鸭星状病毒1型信阳株全基因组扩增及遗传进化分析

doi:10.16431/j.cnki.1671-7236.2026.02.040

Abstract

Abstract:

Objective This study aimed to investigate the evolution characteristics of complete genome of Duck astrovirus type 1 （DAstV1） in Xinyang region，and provide a genomic reference for future studies on its epidemiology， genetic evolution，and pathogenicity. Method Clinical samples from diseased ducks submitted by a commercial farm were screened by PCR/RT-PCR for twelve common avian viruses， including Fowl adenovirus， Avian astrovirus and Duck hepatitis A virus. Liver tissues were collected， sterilized， and inoculated into 10-day-old SPF duck embryos via the yolk-sac route， and passaged four times. Allantoic fluid from each passage was tested for DAstV by RT-PCR. The isolated virus was subjected to whole genome sequencing， and the nucleotide sequences of the ORF1a， ORF1b and ORF2 genes， as well as the amino acid variation sites of their encoded proteins， were analyzed and compared. Result Avian astrovirus was detected in the clinical samples. DAstV1 was consistently positive in allantoic fluids from the 1st to 4th passage duck embryos. The isolate was designated HN24XY06. All fourth-passage embryos died following inoculation， exhibiting poor development and subcutaneous hemorrhage. The genome of HN24XY06 was 7 755 nt in length and contained three open reading frames： ORF1a， ORF1b， and ORF2. Sequence alignment and phylogenetic analysis showed that HN24XY06 belonged to DAstV1 strain and was closely related to the Shandong isolates DAstV-SDZZ and DAstV-SDWF. Sequence analysis of the amino acid sites of the ORF1a， ORF1b and ORF2 proteins showed that these proteins had mutations to varying degrees， mainly in the amino acid sequence encoded by ORF1a. Conclusion A strain of DAstV1 was successfully isolated in this study， thereby enriching the molecular epidemiological data of DAstV1 in Xinyang region and providing a solid foundation for further investigations into its pathogenic mechanisms.

Key words: Duck astrovirus type 1 （DAstV1）; virus isolation and identification; whole genome amplification; phylogenetic analysis

CLC Number:

S852.65^＋9.6

ZHANG Min, LI Yingxiao, ZHANG Lulu, HE Shuhai, QU Zhehui, QIN Dongsheng, LIU Jicheng, JIAO Fengchao. Whole Genome Amplification and Phylogenetic Analysis of Duck Astrovirus Type 1 Xinyang Strain[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(2): 959-972.

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病毒 Viruses	引物序列 Primer sequences （5'→3'）	产物长度 Product length/bp	退火温度 Annealing temperature/℃	参考文献 References
AAstV	F：GAYTGGACNMGNTWYGAYGGNACNATNCC	434	45	Todd等^［7］
	R：YTTNACCCACATNCCRAA	434	45	Todd等^［7］
GAstV	F：ATTCTTGGCTCGGTTGTC	489	54	Zhang等^［3］
	R：CCTGTGTTGCTCCTTCTC	489	54	Zhang等^［3］
DHAV1	F：GGAGGTGGTGCTGAAATAT	214	55	Chen等^［16］
	R：CATGTGCCTGGACAGAT	214	55	Chen等^［16］
DHAV3	F：GAAATCTGCACTCAATGGAGAG	289	55	Chen等^［16］
	R：CCCAGGAAATGATTGGTCAG	289	55	Chen等^［16］
NGPV	F：GACTTAGACAGGCTTATGAAC	781	55	杨晶等^［17］
	R：ATTGCTGGTGAACTGGATT	781	55	杨晶等^［17］
ARV	F：TAGCCACACCCGTGCTAGGA	850	55	杨晶等^［17］
	R：ATAGAGGTRTTRATACCACG	850	55	杨晶等^［17］
DPV	F：ATGTCTAATTCTGGAGGTTC	354	52	杨晶等^［17］
	R：TTATCGCTGATCGTCTG	354	52	杨晶等^［17］
DTMUV	F：GCCACGGAATTAGCGGTTGT	401	55	杨晶等^［17］
	R：TAATCCTCCATCTCAGCGGTGTAG	401	55	杨晶等^［17］
NDV	F：CTGCACATAGAAGAGGAGCTA	427	55	杨晶等^［17］
	R：CTGGTGCTGGTAGTCATGG	427	55	杨晶等^［17］
AIV	F：GGGAGCAAAAGCAGGAGT	1413	53	杨晶等^［17］
	R：GGAGTAGAAACAAGGAGTTTTTT	1413	53	杨晶等^［17］
DuCV	F：TGGGGACCACTCATACGATT	449	55	杨晶等^［17］
	R：ATGCGTGAGAGTTGACGACT	449	55	杨晶等^［17］
FAdV	F：CAACTACATCGGGTTCAGGGATAACTTC	766	55	杨晶等^［17］
	R：CCAGTTTCTGTGGTGGTTGAAGGGGTT	766	55	杨晶等^［17］
DAstV1	F：CTTGGACTGTGGAAGCATATACC	763	55	Wei等^［14］
	R：GTTGAAAACTGCCCTGAAGG	763	55	Wei等^［14］
DAstV2	F：GCAGCGAATGTGGTTGGTTC	401	55	刘宁^［10］
	R：TCTGGAGTCGGCTGGATTCT	401	55	刘宁^［10］
DAstV3	F：AATGTGCCCAGTGTGATCGT	482	55	刘宁^［10］
	R：TTCGTTGCGCATTGTGTCAG	482	55	刘宁^［10］
DAstV4	F：AGGCTGGAAGGGAATTCTATCT	436	53	Li等^［18］
	R：ATGGTGATTCCAGTGTCAGCG	436	53	Li等^［18］
DAstV5	F：AATGTAGGGAGGACCGAAAGTAG	597	55	Zhang等^［3］
	R：GTTTGTCCTAACTCCTATTGG	597	55	Zhang等^［3］

