一株猪圆环病毒2d基因型毒株的全基因组序列及其衣壳蛋白的小鼠免疫原性分析

Investigation of the Genome-wide Sequence and Immunogenicity of Capsid Protein in Mice for a Porcine Circovirus 2d Genotype Strain

旨在确定引起湖北省某规模化猪场断奶仔猪多系统衰竭综合征(postweaning multisystemic wasting syndrome,PMWS)的主要病原。本研究采集该猪场具有PMWS典型临床症状的仔猪组织样品,经PCR检测,发现其存在猪圆环病毒2型(porcine circovirus 2,PCV2)感染。对阳性样品中PCV2全基因组进行扩增和测序后,与国内外流行毒株进行全基因组和ORF2基因的比对,分析其核苷酸同源性。通过猪肾上皮细胞系PK-15对病毒进行分离培养和鉴定,对其核衣壳蛋白Cap的抗原表位和小鼠免疫原性进行分析和评估。病毒分离鉴定结果表明,成功分离到1株PCV2毒株(名称:XY2022;GenBank登录号:OM249965.1),全基因组长度为1767 nt,在PK-15细胞上的毒价为10~(5.70)TCID_(50)·mL^(-1);序列分析结果显示,XY2022属于PCV2d,与参考毒株的全基因组核苷酸相似性为94.3%~99.8%,ORF2核苷酸相似性为89.2%~99.6%;Cap的免疫原性分析和测定结果显示,相比于其他基因型毒株的Cap,XY2022 Cap的线性免疫优势区、线性中和表位和构象中和表位均存在差异位点,PCV2d(XY2022)Cap和PCV2b(WH)Cap均可诱导小鼠机体产生抗PCV2的中和抗体,但2d Cap诱导的中和抗体水平更稳定。本研究为湖北省PCV2的流行病学研究和变异毒株的防控提供了参考,可为研究PCV2致病机制和开发新型疫苗提供材料。

The main objectives of this study were to investigate the main pathogens causing postweaning multisystemic wasting syndrome(PMWS)on a large-scale pig farm in Hubei Province.In this study,tissue samples were collected from piglets with typical clinical symptoms of PMWS in this pig farm,and PCR detection revealed the presence of porcine circovirus type 2(PCV2)infection.The complete genome of PCV2 was cloned and sequenced.Then,the sequence of the complete genome and key genes of the positive samples were compared with other PCV2 strains around the world,and their nucleotide homology was analyzed.The virus strain was isolated,cultured,and identified by using porcine kidney epithelial cell line PK-15,and the epitope and immunogenicity in mice of nucleocapsid protein Cap were analyzed and evaluated.The isolation and identification results showed that we successfully isolated a PCV2 strain(name:XY2022;GenBank accession No.:OM249965.1).The complete genome length of XY2022 was 1767 bp,and the titer of XY2022 on isolated cells was 105.70 TCID 50·mL^(-1).Sequence analysis showed that XY2022 belonged to PCV2d,the nucleotide homology of the reference strains was 94.3%-99.8%,and that of ORF2 was 89.2%-99.6%.The results of immunogenicity analysis and evaluation of Cap showed that XY2022 and other genotypes of PCV2 had different sites in the linear immunodominant region,linear neutralization epitope,and conformational neutralization epitope.Both PCV2d(XY2022)Cap and PCV2b(WH)Cap can induce the production of neutralizing antibodies to inhibit PCV2-infection in mice,but the level of neutralizing antibody induced by 2d Cap is more stable.This study provides a reference for the epidemiological study of PCV2 and the prevention and control of the mutated strains in Hubei Province and provides materials for the study of the pathogenesis of PCV2 and the development of novel vaccines.