Rift Valley fever virus structural and nonstructural proteins: recombinant protein expression and immunoreactivity against antisera from sheep

Abstract

The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA).

FIG. 1.

Baculovirus-expressed Rift Valley fever virus (RVFV) proteins. Proteins were purified and detected using anti-His(C-term)HRP monoclonal antibody. Gne, 54 kD; Gc, 60 kD; NSs, 34 kD; N, 31 kD; NSm, 14 kD; M, marker (A). Monoclonal antibodies against N and Gn, IDE8 and 4D4, respectively, were used to confirm expression of the respective proteins (B). (C) Coomassie Blue staining of the purified proteins. N, nucleoprotein; NSs, nonstructural protein S segment; NSm, nonstructural protein m segment; Gne, ectodomain of amino-terminal glycoprotein; Gc, carboxy-terminal glycoprotein; M, molecular weight marker.

FIG. 2.

Immunofluorescence antibody assay confirms expression of the recombinant proteins Gne and N in Sf9 cells. Monoclonal antibodies 4D4 and ID8 were used to detect expression of Gne and N, respectively. A specific green fluorescent signal around the nucleus of Gne-expressing cells is seen indicating that the recombinant protein is within the cell. Anti-Gn control, noninfected Sf9 cells stained with Gn monoclonal antibody (4D4); anti-N control, noninfected Sf9 cells stained with N monoclonal antibody (ID8) show negative staining. The cell nucleus is stained in blue color.

FIG. 3.

In vitro glycosylation assay of Rift Valley fever glycoproteins, Gc and Gne. (A) Treatment of Gc-recombinant baculovirus-infected Sf9 cells with varying concentrations of tunicamycin (0.5 μg/mL to 10 μg/mL) resulted in inhibition of glycosylation shown by a shift in electrophoretic migration. (B) Similar treatment of Gne (8 μg/mL and 10 μg/mL) resulted in marginal molecular weight shift (compare with nontreated, nt), since the protein has one putative N-glycosylation site; Gc has four putative N-glycosylation sites. Treatment of baculovirus expressed sheep prion protein (PrP) with varying concentrations of tunicamycin (1 μg/mL to 10 μg/mL) resulted in inhibition of glycosylation of the protein(C). nt, nontreated controls; m, molecular weight marker.

FIG. 4.

Western blot showing a sample of immunoreactivity of purified baculovirus-expressed proteins with RVFV antisera from sheep. Reactivity shows that the proteins were expressed in the correct conformation. Gne, ectodomain of amino-terminal glycoprotein; N, nucleoprotein; NSs, nonstructural protein S segment; Gc, carboxy-terminal glycoprotein; CL, noninfected cell lysate; m, molecular weight marker; pv, postvaccination; pi, postinfection.

FIG. 5.

Reactivity of recombinant RVFV proteins, nucleoprotein, N (A), nonstructural protein, NSs (B), glycoprotein, Gne (C), glycoprotein, Gc (D), and nonstructural protein NSm (E) with antisera from MP12 vaccinated sheep. Day 0, prevaccination sera; PC, positive control sera derived from sheep challenged with RVFV wild-type, ZH501; P1–P6, day-28 sera from sheep vaccinated with MP12 RVFV strain (Laramie, WY); P7–P10, day-28 sera from sheep infected with the wild-type virus (ZH501). Asterisks (*) denote level of statistical significance and show that differences in optical density (OD) values of sera tested for each of the time-points was significantly different (p<0.05) from day 0 (prevaccination) sera. Cutoff OD value for each enzyme-linked immunosorbent assay (ELISA) was determined by addition of 2 standard deviations to the mean OD value of serum obtained from prevaccinated/noninfected sheep. N, 0.320; NSs, 0.358; Gn, 0.395; Gc, 0.387; NSm, 0.208.

FIG. 5.

Reactivity of recombinant RVFV proteins, nucleoprotein, N (A), nonstructural protein, NSs (B), glycoprotein, Gne (C), glycoprotein, Gc (D), and nonstructural protein NSm (E) with antisera from MP12 vaccinated sheep. Day 0, prevaccination sera; PC, positive control sera derived from sheep challenged with RVFV wild-type, ZH501; P1–P6, day-28 sera from sheep vaccinated with MP12 RVFV strain (Laramie, WY); P7–P10, day-28 sera from sheep infected with the wild-type virus (ZH501). Asterisks (*) denote level of statistical significance and show that differences in optical density (OD) values of sera tested for each of the time-points was significantly different (p<0.05) from day 0 (prevaccination) sera. Cutoff OD value for each enzyme-linked immunosorbent assay (ELISA) was determined by addition of 2 standard deviations to the mean OD value of serum obtained from prevaccinated/noninfected sheep. N, 0.320; NSs, 0.358; Gn, 0.395; Gc, 0.387; NSm, 0.208.