猪圆环病毒2型2d基因型毒株的全基因组序列分析

Genomic sequence analyses of the 2d genotype strain of porcine circovirus type 2

根据Gen Bank中猪圆环病毒2型(PCV 2)基因序列,设计特异性引物,用PCR方法从福州郊区某猪场疑似PMWS仔猪病料处理后接种育传三代的PK-15细胞中扩增1个分离毒株的全基因组,并将其克隆到p MD-18T载体上,对鉴定为阳性的重组质粒送检测序。对该分离株的基因全序列及其开放阅读框2(ORF2)进行同源性分析及遗传进化关系分析。PCR鉴定结果显示,本试验中分离到的病毒属于PCV2,命名为PCV2-FZ株。全基因组序列分析显示,分离株全基因组大小为1 767 bp,含有11个开放性阅读框,其中ORF2大小为705 bp,共编码234个氨基酸。PCV2-FZ株与参考毒株基因全序列的核苷酸同源性为94.3%~99.4%,与SD毒株同源性最高,为99.4%;与DK1980PMWSfree和DK1990PMWSfree毒株同源性最低,为94.3%。分离株与参考毒株ORF2核苷酸同源性为89.2%~99.4%,其中与SD毒株同源性最高,为99.4%,与DK1980PMWSfree毒株和DK1987PMWSfree毒株同源性最低,均为89.2%。全基因组与ORF2序列遗传进化树分析均表明,PCV2-FZ株属于PCV2d基因型。本研究从福建省首次分离并鉴定了1株PCV2d型毒株,这对PCV2流行病学研究和遗传变异分析具有重要意义。

To isolate and identify porcine circovirus type 2（PCV2） from a pig farm in the outskirt of Fuzhou eity,Fujian Province,the lung,brain and spleen samples from suspected sick pigs were collected. After homogenization and sterilization,the supernatants were inoculated into PK-15 cells for passage of three generations. The viral DNAwas extracted as template for specific PCR amplification of PCV2. After that,we designed a pair of primers to amplify PCV2 complete gene,and cloned the product into pMD-18T vector,and the positive recombinant plasmids were sequenced. The obtained sequence was subject to homology analysis and genetic evolution analysis. PCR test and electron microscopy observation showed that the isolated virus in our experiment belonged to PCV2 and was named as PCV2-FZ strain. The sequence analysis showed that the whole genome size of PCV2-FZ strain was 1767 bp,and it contained 11 open reading frames（ORFs）,of which the ORF2 was 705 bp and encoded a total of 234 amino acids. The nu-cleotide identity of complete gene between PCV2-FZ strain and other reference strains ranged from 94. 3% to 99.4%,and PCV2-FZ strain had a highest homology（99.4%） with SD strain and had a lowest homology （94.3%） with DK1980PMWSfree strain and DK1990PMWSfree. The ORF2 of PCV2-FZ strain shared from 89.2%to 99.4% nucleotide identity with those of other reference strains,with a highest identity （99.3%） with SD strain and a lowest identity （89.2%0） with DK1980PMWSfree strain and DK1987PMWSfree strain. The phylogenetic analysis of both the whole genome and ORF2 sequence showed that PCV2-FZ strain belonged to the genotype of PCV2d. In summary,a strain of PCV2d was firstly isolated and identified from the region of Fujian province in our study,which was of great significance in epidemiological studies and genetic variation analysis of PCV2.