Isolation And Genomic Sequence Analysis Of Porcine Circovirus Type 2(PCV2) HeNan Strain And Construction Of The Chimeric Infectious DNA Clone Of Porcine Circovirus Ⅱ(PCV2-1)

Porcine Circovirus(PCV) is a small icosahedral nonenveloped virus with a single-stranded circular DNA genome, which belongs to the Circoviridae family. PCVl was first discovered in 1974 by Tisher as a noncytopathic contaminant of the porcine kidney cell culture PK-15 (ATCC-CCL33). Its complete genome was composed of 1759 nucleotides. However, PCV2 has been associated with postweaning multisystemic wasting syndrome (PMWS), which genome consists of 1767 or 1768 nucleotides.The nucleic acid framents of PCV2 were detected from the tissues of clinical pigs with PMWS. The positive samples were homogenized in TE (PH8.0) and filtered. The suspension was inoculated in PCVl free PK-15 cells. After 12 passages, one isolate of porcine circovirus type 2 was isolated and named PCV2 HeNan. Purified virus was observed in transmission electron microscope. Virus was a 17nm in diameter and icosahedral virion. The result of sequence and analysis showed that HeNan isolate's complete genome was composed of 1767 nucleotides containing 11 ORFs. Compared with PCVl and PCV2 reference strains from GenBank, genome of HeNan isolate shared 76.2 % -77.3 % homology with PCVl, 95.5 % -99.6 % homology with PCV2.Three pairs of specific primers were designed according to the published PCV genome sequence in GenBank. The primer pair, P1 and P2, was used to amplify the full-length genome DNA by PCR from the cells which were infected by PCV2 (GenBank NO.AY291318). The PCR product was cloned into pBluescript II SK(+) (pSK) vector to construct the recombinant plasmid designated as SK-PCV2. The PCR primer pair, P5 and P6, amplified the pSK vector and the PCV2 genome without the ORF2 gene (SK-PCV2â–³ORF2), a fragment of 4023bp, using the plasmid of SK-PCV2 as the template and introduced Nhe I and Sph I restriction enzyme sites; the primer pair of P3 and P4 amplifed the ORF2 gene of PCVl, a fragment of 702 bp, and introduced Nhe I and Sph I restriction enzyme sites. The SK-PCV2â–³ORF2 and PCVl ORF2 which were digested by Nhe I and Sph I , were ligated to construct the chimeric plasmid of SK-PCV2-1. By Sac II digestion digestion, the PCV2-1 genome was excised and dimerized to produce the reciprocal chimeric plasmid of SK-PCV2-1(DB). Transfect PK-15 cells with SK-PCV2-1(DB) in vitro. After four passages, the infectivity of the cloned chimeric PCV2-1 genome was confirmed by RT-PCR and IFA.In this study, one isolate of porcine circovirus type 2 was isolated from the tissues of clinical pigs with PMWS and its full length of viral genome was amplified by PCR, the nuclectide sequences were determined. Phylogenetic tree analysis showed there was correlation between PCV2 isloates and geographic regions. Consequently, we constructed the chimeric infectious DNA clone of porcine circovirusll (PCV2-1). All of these achievements will give some help on studying epidemiology, vaccine, diagnosis and control of PCV2 in China.