三株新型鸭源微RNA病毒分离毒株的全基因组序列分析

doi:10.11843/j.issn.0366-6964.2024.07.026

摘要/Abstract

摘要：

分离新型鸭源微RNA病毒进行全基因组测序并进行遗传进化分析。对本实验室2021年不同来源的种鸭和肉鸭病料进行PCR检测，初步确定存在一种未知分类的新型微RNA病毒感染。取病死鸭病料组织处理后接种SPF鸡胚分离病毒，设计引物对分离到的病毒进行PCR检测，通过重叠PCR方法进行全基因组扩增测序。将分离病毒各蛋白氨基酸序列两两比对，同时选取GenBank数据库中微RNA病毒代表毒株序列绘制系统进化树，并对主要蛋白P1、2C、3D序列比对分析。结果显示：共分离到三株微RNA病毒，分别命名为21101株、21016株和21075株(GenBank登录号：OQ927377~OQ927379)。基因组长度分别为7 445、7 445和7 447 bp，均包含一个编码2 141个氨基酸的开放阅读框(ORF)，可划分为P1、P2、P3三个部分，符合微RNA病毒序列特征。基于全基因组序列遗传进化分析发现，三株分离病毒与本实验室前期分离的Duck/FC22/China/2017(GenBank登录号：MN102111)毒株及上海兽医研究所分离的Duck/AH15/CHN/2015(GenBank登录号：MT681985)位于同一分支，与鸭甲型肝炎病毒(Duck hepatitis A virus，DHAV)遗传距离最近。分离的三株鸭源微RNA病毒进行全基因组测序及遗传进化分析发现，与目前已知的两株微RNA毒株为同一类新型鸭源微RNA病毒。

关键词: 新型微RNA病毒, 分离鉴定, 基因组测序, 遗传进化分析

Abstract:

The aim of this study was to isolate novel duck-derived picornavirus for whole genome sequencing and genetic in order to perform whole genome sequencing and genetic evolutionary study. In 2021, we identified the presence of an unknown class of novel picornavirus infection in the diseased materials of breeding ducks and meat ducks submitted for inspection. By inoculating the diseased material into specific pathogen-free (SPF) chicken embryos, we isolated the virus. And the isolated viruses were further detected by PCR with primers designed from sequences available and sequenced the whole genome by overlapping PCR method. The amino acid sequences of each protein of the isolated virus were compared. At the same time, the sequences of the representative strains of small RNA viruses in the GenBank database were selected to draw the phylogenetic tree, and the sequences of the main proteins P1, 2C and 3D were analysed in comparison with each other. Three small RNA virus strains were isolated and named 21101, 21016 and 21075 (GenBank ID: OQ927377-OQ927379), with genome lengths of 7 445, 7 445, and 7 447 bp. These sequence encompasses an open reading frame (ORF) that encodes a total of 2 141 amino acids. The ORF can be divided into three distinct parts: P1, P2 and P3. These protein divisions align with the characteristic features observed in picornaviruses. The genetic evolutionary analysis, conducted through the utilization of whole genome sequences, demonstrated that the three isolates were positioned within the same branch as the Duck/FC22/China/2017 strain (GenBank ID: MN102111) previously separate inour laboratory and Duck/AH15/CHN/2015 strain (GenBank ID: MT681985) at the Shanghai Veterinary Research Institute. Furthermore, these isolates exhibited the highest degree of genetic similarity to the Duck hepatitis A virus (DHAV). Three strains of duck-derived picornavirus were isolated, and whole genome sequencing and genetic evolution analysis revealed that the three isolated viruses belong to the same class of the two currently known novel duck-derived picornavirus.

Key words: novel picornavirus, isolation and identification, genome sequence, genetic evolutionary analysis

中图分类号:

S852.659.6

李继桐, 朱彤, 吕俊峰, 高月花, 胡峰, 于可响, 宋敏训, 王建琳, 李玉峰. 三株新型鸭源微RNA病毒分离毒株的全基因组序列分析[J]. 畜牧兽医学报, 2024, 55(7): 3075-3084.

Jitong LI, Tong ZHU, Junfeng LÜ, Yuehua GAO, Feng HU, Kexiang YU, Minxun SONG, Jianlin WANG, Yufeng LI. Isolation and Identification of Novel Picornavirus from Ducks and Whole Genome Sequence Analysis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3075-3084.

