四川地区1株鲤春病毒血症病毒的分离鉴定与全基因组分析

doi:10.3969/j.issn.1004-1524.20240655

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (7): 1430-1440.DOI: 10.3969/j.issn.1004-1524.20240655

四川地区1株鲤春病毒血症病毒的分离鉴定与全基因组分析

祁英吉^a(), 刘舒亚^a, 兰寒韵^a, 王雪梅^a, 孙甜甜^a, 李书含^a, 黎秋男^a, 黄小丽^b, 耿毅^a, 陈德芳^b, 欧阳萍^a^,^*()

四川农业大学 a. 动物医学院; b. 动物科技学院,四川成都 611130

收稿日期:2024-07-19 出版日期:2025-07-25 发布日期:2025-08-20
作者简介:祁英吉(2003—),女,安徽宣城人,本科生,研究方向为水生动物疾病。E-mail: 1251138068@qq.com
通讯作者: ^*欧阳萍, E-mail: ouyang.ping@live.cn
基金资助:
四川农业大学科研兴趣培养项目(20241107)

Isolation, identification and genome-wide analysis of Spring viremia of carp virus in Sichuan Province of China

QI Yingji^a(), LIU Shuya^a, LAN Hanyun^a, WANG Xuemei^a, SUN Tiantian^a, LI Shuhan^a, LI Qiunan^a, HUANG Xiaoli^b, GENG Yi^a, CHEN Defang^b, OUYANG Ping^a^,^*()

College of Veterinary Medicine; b. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China

Received:2024-07-19 Online:2025-07-25 Published:2025-08-20

摘要/Abstract

摘要： 2022年3月四川省乐山市某养殖场的鲤出现大面积死亡,累计死亡率80%。为研究此次疾病的病因和流行规律,将病料解剖,进行病理学检查、细菌学检查、PCR鉴定,以及病毒分离鉴定与系统进化分析。结果显示,病理学病变主要在鳃和肠道,肠道充血、出血,上皮细胞坏死脱落;鳃小片上皮细胞增生、坏死、脱落。根据病理学诊断初步判断患病鱼为鲤春病毒血症病毒(Spring viremia of carp virus, SVCV)感染。RT-PCR检测结果显示,在自然发病鲤、人工感染发病鲤的内脏组织和细胞培养病毒液中均扩增出SVCV的G蛋白基因序列。细胞培养病毒液的超薄切片电镜观察结果显示,病毒粒子长80~150 nm,宽40~60 nm,呈子弹状。人工感染细胞培养病毒液的健康鲤出现与自然发病鲤相似的临床症状,感染组累计死亡率为65%。通过鲤上皮瘤细胞(epithelioma papulosum cyprini cell, EPC细胞)培养分离得到1株病毒,全基因组测序与系统进化分析结果表明,该分离株与SVCV的同源性最高,将该分离株命名为SVCV-2022。结合本次疾病的流行特点与病理诊断、病毒分离鉴定和人工感染试验,确定此次传染病的病原为SVCV。

关键词: 鲤春病毒血症病毒, 鲤, 累计死亡率, 系统进化分析

Abstract:

In March 2022, large scale death of carp from a farm in Leshan City, Sichuan Province, suddenly developed acute mortality with a cumulative mortality rate of 80%. To study the etiology and epidemiological pattern of this disease, the diseased material was subjected to autopsy, pathological examination, bacteriological examination, PCR identification, virus isolation and identification and phylogenetic analysis. The results showed that histopathological lesions were mainly in the gills and intestines, intestinal congestion and hemorrhage, with necrosis and shedding of epithelial cells; and hyperplasia, necrosis and shedding of epithelial cells in gill lamellae. Based on pathological diagnosis, it was preliminarily determined that the disease was caused by Spring viremia of carp virus(SVCV) infection. RT-PCR detection results showed that the G protein gene sequence of SVCV was amplified in the visceral tissues and cell culture virus solution of naturally infected carp and artificially infected carp. Electron microscopic observation of ultrathin sections of cell cultures virus solution showed that the viral particles were 80-150 nm long and 40-60 nm wide, and were bullet-shaped. Artificially infected healthy carp showed clinical symptoms similar to those of naturally infected carp, with a cumulative mortality rate of 65% in the infected group. A virus strain was isolated from epithelioma papulosum cyprini cell(EPC), and whole genome sequencing and sequence analysis were performed on the isolate, which was named SVCV-2022 after sequencing conformation of the highest homology with SVCV. Combining the epidemiological and pathological characteristics of the disease, the isolation and identification of the virus, the results of the artificial infection test and transmission electron microscopy, the pathogen of the epidemic was identified as SVCV.

