Viruses and cells
Anti-CCHFV and isotype antibodies
Mice
Passive protection experiments
Histology
In situ hybridization
Confocal microscopy
Microscopy
Cloning
GP38 purification
GP38 capture ELISA
GP38his ELISA
DNA vaccination
Western blot
VLP production
Immunogold staining and electron microscopy of VLPs
Antibody competition
Statistical analysis
293T cell monolayers were transfected in T25 flasks with the indicated plasmids using FuGENE 6 (Promega). Transfected cells were incubated for 72 hours in a 37°C incubator with 5% CO2. Cells were detached with gentle tapping, were pelleted by centrifugation at 750g, and resuspended in 200 μl of fluorescence-activated cell sorting (FACS) buffer (PBS and 5% FBS). Cells were incubated (1:100 dilution) with mAbs and incubated for 1 hour at room temperature. Cells were then pelleted by centrifugation at 750g and washed three times with FACS buffer. Cells were then incubated with goat anti-mouse Alexa Fluor 488–conjugated secondary antibody (1:500; Invitrogen) for 30 min at room temperature, washed three times, and resuspended in 1 ml of FACS buffer. Flow cytometry was performed on a FACSCalibur flow cytometer (Becton Dickinson). Data were collected and analyzed using FlowJo software (Tree Star Inc., Ashland, OR). A total of 10,000 cells were analyzed for each sample using a live gate.