| Porcine circovirus type 2 (PCV2) is a small non-enveloped single-stranded circular DNA virus classified in the Circoviridae family. PCV2 was associated with various other clinical syndromes. It has caused great damage to pig breeding. Therefore, it has got more and more attention.PCV2 was first identified in China in 2000, and since then, the distribution of PMWS has spread all over country, caused significant economic losses and presented a threat to the pig industry. In this study, some PCV2-suspected samples sent to the lab from some provinces in China between 2008 and 2009 were detected, then PCV2 was isolated and the evolution of the genetic diversity within complete genome was analyzed,hoping to understand the epidemic condition of PCV2 for a better control and preventive step of PCV2 related disease. The results followed as below:1. Isolation and identification of PCVThe PCV2 positive samples detected by PCR were inoculated to PK-15 cells without PCV contamination. PCV2 can be detected by PCR in the cells of 4 or 6 generation. The results indicated that the virus obtained in the experiment was PCV2, and 17 strains of PCV2 were isolated from different regions in China, named: SC-10,SD,ZJ-38,HUN,HUN-2,HUN-11,HUB-5,FJ-3,FJ-4,JX-1,JX-2,HLJ-7,HLJ-10,LN-3,LN-19,YN-8 and SX-1 respectively.2. Sequencing and analyses of PCV complete genome sequencesAccording to the sequence of porcine circovirus type 2(PCV2) published in GenBank,a pair of specific primers were designed and synthesized to amplify the complete genome of PCV2 by polymerise chain reaction(PCR) assay. With this PCR assay, in this research, seventeen PCV2 complete gene fragments were amplified from SICHUAN,SHANDONG,ZHEJIANG,HUNAN,HUBEI,FUJIAN,JIANGXI,HEILONGJIANG,LIAONING,YUNNAN,SHANXI and so on. The DNA fragments were successfully cloned into pGEM-T easy vector, which was confirmed by restrict enzyme identification and sequence analysis. After sequence identification,The complete genome fragments witch were the same as expect were finally got and each of the isolates was 1767bp in length.In this research, we analyzed nucleotide sequences of 17 strains of PCV1 and 93 strains of PCV2 by CLUSTAL and DNAstar software。The results show that The 17 PCV1 strains shared 99.1%-100% nucleotide identity to each other; The 93 PCV2 strains shared 94.7%-100% nucleotide identity to each other. The total nucleotide sequence variation between PCV1 and PCV2 sequences was 76.1%-78%. PCV1 and PCV2 sequences are obviously divided into two different branchs at the phylogenetic tree. By statistic, we discover that,although several years have been pasted, the sequences homology of PCV2 don,t have large changes, the results show that,PCV2 is very conserved. The two open reading frames(ORFl and ORF2) and deduce their amino acid sequence of PCV2 was compared between 17 strains in our lab and several strains from China in Genbank by DNAstar software, the result showed that the ORF1 of all the isolates showed 96.4% to 100% nucleotide sequence identidy and 97.5% to 100% amino acid identidy. The result showed a high degree of homology of ORF1. The ORF2 of all the isolates had 92.6% to 100% nucleotide sequence identidy and 91.1% to 100% amino acid identidy. Sequence analysis of ORF1 and ORF2 genes of all the strains revealed that the extent of nucleotide variation for the ORF2 was greater than for the ORF1.The amino acid sequences alignment of the PCV2 capsid protein identified three major regions of amino acid heterogeneity. Two of which correspond with dominant immunoreactive areas. It may contribute to generate different pathogenicity.
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