Expression and self-assembly in baculovirus of porcine enteric caliciviruscapsids 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 bu t 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, 120:1 15-122, 1991). Cloning and sequence analysis of the PEC Cowden full-length genome revealed that it is most closely related genetically to the human Sa pporo-like viruses. In this study, the complete PEC capsid gene was subclon ed into the plasmid pBlueBac4.5 and the recombinant baculoviruses were iden tified by plaque assay and PCR. The PEC capsid protein was expressed in ins ect (Sf9) cells inoculated,vith the recombinant baculoviruses, and the reco mbinant capsid proteins self-assembled into virus-like particles (VLPs) tha t were released into the cell supernatant and purified by CsCl gradient cen trifugation, The PEC VLPs had the same molecular mass (58 kDa) as the nativ e virus capsid and reacted with pig hyperimmune and convalescent-phase sera to PEC Cowden in enzyme-linked immunosorbent assay (ELISA) and Western blo tting. The PEC capsid VLPs were morphologically and antigenically similar t o the native, virus by immune electron microscopy, High titers (1:102,400 t o 204,800) of PEG-specific antibodies were induced in guinea pigs inoculate d with PFC VLPs, suggesting that the VLPs could be useful for future candid ate 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 inf ected with recombinant baculoviruses expressing PEC capsids, followed by ce ll fixation with formalin, For the VLP ELISA, the VLPs were used for the co ating antigen. Our data indicate that both tests were rapid, specific, add reproducible and might be used for large-scale serological investigations o f PEC antibodies in swine.