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

Abstract

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.

FIG. 1

Expression of rPEC capsids detected by immunoblotting in Sf9 cells infected with each recombinant baculovirus clone. Top, supernatant; bottom, cell lysates. The far left lane shows the prestained low-range molecular weight markers. Lanes 1 to 8 were each loaded with supernatant (top) or cell lysates (bottom) from Sf9 cells infected with an individual recombinant baculovirus clone.

FIG. 2

Dynamics of rPEC capsid production in Sf9 cells after inoculation with recombinant baculoviruses. The rPEC capsid proteins in the supernatants and cell lysates were detected by an Ag ELISA.

FIG. 3

CsCl gradient fractions examined by SDS-PAGE and immunoblotting. Lanes: 1, low-range molecular weight marker; 2, Sf9 cell extract; 3, wild-type PEC; 4, tissue culture PEC; 5 to 16, CsCl gradient fractions no. 1 to 12. The density of the VLP peak fraction was ∼1.33 g/cm³.

FIG. 4

IEM of the native PEC Cowden strain (a) and the rPEC VLP (b) incubated with hyperimmune anti-PEC serum, followed by negative staining. Bar, 100 nm.

FIG. 5

Distribution of PEC antibody titers in sow sera detected by VLP ELISA. The 30 sow sera were collected from a swine herd with PEC-associated postweaning diarrhea. Enteric caliciviruses were detected by RT-PCR in fecal samples from the normal and diarrheic postweaning pigs in the same swine herd.