猪圆环病毒2型湖南株的分离鉴定与进化分析

Identification and genetic variation analyses of porcine circovirus type 2 isolated from Hunan Province

为了解湖南某规模猪场引起繁殖障碍主要病原的感染情况,本研究通过荧光定量PCR检测了血液样品中的6种病原,发现存在PCV2感染。对阳性样品进行全基因组扩增、克隆和测序后,与国内外其他PCV毒株进行同源性比对,绘制系统遗传进化树,并通过猪肾细胞系对病毒进行分离培养及免疫荧光鉴定。结果显示,样品中分离的2株毒株均为PCV2d型,基因组全长均为1767 bp,其核苷酸同源性为99.7%,四处差异均位于ORF2区,其中命名为NX-P3-1毒株的第97位精氨酸突变为甘氨酸,位于衣壳蛋白T细胞表位区;与其他毒株相比,与海南HN1407株同源性最高,分别为99.7%和99.6%,遗传亲缘关系较为接近;PCV2d型毒株之间的差异主要位于B细胞表位区;免疫荧光鉴定显示NX-P3-1毒株可稳定感染和增殖。本研究为分析湖南PCV2流行情况提供了参考依据。

To investigate the infection of the main pathogens causing reproductive failure in a large-scaled pig herd in Hunan Province,six pathogens were detected in blood samples by real-time fluorescence quantitative PCR.The detection showed that porcine circovirus type 2(PCV2)infection was positive in the herd.The complete genome of the candidate pathogen was amplified from the positive samples and cloned for sequencing.Sequence alignment and phylogenetic analysis were performed between PCV strains from China and abroad.Virus strains were isolated and purified using a porcine kidney cell line,and immunofluorescence staining was performed to identify the isolated strains.The results showed that two PCV2 d virus strains were obtained,and their genomes were 1767 bp,sharing 99.7%of identity.Only four nucleotides were different between the two isolated strains,which located in the ORF2 region of PCV2.Glycine at position 97 of the isolated strain designated as NX-P3-1 was mutated to arginine,which located in the epitopes of capsid protein T cells of ORF2.Compared with other strains,the two isolated strains shared 99.7%and 99.6%of homology with the Hainan HN1407 strain,showing the closest genetic relationship.The difference between PCV2 d strains mainly located in B cell epitopes.Immunofluorescence assay showed that only the NX-P3-1 strain exhibited stable infection and proliferative activity.The present study can supply a foundation for analysis of the prevalence of PCV2 in Hunan Province.