引物名称 Primer name	引物序列 Primer sequences （5'→3'）	产物长度 Product length/bp
P1F	CCGAARTGGGCGAGTC	919
P1R	CCAGGTGTCAACAATCATGC	919
P2F	GATGTTTATGGTGAGTTGTACACWG	972
P2R	GCTAGATGGTATTATGCCTCTTG	972
P3F	TTGCAGCTCACTCTGGCATA	666
P3R	TGATGTCACTCCCTGTGAASC	666
P4F	CTCGCGAAAGAAGTTAAGAGTG	971
P4R	CCTTCTTCATCGATGTCATTATCC	971
P5F	CAGAGAATGCTTGATGAAGGY	1 297
P5R	TTWGGATACGCTGGTGTTGA	1 297
P6F	CTTGGACTGTGGAAGCATATACC	763
P6R	GTTGAAAACTGCCCTGAAGG	763
P7F	GTTACCAACTGGAGAAGTCTGTCA	903
P7R	TCCAAGTGTGACCACTGTTGTC	903
P8F	GACAACAGTGGTCACACTTGGA	1 082
P8R	CCATGTGTGGTGTATATTGTGATR	1 082
P9F	GAGGTGTTCTGGGCAGTTTCAA	1 113
P9R	CTCCATCATCATCCTCTCACACTG	1 113
P10F	AGTGTGAGAGGATGATGATGGA	240
P10R	TGTGACCCACATGGTGATTTC	240
3'-Race-P1F	AGTGTGAGAGGATGATGATGGA	240
3'-Race-P1R	GACTCGAGTCGACATCGA（T）18	240
3'-Race-P2F	ACAGCTGCACTTTCTCATGC	236
3'-Race-P2R	GACTCGAGTCGACATCGA	236

毒株 Strains	分离年 Year	类型 Type	分离地 Location	GenBank登录号 GenBank accession No.
DAstV-SDZZ	2020	DAstV1	中国	MW217578.1
DAstV-SDWF	2021	DAstV1	中国	OL652659.1
HB2015	2015	DAstV1	中国	KY646154.1
DA08	-	DAstV1	中国	FJ919227.1
DA07	-	DAstV1	中国	FJ919226.1
D51	2012	DAstV1	中国	MH712856.1
WF1201	2012	DAstV1	中国	JX439643.1
D390	2013	DAstV1	中国	MN149395.1
D120	2013	DAstV1	中国	MN149393.1
D17	2013	DAstV1	中国	MN149392.1
C-NGB	2008	DAstV1	中国	NC_012437.1
AAstV/Chicken/CHN/2018/CZ1801	2018	AAstV	中国	MN807051.1
TAstV/VA/99	－	TAstV	美国	EU143851.1
TAstV/CA/00	－	TAstV	美国	EU143844.1
YP2	－	DAstV4	中国	JX624774.1
TAstV/AK/98	－	TAstV	美国	EU143843.1
X1257	2021	DAstV	中国	OR924091.1
C1365	2023	DAstV	中国	OR902760.1
C1388	2023	DAstV	中国	OR902759.1
X2250	2021	DAstV	中国	OR924090.1
CPH NM1002	2021	DAstV3	中国	OR924089.1
CPH	2013	DAstV3	中国	KJ020899.1
JM	2021	DAstV5	中国	OM095382.1
SL1	2012	DAstV2	中国	KF753804.1
SL5	2012	DAstV2	中国	KF753807.1
Du/Egy/Gharbia/2021/DAstVs	2021	DAstV	埃及	PP512783.1
JSHA	2016	GAstV	中国	MK125058.1
GD	2017	GAstV	中国	MG934571.1
GAstV-SDHZ	2022	GAstV	中国	OP946449.1
AstV/Goose/2018/HLJ01	2018	GAstV	中国	MN175321.1
AstV/Goose/Guangdong/1812LMG/2018	2018	GAstV	中国	MN127959.1

