两株新型鹅星状病毒的分离鉴定及其致病性

doi:10.3969/j.issn.1004-1524.20241082

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (12): 2458-2467.DOI: 10.3969/j.issn.1004-1524.20241082

两株新型鹅星状病毒的分离鉴定及其致病性

丁莹莹¹^,²(), 杨林平¹, 杨庆¹, 张子晨¹, 蔡霖颖¹, 杨康¹, 李琛¹, 刘惠文¹, 鲍光彬¹, 王晴¹, 王桂军¹^,²^,^*()

1.安徽农业大学动物科技学院,安徽合肥 230036
2.兽医病理生物学与疫病防控安徽省重点实验室,安徽合肥 230036

收稿日期:2024-12-12 出版日期:2025-12-25 发布日期:2026-01-09
作者简介:丁莹莹(2000—),女,安徽铜陵人,硕士研究生,主要从事畜禽传染病防治研究。E-mail:2548354778@qq.com
通讯作者: *王桂军,E-mail:wgj@ahau.edu.cn
基金资助:
安徽省家禽产业技术体系项目(2023—2024);安徽省高校协同创新项目(GXXT-2022-057)

Isolation, identification and pathogenicity of two novel goose astrovirus strains

DING Yingying¹^,²(), YANG Linping¹, YANG Qing¹, ZHANG Zichen¹, CAI Linying¹, YANG Kang¹, LI Chen¹, LIU Huiwen¹, BAO Guangbin¹, WANG Qing¹, WANG Guijun¹^,²^,^*()

1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
2. Anhui Provincial Key Laboratory of Veterinary Pathobiology and Disease Control, Hefei 230036, China

Received:2024-12-12 Online:2025-12-25 Published:2026-01-09

摘要/Abstract

摘要： 为确定安徽省两处鹅场的鹅群发病原因,从2023年6—7月收集到的2份疑似新型鹅星状病毒(goose astrovirus, GAstV)感染病料(其中一例为70日龄育成鹅)中分离病毒,并进行全基因组测序和动物回归试验。结果表明:共分离鉴定出两株GAstV,分别将其命名为HR2306/1、MG-23株。这两株分离株的基因组全长均为7 175 nt,其开放阅读框2(ORF2)的核苷酸和氨基酸序列与2016—2023年其他分离株的同源性高于97%。ORF2编码的氨基酸序列的比对结果显示,这两个分离株与HR2110/1株(作者团队于2021年分离得到)含有共同的氨基酸变异位点,推测这两个分离株可能是由HR2110/1株演变而来的。动物回归试验结果显示,两株分离株对雏鹅有强致病性,与自然感染的雏鹅的临床表现一致,肾表面出现明显尿酸盐沉积。病理切片和免疫组织化学观察可见明显的病理学变化与星状病毒抗原,表明肾为GAstV的靶器官。上述结果说明,GAstV可以感染70日龄育成鹅,且该病毒对雏鹅仍具有较强的致病力。

关键词: 新型鹅星状病毒, 育成鹅, 分离鉴定, 致病性

Abstract:

To determine the etiology of disease outbreaks in goose flocks at two farms in Anhui Province of China, viruses were isolated from two suspected cases of novel goose astrovirus (GAstV) infection collected between June and July 2023 (one case involving 70-day-old growing geese), followed by whole-genome sequencing and animal regression assays. The results demonstrated the successful isolation and identification of two novel GAstV strains, which were designated as HR2306/1 and MG-23. Genomic sequence analysis revealed that both strains had a complete genome length of 7 175 nucleotides (nt). Their ORF2 sequences exhibited nucleotide and amino acid sequence homologies exceeding 97% compared with strains isolated from 2016 to 2023. The alignment of ORF2 encoding amino acid sequences indicated shared variation sites between these two strains and the strain HR2110/1 (a GAstV strain isolated by the authors’ team in 2021), suggesting that HR2306/1 and MG-23 might be evolved from HR2110/1. Animal regression assays confirmed the high pathogenicity of both strains in goslings, with clinical manifestations consistent with natural infections. Characteristic urate deposits were observed on kidney surfaces, while histopathological sections and immunohistochemical analysis revealed significant pathological changes and astrovirus antigens, identifying the kidney as a target organ of GAstV. These findings indicated that GAstV could infect 70-day-old growing geese and retain strong pathogenicity in young goslings.

Key words: novel goose astrovirus, growing goose, isolation and identification, pathogenicity

中图分类号:

S852.657

丁莹莹, 杨林平, 杨庆, 张子晨, 蔡霖颖, 杨康, 李琛, 刘惠文, 鲍光彬, 王晴, 王桂军. 两株新型鹅星状病毒的分离鉴定及其致病性[J]. 浙江农业学报, 2025, 37(12): 2458-2467.

