Isolation and identification of porcine circovirus type 2 of GD-zq strain and construction of its infectious clone

Abstract:

[objective] To identify and construct an infectious clone for an isolation of porcine circovirus type 2 (PCV2). [Methods] PCR was used to identify an isolate, PCV2 GD-zq strain, from the tissues of clinical pigs with postweaning multisystemic wasting syndrome (PMWS). The complete sequence of the isolate was megaligned with other 5 PCV2 Guangdong isolates (GD-pz, GD-gj, GD-jm, GD-ss and GD-sz) from GenBank by DNAstar. Two copies of the whole genomic sequence was amplified and cloned into pUC18 with the restriction enzymes EcoRⅠ, SalⅠ, SalⅠand Hind Ⅲ, and the positive clone pPCV2-2GD-zq was identified by enzyme analysis. By purification and quantitation, the pPCV2-2GD-zq DNA was transfected to PK-15 cell lines to rescue the infectious PCV2 GD-zq. [Results] PCV2 GD-zq strain was isolated and identified from the lymphonodus of clinical pigs with PMWS. Sequence analysis shows that the isolate’s complete genome was composed of 1 767 nucleotides, which shares 96.0%–99.6% homology between other 5 Guangdong reference strains, and shares 97.1%?99.7% homology in ORF1, 93.2%?99.6% in ORF2, respectively. Amino acid homology alignment shows 98.7%?100% in ORF1 and 93.2%?99.6% in OFR2. Seventy two hours post transfection of pPCV2-2GD-zq, GD-zq strain could be detected by immunofluorescence assay (IFA). [Conclusion] A PCV2 was isolated, and its infectious DNA clone was constructed successfully.