Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2.
Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein.
Lekcharoensuk
Porntippa
P
Department of Microbiology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.
Morozov
Igor
I
Paul
Prem S
PS
Thangthumniyom
Nattarat
N
Wajjawalku
Worawidh
W
Meng
X J
XJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D036022:Capsid Proteins / Q000276:immunology*; D002460:Cell Line; D017925:Circovirus / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000276:immunology; D018411:Spodoptera; D013552:Swine
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins