| Porcine circovirus type2(PCV2) is associated with several clinical manifestations collectively known as porcine circovirus diseases (PCVD) with postweaning multisystemic wasting syndrome (PMWS) as the most significant manifestation. The capsid of PCV2is the only structural protein, which is the primary immunogenic protein, thus has been the target for development of vaccines and serodiagnostic assays for tracking PCV2-specific immune responses. PCV2strains have been classified into two major genotypes (PCV2a and PCV2b) and8genetic clusters:1A to1C (PCV2b) and2A to2E (PCV2a). Despite the existence of difference genotypes, polyclonal antibodies react similarly with all of PCV2strains, indicating that there is only one PCV2serotype. However, antigenic differences exist among different PCV2strains with monoclonal antibodies (MAbs). To date, no studies have been performed to antigenically subtype PCV2strains enclosing eight PCV2clusters. The first study aimed to antigenically subtype PCV2and perform epitopes'competition analysis using MAbs. PCV2-specific neutralizing antibodies play a crucial role on preventing and controlling PCV2infection. The second study is to characterize the molecular basis of conformational neutralizing epitopes based on the antigenic differences among PCV2capsid protein. Since PCV2infection is mostly subclinical, it is important to design a new vaccine that can track the virus's spread and herd level immunity. In the last study, we aimed to identify genomic location that can tolerate small insertion of epitope tag and to produce an epitope-tagged vaccine virus.Firstly, fourteen PCV2strains representative for eight clusters were tested with20MAbs (fifteen of them were generated against PCV2a strain Stoon-1010and5of them against PCV2b strain1147) in immunoperoxidase monolayer assays (IPMA). Four MAbs reacted to all14PCV2strains and one MAb reacted with all strains except for a2C strain. One MAb reacted with all PCV2a strains, except for a2C strain and one MAb reacted with all PCV2b strains, except for a1C strain. Nine MAbs reacted with the strains of1A/1B (PCV2b),2A and2E (PCV2a). Three MAbs only reacted with the strains of2A and2E (PCV2a). One MAb reacted specifically with the strains of1A/1B (PCV2b). This suggests that discrete antigenic differences exist between different PCV2genetic clusters and that these clusters can be discriminated by the use of a panel of universal and cluster-specific MAbs. Six MAbs were selected for cross-competition analysis by a competitive ELISA using PCV2strain Stoon-1010. Six overlapping epitopes were identified on the PCV2capsid protein. The universal MAbs recognized larger epitopes than the cluster-specific MAbs.In the second study, we identified one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of PCV2based on antigenic differences among PCV2strains. MAb8E4reacted with PK-15cells infected with the genotype PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains by the IPMA and a capture ELISA Furthermore, the MAb had the capacity to neutralize PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains. One critical amino acid that determined a conformational neutralizing epitope was identified using MAb8E4 and PCV2infectious clone technique. Amino acid residues47-72in the capsid protein of PCV2a/CL were replaced with the corresponding region of PCV2b/YJ, and the reactivity of MAb8E4was lost. Further experiments demonstrated that one amino acid substitution, the alanine for arginine at position59(A59R) in the capsid protein of PCV2a (CL, LG and JF2) strains, inhibited completely the immunoreactivity of three PCV2a strains with MAb8E4. PCV2b strain48285was also mutated at position59(R59A) in the capsid protein. The reactivity of the mutants was changed from positive to negative with some MAbs. It is concluded that position59in the capsid protein of PCV2is a critical amino acid, which determines one neutralizing epitope of PCV2.Finally, to produce an epitope-tagged vaccine virus strain, a14-amino-acid V5epitope derived from simian parainfluenza virus type5, was inserted into the C terminus of the capsid protein. Recombinant PCV2expressing the V5epitope, recPCV2/CL-V5, was rescued by transfecting an infectious clone into PK-15cells and was characterised by the IPMA, a serum neutralisation assay (SNA), a capture enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The V5epitope was detected in the recombinant marker virus by IPMA and capture ELISA. Furthermore, there was no detectable difference in the antigenicity of the recombinant marker virus compared with the parental virus by IPMA and SNA using PCV2-positive serum and the neutralising monoclonal antibody1D2. However, recPCV2/CL-V5marker virus could be differentiated from the parental virus by PCR, IPMA and capture ELISA The recombinant marker virus was stable on multiplication through10passages in PK-15cells, with a maximum titer of106.2550%tissue culture infective dose (TCID50)/mL. BALB/c mice were inoculated with the recombinant or parental virus via the intranasal and intraperitoneal routes. The parental and recombinant viruses both could replicate in mice, cause microscopic pathological changes, and induce mice to generate anti-PCV2antibodies. Furthermore, the recombinant marker virus could also induce anti-V5epitope tag antibodies. These results indicated that V5epitope could be displayed on the surface of the capsid protein by inserting its gene just before stop codon of open reading frame2. More importantly, insertion of the V5epitope did not seem to interfere with biological characterisation of the recPCV2/CL-V5marker virus.This study provides valuable information for further in-depth mapping of the conformational neutralizing epitope, understanding antigenic difference among PCV2strains, development of a universal diagnostic tests and a epitope-tagged vaccine for control of PCV2-associated disease.
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