Study On Molecular Epidemiology Of Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) and other related diseases in pigs. This study focused on molecular epidemiology of Chinese PCV2 including analysis of genotype for prevailing strains, transcriptional differences of the dominant and nondominant genotypic strains and molecular mechanism of prevailing for the dominant genotypic strain, as well as the evolution of ORF1 and ORF2 gene of PCV2.Whole ORF2 genes of 173 positive clinical PCV2 samples from Beijing and other areas were amplified by PCR and followed by restriction fragment length polymorphism (RFLP) analysis. They could be divided into 9 different genotypes (A~I), of which 0.6% were CHN-2A, 0.6% were CHN-2B, 0.6% were CHN-2C, 5.8% were CHN-2D, 8.6% were CHN-2E, 2.3% were CHN-2F, 2.3% were CHN-2G, 60.7% were CHN-2H, and 18.5% were CHN-2I. The CHN-2H was the dominant genotype of PCV2 among genotypes in China. Sequences analysis of ORF2 genes indicated that Chinese PCV2 strains were closely related to each other (89.2~99.4% nucleotide identity) but also to other PCV2 strains originating from Canada, US, Europe and Asia. There was no close relationship between the genotype of Chinese PCV2 and geographic origin. The amino acid sequence alignment of the capsid protein encoded by ORF2 gene showed Chinese PCV2 strains exhibited three major regions of greater heterogeneity at residues 57-90, 121-136 and 180-191.The RNAs synthesized in PK15 cells of GD and BF strain, representative strain of PCV2 dominant genotype (CHN-2H) and nondominant genotype (CHN-2A) respectively, were characterized by Northern blot and RT-PCR. The transcriptional analysis showed that not only quantitative but also qualitative differences among the transcripts existed between CHN-2H and CHN-2A. They commonly shared the replication initiator protein (Rep) RNA and the viral capsid protein (CR) RNA. CHN-2A had more abundant Rep but shorter CR expression than CHN-2H. Moreover, CHN-2A had the other RNA (designated AD and Reps), but CHN-2H had none.According to the different transcripts between the dominant genotype (CHN-2H) and nondominant genotype (CHN-2A), four infectious molecular clones of the nondominant genotype (CHN-2A) were constructed by site-directed mutagenesis technique. The rescue viruses obtained by transfection and passage in PK15 cells were analyzed with immunochemical staining and PCR. All mutant plasmids were able to synthesize DNA of PCV2. Three amino acids mutation in CR did not affect viral protein synthesis. Whereas, three amino acids mutation in Rep, altering the consensus dinucleotides at the splice junctions of the Reps and clearing away of AD did have effect on viral protein synthesis, caused great reduction of viral protein synthesis. These results indicated that the dominant PCV2 (CHN-2H) could not process efficiently viral protein synthesis in vitro which was associated with specific amino acids mutation in Rep and lack of Reps and AD transcript.The genomic sequences of PCV2 BF strain at various passages in PK15 cells and clinical sera samples were detected by PCR. It was shown that ORFl gene of PCV2 was unstable and easy to loseand degrade. In contrast, the ORF2 gene was relatively stable, exhibiting that complete or partial ORF2 gene might integrate with PK15 cell genome in vitro, and recombine with genome of adenovirus and/or retrovirus in vivo to generate the PCV2-like agents.