DING Yingying, YANG Linping, YANG Qing, ZHANG Zichen, CAI Linying, YANG Kang, LI Chen, LIU Huiwen, BAO Guangbin, WANG Qing, WANG Guijun. Isolation, identification and pathogenicity of two novel goose astrovirus strains[J]. Acta Agriculturae Zhejiangensis, 2025, 37(12): 2458-2467.

图/表 11

表1 用于全基因组测序的引物的基本信息

Table 1 Basic information of primers for whole genome sequencing

引物名称 Primer name	引物序列(5'→3') Primer sequences (5'→3')	引物位置 Primer location
GASTV-1-F	AAACAGCGATATGGCGGC	1~18
GASTV-1-R	TGTACGACTTAGTCCAAGGAACG	823~845
GASTV-2-F	CACAGAGGCTACAATGCT	378~395
GASTV-2-R	CCTTCAACAACGACAATGG	1 665~1 683
GASTV-3-F	CAGTCCCTGTACAGATTTTA	1 446~1 465
GASTV-3-R	TCAACTTGTTCATCCTTTAC	2 792~2 811
GASTV-4-F	AGATTGATGAAGCCATTGAG	2 604~2 623
GASTV-4-R	CAGCCCGCCGTTCTGTCTGT	3 939~3 958
GASTV-5-F	AGGCTGTATCAGATATTGAT	3 755~3 774
GASTV-5-R	TCATTTTGTCATTAACGGG	5 014~5 032
GASTV-6-F	TCTGGTGAGTGGCGGACCGA	4 799~4 818
GASTV-6-R	TGTCATAACAGCCCACCAATTGTGT	6 273~6 297
GASTV-7-F	ACAACTGGACAAGGTACC	6 028~6 045
GASTV-7-R	TTTGCGGATTTTAAATGC	7 131~7 148

图1 病例1(A~D)与病例2(E~H)临床发病鹅的剖检结果

Fig.1 Anatomical changes of clinically ill geese in case No. 1 (A-D) and case No. 2 (E-H)

图2 鹅星状病毒(GAstV)的反转录聚合酶链反应(RT-PCR)检测结果 M,DL2000 marker;1,GAstV阴性对照;2,病例1样品;3,病例2样品。

Fig.2 Reverse transcriptase-polymerase chain reaction (RT-PCR) results of goose astrovirus (GAstV) M, DL2000 marker; 1, GAstV negative control; 2, Sample of case No.1;3: Sample of case No.2.

图3 HR2306/1、MG-23株接种3代的鹅胚病变图 A,对照组;B,接种HR2306/1株的鹅胚;C,接种MG-23株的鹅胚。

Fig.3 Pathological changes of goose embryo inoculated with HR2306/1 and MG-23 strains for 3 generations A, Goose embryo of control group; B, Goose embryo inoculated with HR2306/1 strain; C, Goose embryo inoculated with MG-23 strain.

图4 F3代鹅胚的GAstV RT-PCR检测结果 M,DL2000 marker;1,阴性对照;2,接种HR2306/1株的F3代鹅胚尿囊液;3,接种MG-23株的F3代鹅胚尿囊液。

Fig.4 RT-PCR results of GAstV in F3 generation goose embryos M, DL2000 marker; 1, Negative control; 2, Allantoic fluid of F3 goose embryo inoculated with HR2306/1 strain ;3: Allantoic fluid of F3 goose embryo inoculated with MG-23 strain.

图5 LMH细胞接种鹅星状病毒后F3代的细胞病变情况(100×) A,对照组;B,HR2306/1株病毒液接种LMH细胞72 h;C,MG-23株病毒液接种LMH细胞72 h。

Fig.5 Cytopathic effect of F3 generation LMH cells inoculated with GAstV (100×) A, Control group; B, LMH cells inoculated with HR2306/1 viral fluids for 72 h; C, LMH cells inoculated with MG-23 viral fluids for 72 h.

图6 LMH细胞GAstV的RT-PCR检测结果 M,DL2000 marker;1,MG-23株接种后的 F3代病毒液检测样;2、3:阴性对照;4,HR2306/1株接种后的F3代病毒液检测样。

Fig.6 RT-PCR result of GAstV on LMH cells M, DL2000 marker; 1, Fluid sample of F3 generation with inoculation of MG-23 strain; 2, 3, Negative control; 4, Fluid sample of F3 generation with inoculation of HR2306/1 strain.

