Expression and self-assembly in baculovirus of porcine enteric calicivirus capsids into virus-like particles and their use in an enzyme-linked immunosorbent assay for antibody detection in swine.
Porcine enteric calicivirus (PEC) causes diarrhea and intestinal lesions in pigs. PEC strain Cowden grows to low to moderate titers in cell culture but only with the addition of intestinal contents from uninfected gnotobiotic pigs (W. T. Flynn and L. J. Saif, J. Clin. Microbiol. 26:206--212, 1988; A. V. Parwani, W. T. Flynn, K. L. Gadfield, and L. J. Saif, Arch. Virol. 120:115--122, 1991). Cloning and sequence analysis of the PEC Cowden full-length genome revealed that it is most closely related genetically to the human Sapporo-like viruses. In this study, the complete PEC capsid gene was subcloned into the plasmid pBlueBac4.5 and the recombinant baculoviruses were identified by plaque assay and PCR. The PEC capsid protein was expressed in insect (Sf9) cells inoculated with the recombinant baculoviruses, and the recombinant capsid proteins self- assembled into virus-like particles (VLPs) that were released into the cell supernatant and purified by CsCl gradient centrifugation. The PEC VLPs had the same molecular mass (58 kDa) as the native virus capsid and reacted with pig hyperimmune and convalescent-phase sera to PEC Cowden in enzyme-linked immunosorbent assay (ELISA) and Western blotting. The PEC capsid VLPs were morphologically and antigenically similar to the native virus by immune electron microscopy. High titers (1:102,400 to 204,800) of PEC-specific antibodies were induced in guinea pigs inoculated with PEC VLPs, suggesting that the VLPs could be useful for future candidate PEC vaccines. A fixed-cell ELISA and VLP ELISA were developed to detect PEC serum antibodies in pigs. For the fixed-cell ELISA, Sf9 cells were infected with recombinant baculoviruses expressing PEC capsids, followed by cell fixation with formalin. For the VLP ELISA, the VLPs were used for the coating antigen. Our data indicate that both tests were rapid, specific, and reproducible and might be used for large-scale serological investigations of PEC antibodies in swine.
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691, USA.
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Guo
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Qian
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Chang
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LJ
Saif
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002139:Caliciviridae / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017250:Caliciviridae Infections / Q000662:veterinary / Q000821:virology; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D011994:Recombinant Proteins / Q000276:immunology; D018411:Spodoptera; D013552:Swine; D013553:Swine Diseases / Q000821:virology*; D014771:Virion / Q000276:immunology*; D019065:Virus Assembly
D000914:Antibodies, Viral; D011994:Recombinant Proteins