猪圆环病毒2型不同基因型毒株感染性克隆的构建及拯救毒株的体外生物学特性

Construction of Infectious Clone for Different Genotype Strains of Porcine Circovirus Type 2 and Characterization of the Rescued Viruses in vitro

【目的】中国猪群中猪圆环病毒2型(PCV2)感染存在不同基因型毒株(PCV2a、PCV2b和PCV2d),有必要阐明不同基因型PCV2毒株的致病性差异。【方法】采用感染性克隆技术构建了不同基因型代表性毒株,对拯救的毒株进行体外生物学特性试验。【结果】构建了4个不同基因型的PCV2感染性分子克隆,经转化细胞后拯救4个克隆毒株,经核酸序列分析、病毒形态学观察、免疫过氧化物酶单层细胞试验(IPMA)等证实属于不同基因型的PCV2毒株;拯救的毒株均通过细胞连续传10代,病毒增殖能力稳定,毒价均可达到105.0TCID50.mL-1;用具有中和病毒活性的单克隆抗体(1D2株)进行抗原捕获ELISA检测,拯救的PCV2a/rCL毒株与该单抗产生特异性反应,另3个克隆毒株(PCV2b/rYJ、rJF和PCV2d/rBDH)与该单抗均无反应,表明不同基因型代表毒株抗原性不同。【结论】中国猪群中PCV2流行毒株基因组及ORF2长度存在差异,其病毒抗原特性不同,构建和拯救了4个代表不同基因型PCV2克隆毒株,为进一步研究其致病性差异奠定了基础。

【Objective】 Different genotypes（PCV2a,PCV2b and PCV2d） of porcine circovirus type 2（PCV2） are present in clinical PCV2 infections in China,and it is necessary to elucidate the pathogenic difference among different genotypes of PCV2.【Method】Representative strains of different genotypes of PCV2 were constructed by infectious molecular clone and biologic characterization of the rescued viruses were identified in vitro.【Result】 Four infectious clones for different genotypes of PCV2 were constructed and four viruses of different genotypes were rescued after transfection into PK15 cells.The rescued viruses were PCV2 after identified by nucleotide sequence analysis,morphology of the viruses and immunoperoxidase monolayer assay（IPMA）.The rescued viruses propagated stably after consecutive incubation for 10 passages,and virus propagation reached up to peak at 72 h post infection（PI）,and the virus titers were up to 105.0TCID50/mL.By using neutralizing 1D2 monoclonal antibody（mAb） of PCV2,the antigen capture ELISA showed that only rescued PCV2a/rCL had reactivity with 1D2 mAb,and however,another three rescued strains（PCV2b/rYJ,PCV2b/rJF and PCV2d/rBDH） did not,which indicated the antigenic difference among the rescued viruses of different genotypes.【Conclusion】Conclusion could be draw from this study that PCV2 prevailing has differences in genomic and ORF2 gene length and antigen in swine herds.Four representative clones for different genotypes were constructed and rescued,which will facilitate further studies on the pathogenic difference resulted from different genotypes of PCV2.