图7 基于星状病毒ORF2序列构建的系统发育树 “”标注序列为本研究分离毒株HR2306/1;“”标注序列为本研究分离毒株MG-23;“”标注序列为作者团队分离的其他毒株。GAstV-2,鹅星状病毒2型;TAstV-2,火鸡星状病毒2型;CAstV-1,鸡性状病毒1型;GAstV-1,鹅星状病毒1型;TAstV-1,火鸡星状病毒1型;ANV,禽肾炎病毒;HAstV,人星状病毒。

Fig.7 Phylogenetic tree based on the ORF2 sequence of astrovirus “ ” indicates the sequence of the isolated virus strain HR2306/1; “” indicates the sequence of the isolated virus strain MG-23; “” indicates the sequences of the other isolated virus strain by the authors’ team.GAstV-2,Goose astrovirus type 2; TAstV-2, Turkey astrovirus type 2; CAstV-1, Chicken astrovirus type 1; GAstV-1, Goose astrovirus type 1; TAstV-1, Turkey astrovirus type 1; ANV, Avian nephritis virus; HAstV, Human astrovirus.

表2 核苷酸和氨基酸序列的同源性与ORF2编码氨基酸的变异位点分析

Table 2 Analysis of nucleotide and amino acid sequences homology and variant sites of ORF2-encoded amino acids

年份 Year	分离毒株 Virus strains	核苷酸序列同源性 Nucleotide homology/%						氨基酸序列同源性 Amino acid homology/%		突变位点及其氨基酸 Mutation site and amino acid
		HR2306/1			MG-23			HR2306/1	MG-23	HR2306/1						MG-23
				ORF1a	ORF1b	ORF2	ORF1a	ORF1b	ORF2			26	228	380	608	614	630	228	608	614	695
												Q	T	S	T	T	A	S	T	T	T
2023	GXNN	98.6	99.3	98.3	98.4	99.2	98.7	99.0	99.4	H	S	N	T	T	T	S	T	T	A
	Q2001	97.3	99.0	97.6	97.0	98.6	97.5	97.9	98.2	H	A	N	S	A	T	A	S	A	A
	XJHT-2	98.3	99.2	99.1	97.8	98.9	99.0	99.4	99.7	H	T	N	T	T	T	T	T	T	S
2022	SD05	97.5	98.8	98.0	97.2	98.3	97.8	98.0	98.3	H	A	N	S	A	T	A	S	A	A
	GD2105	96.9	98.7	97.3	96.3	98.2	97.2	97.4	97.9	H	S	N	S	N	T	S	S	N	A
2021	HR2105/1	99.2	99.5	98.7	98.6	98.9	98.4	99.0	99.0	H	A	N	S	N	A	A	S	N	A
	HR2105/2	98.6	98.5	98.2	97.9	98.0	97.9	98.4	98.9	H	S	N	S	N	T	S	S	N	A
	HR2110/1	99.4	99.5	99.3	98.5	99.0	98.8	99.6	99.4	Q	T	N	T	T	T	T	T	T	A
	ZY02	98.6	98.7	98.0	98.0	98.2	97.7	97.9	98.3	H	S	N	S	N	T	S	S	N	A
	ZM	98.7	98.6	98.3	98.1	98.1	98.0	98.6	99.0	H	S	N	S	N	T	S	S	N	A
	GD2104	98.8	99.3	98.5	99.2	99.3	98.7	98.9	99.3	H	S	N	S	N	T	S	S	N	A
2020	HNKF-1	99.0	99.4	99.1	98.6	99.0	98.9	99.0	99.3	H	A	N	S	A	T	A	S	A	A
	HNSQ-6	98.9	99.4	99.1	98.5	98.9	98.9	99.0	99.3	H	A	N	S	A	T	A	S	A	A
2019	DY-19	99.0	99.5	98.8	98.6	99.0	98.6	98.4	98.7	H	A	N	S	A	T	A	S	A	A
	SDXT	96.3	98.2	98.2	96.0	97.8	97.9	98.0	98.3	H	A	N	S	A	T	A	S	A	A
2018	AHAU2018	97.5	98.7	98.0	97.1	98.1	97.8	97.9	98.2	H	A	N	S	A	T	A	S	A	A
	CXZ	96.7	98.5	98.3	96.3	98.0	98.0	98.2	98.4	H	A	N	S	A	T	A	S	A	A
	AHAU1	97.4	98.5	98.1	96.6	98.0	98.0	98.0	98.6	H	A	N	S	A	T	A	S	A	T
2017	SD01	97.8	98.5	97.8	97.3	98.1	97.5	97.4	97.7	H	A	N	S	A	T	A	S	A	A
	GD	97.5	98.5	97.6	96.9	98.0	97.4	98.0	98.3	H	A	N	S	A	T	A	S	A	A
	SDPY	97.1	98.6	98.2	96.7	98.2	98.1	98.4	99.0	H	A	N	S	A	T	A	S	A	T
2016	JSHA	97.2	98.6	98.0	96.9	98.0	97.7	98.0	98.3	H	A	N	S	A	T	A	S	A	A

