

Isolation, Identification, and Whole Gene Sequence Analysis of Two J Subtype Avian Leukemia Virus

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