Key words: Spring viremia of carp virus, carp, cumulative mortality rate, phylogenetic analysis

中图分类号:

S941.416

祁英吉, 刘舒亚, 兰寒韵, 王雪梅, 孙甜甜, 李书含, 黎秋男, 黄小丽, 耿毅, 陈德芳, 欧阳萍. 四川地区1株鲤春病毒血症病毒的分离鉴定与全基因组分析[J]. 浙江农业学报, 2025, 37(7): 1430-1440.

QI Yingji, LIU Shuya, LAN Hanyun, WANG Xuemei, SUN Tiantian, LI Shuhan, LI Qiunan, HUANG Xiaoli, GENG Yi, CHEN Defang, OUYANG Ping. Isolation, identification and genome-wide analysis of Spring viremia of carp virus in Sichuan Province of China[J]. Acta Agriculturae Zhejiangensis, 2025, 37(7): 1430-1440.

图/表 11

图1 患病鲤的临床症状和剖检病变 A-1,体表出血;A-2,鳃丝苍白,点状出血;A-3,血样腹水,肠系膜点状出血;A-4,肛门红肿,尾鳍部位点状出血;A-5,肠道充血红肿;B-1,眼球突出;B-2,鳃丝苍白;B-3,脾脏肿大,有大量腹水;B-4,腹部膨大,肛门红肿;B-5,鱼鳔点状出血。

Fig.1 Clinical symptoms and necropsy lesions of infected carp A-1, Superficial hemorrhage; A-2,Gill filaments pale with punctate hemorrhage; A-3, Hemoid ascites, mesenteric punctate hemorrhage; A-4, Redness and swelling in the anus and punctate hemorrhage in the caudal fin; A-5, Intestinal congestion, redness and swelling; B-1, Exophthalmos; B-2, Gill filaments pale; B-3, Spleen enlargement, ascites; B-4, Abdominal distension, redness and swelling in the anus; B-5, Punctate hemorrhage in swim bladder.

图2 自然感染SVCV 鲤的组织病理学损伤(HE染色) A,肾小管上皮细胞变性、坏死( ),管腔内出现均质、红染、半透明滴状物( ); B,肝出血,肝细胞空泡变性;C,肝细胞存在核溶解、核固缩及核碎裂现象( ); D,脾出血,分布大量含铁血黄素,网状内皮细胞增生;E,肠道充血出血,肠道上皮细胞坏死脱落;F,鳃小片上皮细胞增生、坏死( ),上皮细胞气球样变( )。

Fig.2 Histopathological damage of naturally infected SVCV carp(HE staining) A, Degeneration and necrosis of renal tubular epithelial cells( ), homogeneous, red-stained, translucent drops appeared in the lumen( ); B, Hepatic hemorrhage, vacuolar degeneration of hepatocytes; C, Nucleolysis, nuclear solidification and nuclear fragmentation of hepatocytes( ); D,Splenic hemorrhage, distribution of large quantities of hemosiderin, reticuloendothelial cell proliferation; E, Intestinal congestion and hemorrhage, necrosis and shedding of intestinal epithelial cells; F, Hyperplasia and necrosis of epithelial cells in gill lamellae( ),epithelial cell ballooning( ).

图3 SVCV G蛋白基因的 RT-PCR扩增 A,第一轮 PCR产物(714 bp);B,第二轮 PCR产物(606 bp);M,DL 2 000 DNA marker;“+”,阳性对照;“-”,阴性对照;1、2、3、4,送检鲤。

Fig.3 RT-PCR amplification of the SVCV G protein gene A, The first round PCR product(714 bp); B, The second round PCR product (606 bp); M, DL 2 000 DNA marker; “+”, Positive control; “-”, Negative control; 1, 2, 3 and 4, Carp sent for inspection.

