Esf 921

CI-MPR constructs were expressed in a Spodoptera frugiperda cell line (Sf9, Expression Systems) using serum-free medium (ESF921, Expression Systems) and purified as previously described^{24 (link)}. Media containing the secreted CI-MPR constructs were dialyzed against buffer containing 20 mM Tris and 300 mM NaCl (pH 8.0). Dialyzed medium was passed over nickel-nitrilotriacetic acid-agarose resin (Clonetech), washed, and then eluted with buffer containing 20 mM Tris, 150 mM NaCl, and 400 mM imidazole (pH 8.0). Following nickel chromatography purification, the protein was concentrated and then buffer exchanged on a sizing column (Sephacryl S-300, GE Healthcare) equilibrated in 20 mM HEPES and 150 mM sodium acetate. The proteins were concentrated, and those proteins containing a C-terminal AviTag were biotinylated using BirA ligase (BirA500, Avidity) per the protocol provided by the manufacturer.

Vero E6 and C6/36 cells were obtained from American Tissue Culture Collection. Vero E6 cells were maintained in DMEM/10% fetal bovine serum (Wisent) in vent cap flasks (Corning) at 37°C in a 5% CO₂ incubator. The C6/36 cells were grown in ESF-921 (Expression Systems) medium mixed with EMEM in 1∶1 ratio, supplemented with 10% fetal bovine serum (Wisent)/2.5% HEPES (25 mM final)/1% sodium pyruvate (1 mM final)(Sigma Aldrich)/1% nonessential amino acids (Wisent) at 28°C in phenolic cap or plug seal cap flasks (Corning).
Stock of RVFV strain ZH501, kindly provided by Dr. Heinz Feldmann (National Microbiology Laboratory, Winnipeg), was prepared in Vero E6 cells and plaque titrated as follows: 400 µl/well of tenfold serially diluted samples in DMEM were incubated on confluent monolayers of Vero E6 cells in 12 well plates in triplicates at 37°C in 5% CO₂ for 1 h. The inoculum was replaced by 1.75% carboxymethyl cellulose (CMC overlay) (Sigma-Aldrich, St. Louis, MO) in DMEM/0.3% BSA (Wisent) supplemented with 25 mM HEPES (Sigma-Aldrich), 100 µg/ml of Streptomycin and 100 IU/ml of Penicillin (Wisent), and incubated for 4 days at 37°C, 5% CO₂. Formalin (10%) fixed plates were stained with crystal violet (0.5% w/v in 80% methanol in PBS), and virus titer determined in PFU/ml.