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名称Name	序列(5′→3′) Sequences	片段大小/bp Fragment length
DHAV1F	GACTGTGCAACACGCTTCAAC	473
DHAV1R	AATCTACTTCATCCCCAGACTG
DHAV3F	TGTGTATCTTATGAGCAGGCCA	646
DHAV3R	AGCCCAACACGGCAAGCAC
DTMUVF	AGGAATTCATGTCTAACAAAAAACCAGG	360
DTMUVR	CCCTCGAGCAGCCCAGCAACTATCG
DAstVF	GTTGGACHCCCTTCTWTGG	519
DAstVR	GCTTCACCCACATDCCAAA
MDPVF	GGAAATTGGCATTGCGATTC	390
MDPVR	CCGAGCCCAGGACATACG
AIVF	TTCTAACCGAGGTCGAAAC	230
AIVR	AAGCGTCTACGCTGCAGTCC
MDRVF	CTATCAGCACAGTGAGCAGC	188
MDRVR	GTGCGTGTTGGAGTTTCCCG
NDVF	ATATGAGACGGCAGATGAGTC	540
NDVR	CAGTGTTCCTGATGTATGAGTC
DPVF	TCCTGGAACAATCACAAC	690
DPVR	TCGCCTGCCAACTTAT
PIVF	GGTGGYATGTGTTCAGGYTC	588
PIVR	TTYAGGGTATCCYRCAACAC

名称Name	序列(5′→3′) Sequences	片段大小/bp Fragment length
FC22-F	CGGGGTGTAGTAGCGAGTC	133
FC22-R	GGAAGTTAGAGGTCTGGGTATTGG

名称Name	序列(5′→3′) Sequences	片段大小/bp Fragment length
CX1-F	TTTGAAAACTGGCCCCCCTGGGGA	2 279
CX1-R	ACACTAGATGACGTGTGGCTAGCTC
CX2-F	GCTCAGCACTCGTGCTGACCTAGG	2 352
CX2-R	TAAAAAGTGCCTTTCTGTTCTAGGG
CX3-F	TCTTTCTTTGCAAACATGATTGC	2 140
CX3-R	TCCTCTTGTAGATGTTTCCCATAACC
CX4-F	TTCTTCCAATTGATTGGTCAAC	1 448
CX4-R	TTTTTTTTTTTTTTTAAAAGTGTAGGGAAA

毒株 Strain	氨基酸位置 Position of amino acids
毒株 Strain	151	160	278	309	352	373	597	701	725	1 355	1 467	2 016	2 018
21101	I	Y	R	D	T	A	V	F	D	I	H	R	K
21016	I	Y	R	D	T	A	V	F	D	I	Y	K	K
21075	V	F	K	E	N	V	I	L	E	V	H	K	I

微RNA病毒科 Picornaviridae				氨基酸序列相似性Pairwise amino acid sequence identity
属 Genus	种 Species	登录号 GenBank No.	来源 Source	21101				21016				21075
属 Genus	种 Species	登录号 GenBank No.	来源 Source	P1	2C	3D	多聚蛋白 Polyprotein	P1	2C	3D	多聚蛋白 Polyprotein	P1	2C	3D	多聚蛋白 Polyprotein
Aalivirus (Avihepatovirus/Avisivirus-like virus)	Duck aalivirus 1	NC023985	Duck	33.2	42.7	43.8	38.2	33.2	42.7	43.8	38.2	33.1	42.7	43.8	38.1
禽肝病毒属 Avihepatovirus	Duck hepatitis A virus 1	NC008250	Duck	37.0	43.3	43.8	41.1	37.0	43.3	43.8	41.1	37.3	43.3	43.8	41.2
禽肝病毒属 Avihepatovirus	Duck hepatitis A virus 2	OQ862826	Duck	31.0	41.4	44.9	42.6	31.0	41.4	44.9	42.5	31.1	41.4	44.7	42.7
禽肝病毒属 Avihepatovirus	Duck hepatitis A virus 3	KU860089	Duck	36.9	44.5	43.8	41.2	36.9	44.5	43.8	41.3	37.3	44.5	43.6	41.3
Avisivirus	Turkey avisivirus	KC614703	Turkey	34.5	40.2	37.4	34.6	34.5	40.2	37.4	34.6	34.7	40.2	37.4	34.7
未指定 Unassigned	Red-crownedcrane picornavirus	KY312544	Crane	33.0	48.5	48.4	41.8	33.0	48.5	48.4	41.8	33.0	48.5	48.2	41.7
嵴病毒属 Kobuvirus	Porcine kobuvirus	EU787450	Porcine	10.3	21.0	17.4	16.0	10.3	21.0	17.0	16.1	10.3	21.0	17.2	16.1
Megrivirus	Turkey hepatitis virus	HM751199	Turkey	12.3	23.4	18.3	15.5	12.3	23.4	18.3	15.5	12.1	23.4	18.1	15.4
震颤病毒属 Tremovirus	Avian encephalomyelitis virus	NC003990	Avian	12.9	21.4	19.4	16.1	12.9	21.4	19.6	16.2	13.1	21.4	19.6	16.1
Orthoturdivirus	Turdivirus 1	GU182406	Birds	11.7	23.2	15.0	17.1	11.7	23.2	15.0	17.1	11.5	23.2	15.0	17.0
肠道病毒属 Enterovirus	Poliovirus	NC002058	Human	10.8	22.6	20.0	15.0	10.8	22.6	20.0	15.1	10.9	22.6	20.0	15.0
口蹄疫病毒属 Aphthovirus	Foot-and-mouth disease virus	NC002554	Porcine	9.3	9.7	15.6	11.1	9.3	9.7	15.6	11.2	9.6	9.7	15.6	11.2