Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 ...Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 gene has been identified as an immunogenic protein.Currently,there is no immunofluorescence assay(IFA)available for serological diagnosis.Here,the N-terminal 33 amino acids of Cap protein were predicted to serve as a PCV3 nuclear localization signal(NLS).Two types of recombinant plasmids were constructed for recombinant protein expression in 5f9 cells by using a baculovirus expression system:plasmid rvBac-Pc for full-length Cap protein expression and rvBac-Sc for Cap protein expression with a honeybee melittin signal peptide in place of the predicted NLS sequence.Expression of the nuclear localization sequences was further analyzed by IFA.Strong and specific fluorescence signals were observed in the nucleus of rvBac-Pc-transfected cells and in the cytoplasm of rvBac-Sc-transfected cells.No cross-reactivity was observed with porcine circovirus type 2,porcine pseudorabies virus,classical swine fever virus,or porcine reproductive and respiratory syndrome virus.In summary,we developed two fluorescence detection modes for Cap protein that can be used to detect PCV3 antibodies.This method is suitable for the diagnosis and epidemiological investigation of PCV3.This study provides a reliable detection method for monitoring PCV3 antibody level in pigs in the future.展开更多
【目的】猪圆环病毒3型(porcine circovirus type 3,PCV3)与母猪繁殖障碍、仔猪皮炎及心肌炎等疾病的发生密切相关。病毒在细胞上难以稳定传代,阻碍了PCV3动物感染模型的构建及其致病性的研究。【方法】利用PCV3阳性断奶仔猪的淋巴结病...【目的】猪圆环病毒3型(porcine circovirus type 3,PCV3)与母猪繁殖障碍、仔猪皮炎及心肌炎等疾病的发生密切相关。病毒在细胞上难以稳定传代,阻碍了PCV3动物感染模型的构建及其致病性的研究。【方法】利用PCV3阳性断奶仔猪的淋巴结病料接种PK-15细胞进行病毒分离,通过PCR、间接免疫荧光试验和透射电镜观察等方法对毒株进行鉴定,成功分离到一株PCV3毒株(PCV3-HK株),并测定了该毒株的全基因组。使用毒价为106.4TCID50/mL的第15代病毒细胞培养物在接种21日龄未断奶仔猪,通过临床症状、体重变化、排毒情况和病理变化等评价该毒株的致病性。【结果】病毒分离鉴定结果表明,PCV3-HK株基因组全长2000 bp,属于PCV3c基因亚型,病毒粒子为圆球形、无囊膜、直径约15-20 nm的颗粒,能够与PCV3 Cap蛋白单克隆抗体发生特异性反应;仔猪感染试验结果显示,PCV3-HK株感染猪出现皮炎、增重缓慢甚至消瘦等症状,以及淋巴结水肿、间质性肺炎等剖检变化;在感染的第7-21天,口拭子和肛拭子中均可检测到病毒核酸,并出现病毒血症。该病毒感染仔猪的心、肺、肾和淋巴结等器官,尤其在肾脏和淋巴结中观察到炎性细胞浸润、坏死细胞聚集等病理变化。【结论】本研究分离到一株PCV3c毒株,建立了仔猪感染模型,为PCV3致病机制研究和疫苗研发提供了关键材料。展开更多
基金This work was supported by a grant from the China Agriculture Research System(CARS-35).
文摘Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 gene has been identified as an immunogenic protein.Currently,there is no immunofluorescence assay(IFA)available for serological diagnosis.Here,the N-terminal 33 amino acids of Cap protein were predicted to serve as a PCV3 nuclear localization signal(NLS).Two types of recombinant plasmids were constructed for recombinant protein expression in 5f9 cells by using a baculovirus expression system:plasmid rvBac-Pc for full-length Cap protein expression and rvBac-Sc for Cap protein expression with a honeybee melittin signal peptide in place of the predicted NLS sequence.Expression of the nuclear localization sequences was further analyzed by IFA.Strong and specific fluorescence signals were observed in the nucleus of rvBac-Pc-transfected cells and in the cytoplasm of rvBac-Sc-transfected cells.No cross-reactivity was observed with porcine circovirus type 2,porcine pseudorabies virus,classical swine fever virus,or porcine reproductive and respiratory syndrome virus.In summary,we developed two fluorescence detection modes for Cap protein that can be used to detect PCV3 antibodies.This method is suitable for the diagnosis and epidemiological investigation of PCV3.This study provides a reliable detection method for monitoring PCV3 antibody level in pigs in the future.
