2株猪圆环病毒2型的分离鉴定及全基因组序列分析

Isolation,identification,and genomic sequence of two PCV2 isolates

【目的】对河南部分地区疑似猪圆环病毒2型（Porcinecircovirustype2，PCV2）感染猪进行病毒分离鉴定，并对分离株的全基因组序列进行分析。【方法】对郑州、焦作两地疑似PCV2感染的猪淋巴结、脾脏和肺脏组织进行PCR检测，对检测为阳性的病料进行PCV2的分离，利用间接免疫荧光试验和PCR扩增进行初步鉴定。用PCR扩增PCV2全基因，经克隆、测序后，用MEGA5．1软件对克隆的序列与GenBank上获取的其他PCV2全基因组进行序列比对。【结果】分离到2株病毒，分别命名为ZHENGZ-12株和JIAOZ-12株。间接免疫荧光试验结果显示，感染病毒的细胞出现了特异的亮绿色荧光。2个分离株全基因组长度均为1767bp，与国内外PCV2参考毒株核苷酸序列同源性为92．6％～99．9％，2个毒株之间的核苷酸序列同源性为96．0％，与PCV1核苷酸序列同源性分别为74．6％和77．7％。系统进化树分析结果显示，2个PCV2分离株同国内外分离株的亲缘关系很近；PCV2核苷酸序列比较稳定，其进化不存在明显的地域相关性。【结论】初步证实分离病毒为PCV2，ZHENGZ-12株基因型为PCV-2a，JIAOZ-12株基因型为PCV-2b。

[Objective] This study aimed to isolate and identify the suspect viruses of pigs affected by PCV2 in regions of Henan, and analyzed the whole genome sequences of isolates. [Method] Polymerase chain reaction （PCR） was used to detect the DNA of suspect PCV2 lymph nodes,lungs and spleens of bor- derline cases from Zhengzhou and Jiaozuo before the PCR positive samples were used to isolate PCV2. The PCV2 isolates were identified by PCR and indirect fluorescent antibody test. PCR products of complete ge- nomes of two PCV2 isolates were cloned and sequenced. The complete genome nucleotide sequences of these PCV2 isolates were compared with those of PCV2 isolates published previously in GenBank using MEGA5.1 software. [Result] Two PCV2 isolates were successfully isolated. The lengths of both complete genomic sequences were 1 767 bp. Comparison analysis of the complete genomic sequences indicated that they were closely related to each other （96. 0%） and to other PCV2 strains in GenBank （92. 6%- 99.9%）. The sequences identities among PCV2 and PCV1 isolates gene were 74.6% and 77.7% ,respec- tively. Phylogenetic tree showed that two PCV2 isolates were very close to home and abroad strains in genetic relationship. The difference distance of PCV2 nucleotide sequences had no obvious relationship. ICon clusion] The virus isolates were PCV2,and ZHENGZ-12 isolate was genotype PCV-2a and JIAOZ-12 iso late was genotype PCV-2b.