畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (6): 3032-3040.doi: 10.11843/j.issn.0366-6964.2025.06.045

• 研究简报 • 上一篇    

一株具有多位点核苷酸替换的新型猪圆环病毒2d亚型毒株的分离与鉴定

陈云龙1,2, 樊港1,2, 樊心怡1,2, 郭永超1, 张鑫淼1, 王妍1,2*, 张仕强1,2*   

  1. 1. 西北农林科技大学动物医学院/陕西省干细胞工程技术研究中心, 杨凌 712100;
    2. 西北农林科技大学家畜生物学重点实验室, 杨凌 712100
  • 收稿日期:2024-07-10 发布日期:2025-06-25
  • 通讯作者: 王妍,主要从事临床兽医学研究,E-mail:wangyan11@nwsuaf.edu.cn;张仕强,主要从事体细胞重编程与细胞命运决定的相关研究,E-mail:shiqiangzhang@nwsuaf.edu.cn
  • 作者简介:陈云龙(2000-),男,河南驻马店人,主要从事动物疫病及干细胞诱导分化研究,E-mail:931326211@nwafu.edu.cn;樊港(1997-),男,山西运城人,主要从事动物疫病研究,E-mail:15513999792@163.com。陈云龙和樊港为同等贡献作者
  • 基金资助:
    国家自然科学基金(32072856);动物基因工程疫苗国家重点实验室开放课题(SKLGEVV-ZD/ZY-2019);大学生创新创业训练计划项目(202400560A3;202400560AD)

Isolation and Identification of a Novel Porcine Circovirus Type 2d Strain with Multiple Nucleotide Substitutions

CHEN Yunlong1,2, FAN Gang1,2, FAN Xinyi1,2, GUO Yongchao1, ZHANG Xinmiao1, WANG Yan1,2*, ZHANG Shiqiang1,2*   

  1. 1. College of Veterinary Medicine, Northwest A&F University/Engineering Research Center of Stem Cells of Shaanxi Province, Yangling 712100, China;
    2. Key Laboratory of Animal Biology, Northwest A&F University, Yangling 712100, China
  • Received:2024-07-10 Published:2025-06-25

摘要: 猪圆环病毒(porcine circovirus,PCV)是引起猪圆环病毒相关性疾病(porcine circovirus associated diseases,PCVAD)的主要病原。患病猪表现为发育不良、呼吸困难、生殖障碍等症状。目前,PCV多种亚型在中国流行,给养猪业造成了巨大经济损失。为了鉴定一株疑似新型PCV2感染死亡的猪病料,本研究采集疑似PCV感染的病料组织并对病毒进行分离培养,通过PCR,免疫荧光染色和序列测定分析对病毒进行鉴定。并对阳性样品进行全基因组扩增,测序后与GenBank上其他不同国家和地区来源的28株PCV的同源性、系统发育树和重组特性进行分析。使用6周龄BALB/c小鼠模型,感染毒株后观察不同组织病理切片。使用第5代病毒液滴鼻和颈部肌肉注射两种方式感染14周龄健康猪,观察和分析试验猪的体温、增重、病毒血症、各组织病毒载量等评价该毒株的致病性。结果显示,本试验分离到一株PCV2d亚型毒株,命名为SD02,该毒株全基因组大小为1 767 bp (GenBank登录号为OQ730503),其中ORF2大小为705 bp,共编码234个氨基酸。SD02相较于其他已报道的PCV2d毒株,在ORF2编码的Cap蛋白羧基末端增加了1个赖氨酸。SD02株与参考毒株基因全序列的核苷酸同源性为94.2%~99.1%,与22625-33毒株(PCV2d)相似性最高,为99.1%。该毒株感染的小鼠出现明显间质性肺炎和坏死性肝炎等症状。该毒株感染后会引起猪消瘦和生长缓慢等现象,病毒血症存在时间超过21 d,并且具有广泛的组织嗜性,其中各淋巴结病毒载量更高,会引起机体的免疫抑制。本研究为PCV流行病学研究和疫苗开发提供了新的参考。

关键词: 猪圆环病毒2型, 分离鉴定, 遗传变异分析, 致病性

Abstract: Porcine circovirus type 2 (PCV2) is the primary pathogen causing porcine circovirus-associated diseases (PCVAD), which manifest in pigs as poor growth, respiratory difficulties, reproductive disorders, and other symptoms. These diseases have caused significant economic losses to the global pig industry. Currently, multiple subtypes of PCV are prevalent in China, resulting in tremendous economic losses to the pig industry. To identify the tissue sample of a pig suspected of dying from a novel PCV2 infection, we collected tissue samples from pigs suspected of PCV infection and isolated and cultured the virus. The virus was identified through PCR, immunofluorescence staining, and sequencing analysis. Six week old BALB/c mice were used to observe the pathological sections of different tissues after infection with the virus strain. The positive samples underwent whole-genome amplification and sequencing. Homology, phylogenetic trees, and recombination characteristics were analyzed with 28 PCV strains from different countries and regions in GenBank. The fifth-generation virus solution was used to infect healthy 14-week-old pigs through nasal drop administration and intramuscular injection into the neck. The pathogenicity of the virus strain was evaluated by observing and analyzing the pigs’ body temperature, weight gain, viremia, and viral load in various tissues. The results showed that a PCV2d subtype strain, named SD02, was isolated in this experiment. The whole genome size of this strain was 1 767 bp (GenBank accession number OQ730503), of which the ORF2 size was 705 bp, encoding a total of 234 amino acids. Compared to other reported PCV2d strains, SD02 has an additional lysine at the carboxyl terminus of the Cap protein encoded by ORF2. The nucleotide homology of the entire gene sequence between SD02 strain and the reference strain ranges from 94.2% to 99.1%, with the highest homology of 99.1% with the 22625-33 strain (PCV2d). The mice infected with this strain showed obvious symptoms such as interstitial pneumonia and necrotizing hepatitis. After infection with this strain, it can cause symptoms such as emaciation and slow growth in pigs. Viremia lasts for more than 21 days and has a wide range of tissue tropism, with higher viral loads in various lymph nodes, which can cause immune suppression in the body. This study provides new references for PCV epidemiological research and vaccine development.

Key words: porcine circovirus type 2, isolation and identification, analysis of genetic variation, pathogenicity

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