Eucaryotic expression of the nucleocapsid protein gene of porcine circovirus type 2 and use of the protein in an indirect immunofluorescence assay for serological diagnosis of postweaning multisystemic wasting syndrome in pigs.
The purpose of this study was to develop a sensitive, rapid, and inexpensive immunofluorescence assay (IFA) using a recombinant porcine circovirus type 2 (PCV2) nucleocapsid protein for the serological detection of PCV2-specific antibodies in pig sera. The viral nucleocapsid protein encoded by the PCV2 ORF2 gene has recently been identified as the most immunoreactive viral protein that carries type-specific antigenic determinants. The ORF2 sequence of the IAF-2897 strain of PCV2 has been cloned into a pCEP5 eucaryotic expression vector under the control of the cytomegalovirus promoter, downstream of a polyhistidine sequence tag. The recombinant plasmid was used in transfection experiments with human epithelial kidney 293 cells that were further tested, and positive expression of the viral nucleocapsid protein was confirmed by IFA and Western blotting. Strong, specific fluorescence was observed in the nuclei of transfected cells. Test specificity to PCV2 was verified with several related infectious agents. Sensitivity was compared to that of standard IFA using PCV2-infected cells by evaluating the reactivities of 44 field serum samples from pigs on farms with a porcine population suffering from postweaning multisystemic wasting syndrome. The recombinant nucleocapsid-based test was able to detect 15 more positive-testing pigs than the PCV2-based IFA. Therefore, the relative sensitivity of the latter test was estimated at only 57.1% compared to that of the recombinant nucleocapsid-based test. The recombinant fusion protein has been purified by affinity chromatography and is being used to develop further sensitive serological tests.
Racine
Sébastien
S
INRS-Institut Armand-Frappier, Laval, Québec, Canada H7V 1B7.
Kheyar
Ali
A
Gagnon
Carl A
CA
Charbonneau
Benoît
B
Dea
Serge
S
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D015153:Blotting, Western; D018173:Circoviridae Infections / Q000175:diagnosis* / Q000662:veterinary; D017925:Circovirus / Q000276:immunology; D017931:DNA Primers; D004195:Disease Models, Animal; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D013552:Swine; D014162:Transfection; D019282:Wasting Syndrome / Q000175:diagnosis* / Q000276:immunology / Q000662:veterinary
D000914:Antibodies, Viral; D017931:DNA Primers; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins