| Porcine circoviruses belong to the genus Circovims of the family Circoviridae, which comprise two genotypes as porcine circovims type1(PCV1) and porcine circovims type2(PCV2). PCV1has been recognized as the contaminant of PK15cell line and does not induce a disease in swine, while PCV2is the primary etiological agent of postweaning multisystemic wasting syndrome (PMWS). PCV2is considered generally to be one of the primary causative agents threatening swine industry worldwide, which has become a serious economic problem. Recently, to address the current scientific confusion on genotype names, the EU consortium on porcine circovims diseases (www.pcvd.net) proposed a unified nomenclature for PCV2genotypes. To standardise PCV2genotype nomenclature, the EU Consortium proposes naming those genotypes as PCV2a, PCV2b and PCV2c. PCV2a and PCV2b as the major genotypes in the swine herds and PCV2b prevailed as the dominant genotype worldwide since2004. The PCV2c is only detected in Denmark during the1980s, and it was not present to date. Based on the wide exist and serious damage of PCV2infection, this study aimed to systematically investigate the molecular epidemiology, antigenic epitopes as well as the pathogenicity of PCV2in China, which will facilitale the studies on the control and pathogenic mechanism of PCV2.Nineteen PCV2strains were isolated from the detected PCV2positive samples based on the investigation of molecular epidemiology in part regions of China. The19isolates were designated into three genotypes as PCV2a, PCV2b and PCV2d, with the ratio of10.5%,73.7%and15.8%, respectively, of which PCV2d was first reported and isolated as a newly emerging genotype. Besides four PCV2strains were present with the genomic size of1766nt, when a single base was deleted at the position of39for PCV2d genotype or1039for PCV2b/YJ mutant in the genome compared with the sequences with1767nt. Analysis of the ORF2encoded Cap indicate that ORF2gene from four trains were mutated into705nt and708nt resulted from mutation, being one or two amino acids enlongation at the C teminal of ORF2encoded Cap.In addition PCR-RFLP has been established as a useful methodology by using Acc I and Fba I, which will facilitate the clinical genotyping. It was demonstrated that PCV2b has prevailed as the predominant genotype in China and a newly emerging PCV2d, as well as PCV2with mutation of ORF2encoded Cap were first reported.The Capsid protein (Cap) as the main structural protein encoded by PCV2-ORF2is involved in the host immune protective response, so it is very important to identify the antigenic epitopes of PCV2-Cap. In this study, five monoclonal antibodies (MAbs) against recombinant PCV2-ORF2Cap expressed by baculovirus system were generated and preliminary epitope region was localized using the five MAbs after expression of truncated fragments from ORF2gene in Echerichia coli system. Subsequently, peptides were synthesized to further accurately identify epitopes on the Cap. Of the five MAbs, four MAbs were against the same core epitope (26RPWLVHPRHRY36) verified by peptides mapping, locating in nuclear localization signal region at the N terminal of Cap. Besides, another MAb (1D2) could not react with the four truncated fragments only to react with the whole recombinant Cap, and it may be a conformational epitope based on its neutralizing effect against PCV2. It was concluded that an epitope located in nuclear localization signal region of Cap was firstly reported, which will provide a basis for further studies on the functions of Cap as well as on the mechanism of Cap localization into nuclear.Different genotypes (PCV2a, PCV2b and PCV2d) of PCV2are present in clinical PCV2infections in China, and it is necessary to elucidate the pathogenic difference among different genotypes of PCV2. Representative strains of different genotypes of PCV2were constructed by infectious molecular clone and biological characterizations of the rescued viruses were identified in vitro. Four infectious clones for different genotypes of PCV2were constructed and four viruses of different genotypes were rescued after transfection into PK15cells.The rescued viruses were PCV2after identified by nucleotide sequence analysis, morphology of the viruses and immunoperoxidase monolayer assay (IPMA). The rescued viruses propagated stably after consecutive incubation for10passages, and virus propagation reached up to peak at72h post infection (PI), and the virus titers were up to1050TCIDso/mL. By using neutralizing MAb1D2of PCV2, the antigen capture ELISA showed that only rescued PCV2a/rCL had reactivity with1D2MAb, and however, another three rescued strains (PCV2b/rYJ, PCV2b/rJF and PCV2d/rBDH) did not, which indicated the antigenic difference among the rescued viruses of different genotypes. Conclusion could be drawn from this study that PCV2prevailing has differences in genomic and ORF2gene length and antigen in swine herds. Four representative clones for different genotypes were constructed and rescued, which will facilitate further studies on the pathogenic difference resulted from different genotypes of PCV2.More and more studies on natural recombination between different PCV2genotypes have been reported, and however, the relationship between recombinantion and pathogenicity as well as genetic evolution remains limited. In this study, recombination model was performed to mock the natural recombination between different PCV2genotypes on PK15cells in vitro. The PCV2b representative strains PCV2b/JF11and PCV2b/YJ were selected and used to co-infect PK15cells with the PCV2a representative strain (PCV2a/CL1), respectively. PCV2b(JF11)/2a(CL1) and PCV2b(YJ)/2a(CL1) were first obtained as the two recombinant mutants of PCV2. The two PCV2recombinant mutants were both based on the backbones of their parental PCV2b genotype. Replacement of the genomic sequence from1018nt to the end of the viral genome (1767nt and1766nt for PCV2b/JF11and PCV2b/YJ, respectively) of the PCV2b genotype with the corresponding sequence from the PCV2a genotype was confirmed by sequence analysis using the DNAMAN software. The replication efficiencies of the two PCV2recombinant mutants were significantly enhanced and their antigenicities were significantly altered in vitro compared with their parental strains.To evaluate the difference in pathogenicity between different PCV2genotypes, PCV2b mutant and PCV2recombinant mutants, animal infection experiments were performed using PCV2a/rCL for PCV2a genotype representative, PCV2b/rJF for PCV2b genotype representative, PCV2d/rBDH for PCV2d genotype representative, PCV2b/rYJ for PCV2b mutant representative and PCV2b(YJ)/2a(CL1) for recombinant representative. The results showed that more significantly enhanced pathoginicity was recorded for PCV2d/rBDH and PCV2b/rYJ (p <0.05) compared with classical PCV2a and PCV2b genotype, among which no significant difference was present (p <0.05). In addition, the pathogenicity was similar between the recombinant mutant PCV2b(YJ)/2a(CL1) and its parental PCV2a/rCLl, however significantly attenuated virulence was observed when compared with its other parental PCV2b/rYJ(p<0.05).The pathogenic mechanism induced by PCV2remains limited. Based on the non pathogenic PCV1/rLN as the backbone, a serial of chimeric PCVs were constructed and rescued by replacement of the ORF2gene (excluding the region of nuclear localization signal) as well as its overlapping truncated fragments with the corresponding sequence from the PCV2a/rLG. Three chimeric PCV1-2/rCap/W, PCV1-2/rCap/W1and PCV1-2/rCap/W2were obtained followed by verification of IPMA and sequencing, which could facilitate the studies on the pathogenic mechanism of PCV2in the future. |
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