猪圆环病毒Ⅱ型DT株的分离鉴定及动物模型建立

应用PCR方法对江苏省东台市某猪场送检的一头无名高热病死猪的腹股沟淋巴结及肺脏病料进行检测.结果表明:该病料中存在PCV2;病料转染PK-15细胞,盲传3代后,IFA方法检测结果显示有明显的特异性荧光.利用设计的一对特异性引物对PCV2-DT株(东台分离株)全长基因进行扩增,测序结果显示所扩增基因为PCV2全长基因,大小为1 767 bp,包括11个完整的开放阅读框(ORF).PCV2-DT分离株与其他20株PCV2分离株序列比较发现,核苷酸同源性都在95%以上,并与欧美株处于同一分支.PCV2-DTRep、Cap序列同国内分离的16株相比较,核苷酸序列同源性分别为97.5%~100%和94.2%~99.9%.氨基酸序列同源性分别为98.4%~99.7%和93.2%~99.6%.用PK-15细胞从病料中分离的细胞毒接种3头42日龄健康仔猪(血清学检测无PCV1、PPV、PRV等感染),于首次接毒后的第10天出现发热、咳嗽、水样腹泻等症状,食欲几乎废绝,出现了PM-WS症状,初步建立了PMWS动物模型.

Isolation and identification of porcine circovirusⅡ of DT strain and establishment of animal model

PCV2 was detected by PCR from the inguen lymph node and lung of a pig that displayed the clinical symptoms of non-named fever in Dongtai city.Then tissue was transfected to PK-15 cell.After 3 generations,specific fluorescence was detected by using IFA.Meanwhile the full length PCV2 gene was expanded by using a pair of specific primers.The sequence analysis results confirmed that the size of PCV2-DT strain was 1 767 bp with 11 ORFs.PCV2-DT strain was compared with 20 strains that were isolated in different districts in the world,the homology of nucleotide sequence was all above 95 %.PCV2-DT strain and majority strands from American and Europe lied in the same branch.Compared with domestic 16 strains,the homology of the sequence of Rep and their amino acid sequence were 97.5 %~100 % and 94.2 %~99.9 % respectively.The homology of Cap gene and amino acid sequence were 98.4 %~99.7 % and 93.2 %~99.6 % respectively.Three healthy piglets(42 days) that had no PCV1、PRV or PPV were inoculated in the virus separated from tissues.After 10 days,the three piglets displayed typical PMWS symptoms with fever,cough,anemia and athrepsy.