表2 核苷酸和氨基酸序列的同源性与ORF2编码氨基酸的变异位点分析

Table 2 Analysis of nucleotide and amino acid sequences homology and variant sites of ORF2-encoded amino acids

年份 Year	分离毒株 Virus strains	核苷酸序列同源性 Nucleotide homology/%						氨基酸序列同源性 Amino acid homology/%		突变位点及其氨基酸 Mutation site and amino acid
		HR2306/1			MG-23			HR2306/1	MG-23	HR2306/1						MG-23
				ORF1a	ORF1b	ORF2	ORF1a	ORF1b	ORF2			26	228	380	608	614	630	228	608	614	695
												Q	T	S	T	T	A	S	T	T	T
2023	GXNN	98.6	99.3	98.3	98.4	99.2	98.7	99.0	99.4	H	S	N	T	T	T	S	T	T	A
	Q2001	97.3	99.0	97.6	97.0	98.6	97.5	97.9	98.2	H	A	N	S	A	T	A	S	A	A
	XJHT-2	98.3	99.2	99.1	97.8	98.9	99.0	99.4	99.7	H	T	N	T	T	T	T	T	T	S
2022	SD05	97.5	98.8	98.0	97.2	98.3	97.8	98.0	98.3	H	A	N	S	A	T	A	S	A	A
	GD2105	96.9	98.7	97.3	96.3	98.2	97.2	97.4	97.9	H	S	N	S	N	T	S	S	N	A
2021	HR2105/1	99.2	99.5	98.7	98.6	98.9	98.4	99.0	99.0	H	A	N	S	N	A	A	S	N	A
	HR2105/2	98.6	98.5	98.2	97.9	98.0	97.9	98.4	98.9	H	S	N	S	N	T	S	S	N	A
	HR2110/1	99.4	99.5	99.3	98.5	99.0	98.8	99.6	99.4	Q	T	N	T	T	T	T	T	T	A
	ZY02	98.6	98.7	98.0	98.0	98.2	97.7	97.9	98.3	H	S	N	S	N	T	S	S	N	A
	ZM	98.7	98.6	98.3	98.1	98.1	98.0	98.6	99.0	H	S	N	S	N	T	S	S	N	A
	GD2104	98.8	99.3	98.5	99.2	99.3	98.7	98.9	99.3	H	S	N	S	N	T	S	S	N	A
2020	HNKF-1	99.0	99.4	99.1	98.6	99.0	98.9	99.0	99.3	H	A	N	S	A	T	A	S	A	A
	HNSQ-6	98.9	99.4	99.1	98.5	98.9	98.9	99.0	99.3	H	A	N	S	A	T	A	S	A	A
2019	DY-19	99.0	99.5	98.8	98.6	99.0	98.6	98.4	98.7	H	A	N	S	A	T	A	S	A	A
	SDXT	96.3	98.2	98.2	96.0	97.8	97.9	98.0	98.3	H	A	N	S	A	T	A	S	A	A
2018	AHAU2018	97.5	98.7	98.0	97.1	98.1	97.8	97.9	98.2	H	A	N	S	A	T	A	S	A	A
	CXZ	96.7	98.5	98.3	96.3	98.0	98.0	98.2	98.4	H	A	N	S	A	T	A	S	A	A
	AHAU1	97.4	98.5	98.1	96.6	98.0	98.0	98.0	98.6	H	A	N	S	A	T	A	S	A	T
2017	SD01	97.8	98.5	97.8	97.3	98.1	97.5	97.4	97.7	H	A	N	S	A	T	A	S	A	A
	GD	97.5	98.5	97.6	96.9	98.0	97.4	98.0	98.3	H	A	N	S	A	T	A	S	A	A
	SDPY	97.1	98.6	98.2	96.7	98.2	98.1	98.4	99.0	H	A	N	S	A	T	A	S	A	T
2016	JSHA	97.2	98.6	98.0	96.9	98.0	97.7	98.0	98.3	H	A	N	S	A	T	A	S	A	A

图8 发病雏鹅的临床症状与剖检变化

Fig.8 Clinical observation and anatomical changes of diseased goslings

图9 雏鹅肾的苏木精-伊红(HE)染色(A)和免疫组织化学(IHC)染色(B)结果图中黑色箭头指示病理变化与阳性信号。

Fig.9 Hematoxylin-eosin (HE) staining (A) and immunohistochemistry (IHC) staining (B) result of goslings kidney Black arrows point out pathological changes and positive signals.

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两株新型鹅星状病毒的分离鉴定及其致病性

Isolation, identification and pathogenicity of two novel goose astrovirus strains

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