First construction of infectious clone for newly emerging mutation porcine circovirus type 2 (PCV2) followed by comparison with PCV2a and PCV2b genotypes in biological characteristics in vitro.

BACKGROUND

Porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is a serious economic problem in the swine industry. Different genotypes (PCV2a, PCV2b and PCV2d) of the virus are present in the clinical cases in China, and it is necessary to elucidate the pathogenic difference among different genotypes of PCV2. In this study, four strains of different genotypes were isolated, two were ordinary strains and another two were mutation strains, which there are one and two amino acids elongation in the capsid protein (Cap) of PCV2, respectively. Representative strains of different genotypes of the virus were constructed by infectious molecular clone and biological characterization of the rescued viruses were identified in vitro.

RESULTS

Four PCV2 isolates (PCV2a/CL, PCV2b/YJ, PCV2b/JF and PCV2d/BDH) of different genotypes were isolated from the clinical cases of PMWS in China. Four infectious clones of PCV2 were constructed and the rescued viruses were harvested after transfection into PK15 cells. The rescued viruses were verified by nucleotide sequence analysis, morphology of the viruses and immunoperoxidase monolayer assay (IPMA). The rescued viruses propagated stably after consecutive incubation for more than ten passages, and virus propagation reached its peak 72h post infection (PI), and the virus titers were up to 10⁵·⁷ TCID₅₀/ml. By using neutralizing 1D2 monoclonal antibody (mAb) of PCV2, the antigen capture ELISA showed that only the PCV2a/rCL and PCV2b/rJF strains has immunoreactivity with the 1D2 mAb, however, another two rescued strains (PCV2b/rYJ and PCV2d/rBDH) do not, which indicated the antigenic difference among the rescued viruses of different genotypes. In addition, here is the first report of obtaining the newly emerging PCV2 with mutation in vitro by infectious molecular clone technology.

CONCLUSIONS

Conclusions drawn from this study show that PCV2 has prevailing differences in genomic and ORF2 gene length and antigen in swine herds in China. Four representative clones for different genotypes were constructed and rescued, which will facilitate further studies on the pathogenic differences resulting from different subtypes of PCV2.