毒株 Strains	类型 Type	相似性 Similarities /%	毒株 Strains	类型 Type	相似性 Similarities /%
DAstV-SDZZ	DAstV1	97.8	X1257	DAstV	54.4
DAstV-SDWF	DAstV1	97.8	C1365	DAstV	54.4
HB2015	DAstV1	97.6	C1388	DAstV	54.5
DA08	DAstV1	96.0	X2250	DAstV	54.2
DA07	DAstV1	95.4	CPH NM1002	DAstV3	57.3
D51	DAstV1	91.2	CPH	DAstV3	57.3
WF1201	DAstV1	91.2	JM	DAstV5	54.3
D390	DAstV1	91.2	SL1	DAstV2	56.6
D120	DAstV1	91.2	SL5	DAstV2	56.9
D17	DAstV1	91.1	Du/Egy/Gharbia/2021/DAstVs	DAstV	57.4
C-NGB	DAstV1	91.0	JSHA	GAstV	57.4
AAstV/Chicken/CHN/2018/CZ1801	AAstV	57.2	GD	GAstV	57.6
TAstV/VA/99	TAstV	61.1	GAstV-SDHZ	GAstV	57.6
TAstV/CA/00	TAstV	60.9	AstV/Goose/2018/HLJ01	GAstV	57.5
YP2	DAstV4	54.5	Astv/Goose/Guangdong/1812LMG/2018	GAstV	56.7
TAstV/AK/98	TAstV	61.3

毒株 Strains	开放阅读框 Open reading frame	突变位点 Mutational site
C-NGB	ORF1a	Q11E、E17Q^、S25P、P26F、G50D、D50N、S103A、I104L、I143F、M148V、A185V、H192Q、F198Y^、M230I、L230I、E266D^、P283S、K306N、Q346K、H346Y、A361V^、S379T、I381F、I385M、T421K、L423I、T449I、M465I、S493K^、S501F、M504F、I528V、I545V、V565I、V595I、A624V、A651V、S677A、I700V、R747Q、L751P、T752I、V753A^、K757R、D761T、R766K、I817T、T831I、E833K^、I834T、R836G、A840T、A841T、K844N、S846C^、R856G^、E859K^、F862Y、T864A、A871T、T873A、T885A、K884R、E888G^、S971N、A1152V、L1156P、V1161A^、S1192N、I1217V、S1238P
	ORF1b	E19K^、K60R、E108D^、I132M、N172S、K172R、F261Y、N303D、A324V、S3949、T396I、I397V、I437V
	ORF2	P18L、K43R^、H60R、M113L、R137K、R140K、K280T、R307K、N373S、T392A、L427S、R4297P、P437S、S438P^、L461F、N471S、D487S、N488S、V514L、S516A、A520T、R529K、A597E、P602T、F604Y、N620D、S623T、D637E、N639V、V645E、V650G、L663F、E668D、L716P
DAstV-SDWF	ORF1a	E17Q^、F198Y^、R223K、E266D^、A361V^、I385M、M465I、S493K^、V753A^、E833K^、A840T、S846C^、K854E、K856G^、E859K^、E888G^、T896A、F961S、V1161A^、A1233V
	ORF1b	E19K^、E108D^、M324V
	ORF2	K43R^、S438P^、K451Q
DAstV-SDZZ	ORF1a	E17Q^、F198Y^、R223K、E266D^、A361V^、I385M、S387A、I428L、S493K^、V753A^、V808L、E833K^、V838M、A840T、S846C^、R856G^、E859K^、E888G^、I998M、V1161A^、A1233V
	ORF1b	E19K^、R35G、K76Q、E108D^、K228Q、M324V
	ORF2	K43R^、S438P^、N488S

Whole Genome Amplification and Phylogenetic Analysis of Duck Astrovirus Type 1 Xinyang Strain

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