图4 接种患病鲤组织匀浆的鲤上皮瘤细胞 A,对照组细胞形态;B,48 h后出现 CPE的细胞形态。

Fig.4 Epithelioma papulosum cyprini cells inoculated with tissue homogenate of diseased carp A, Cell morphology of control group; B, Cell morphology of CPE appeared 48 h later.

图5 细胞培养物RT-PCR鉴定 A,第一轮 PCR产物(714 bp);B,第二轮 PCR产物(606 bp);M,DL2 000 DNA marker;1,阴性对照;2,阳性对照;3,对照组细胞;4,细胞培养病毒液。

Fig.5 RT-PCR identification of cell cultures A, The first round PCR product(714 bp); B, The second round PCR product (606 bp); M, DL2 000 DNA marker; 1, Negative control; 2, Positive control; 3, Control group; 4, Cell culture virus.

图6 电镜观察结果 A,细胞凋亡和细胞周围大量病毒粒子(白色箭头指向);B,细胞质中聚集的病毒粒子。

Fig.6 Electron microscopic observation results A, Apoptosis and a large number of virions around the cells (white arrow points); B, Virions aggregated in the cytoplasm.

图7 鲤春病毒血症病毒攻毒后的累计存活率

Fig.7 Cumulative survival rate of carp after infection with Spring viremia of carp virus

图8 SVCV-2022(OP495631)分离株基因组结构示意图

Fig.8 Schematic representation of the genome structure of SVCV-2022(0P495631.1) isolate

表1 系统发育树参考病毒株信息

Table 1 Gene information of virus strain for phylogenetic tree

编号 Number	GenBank登录号 GenBank accession number	分离株 Isolate	来源地 Source location	宿主名称 Host name	长度 Length/bp
1	DQ097384.2	A1	中国China	Cyprinus carpio	11 100
2	DQ491000.1	A2	中国China	—	10 990
3	EU177782.1	BJ0505-2	中国北京Beijing,China	—	11 047
4	MT675953.1	shlj4	中国China	Cyprinus carpio	11 029
5	KR012465.1	SH140501	中国上海Shanghai,China	金鱼Goldfish	11 029
6	KR012466.1	SH140502	中国上海Shanghai,China	金鱼Goldfish	11 029
7	KR012467.1	SH140503	中国上海Shanghai,China	金鱼Goldfish	11 029
8	KR012468.1	SH140522	中国上海Shanghai,China	鲤鱼Common carp	11 029
9	KT321307.1	SH150514	中国上海Shanghai,China	金鱼Goldfish	11 029
10	KY475636.1	SH160901	中国China	草鱼Grass carp	11 029
11	KJ513477.1	SVCV-265	中国China	锦鲤Koi	11 029
12	KU230365.1	202238	中国香港Hong Kong,China	Cynops orientalis	10 991
13	MG663514.1	ADC-SVC2016- 1	韩国Republic of Korea	Cyprinus carpio	11 034
14	MG663513.1	ADC-SVC2016-3	韩国Republic of Korea	Micropterus salmoides	11 034
15	MG663512.1	ADC-SVC2016-5	韩国Republic of Korea	Cyprinus carpionudus	11 029
16	NC_002803.1	ATCC VR-1390	南斯拉夫Yugoslavia	Cyprinus carpio	11 019
17	AJ318079.1	Fijan reference strain	南斯拉夫Yugoslavia	鲤Carp	11 019

图9 基于SVCV-2022全基因组序列构建系统发育树

Fig.9 Phylogenetic tree construction based on whole genome series of SVCV-2022

图10 SVCV-2022(OP495631)分离株与参考株核苷酸序列相似性分析

Fig.10 Similarity analysis of nucleotide sequences between isolate and reference strains of SVCV-2022(OP495631)

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四川地区1株鲤春病毒血症病毒的分离鉴定与全基因组分析

Isolation, identification and genome-wide analysis of Spring viremia of carp virus in Sichuan Province of China

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