2.1. Human samples
All the participants provided written informed consent prior to the study. The study was approved by the University of Free State's Health Sciences Research Ethics Committee. A total of 12 serum samples from previously laboratory confirmed CCHFV-infected individuals and 11 CCHFV-negative control samples, isolated from volunteers with no previous history of CCHF virus infection were used in this study. CCHF positive and negative samples were previously tested using commercial Crimean-Congo fever virus Mosaic 2 (IgG) kit (EUROIMMUN) which contains slides for use in the IFA [27]. Each slide contains ten fields, and each field contains three BIOCHIPS, either non-transfected cells or coated with cells expressing the CCHFV NP or the CCHFV Gc and were positive for CCHFV IgG. The 12 positive samples were collected between 2008 and 2011 (Ethics reference number: HSREC 95/2016). The 11 negative samples were collected in 2013 (Ethics reference number: HSREC 95/2016). All samples were heat inactivated at 54ºC for 30 minutes before storing at -80 ºC.
2.2. Cells
For the transfection experiments, adherent HEK-293 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% gamma-irradiated fetal bovine serum (Gibco), 1% L-glutamine (Lonza, Verviers, Belgium), 1% penicillin-streptomycin (Lonza) and 1% non-essential amino acids (Lonza,) and cultivated at 37⁰C, 5%CO2. The 293F (Obtained from Prof. Penny Moore) cells were cultivated in FreestyleTM 293 Expression medium and incubated at 37⁰C, 8% CO2.
2.3. Construct design and cloning
The mammalian cell codon-optimized nucleotide sequence coding for the glycoprotein of the CCHFV isolate SPU187/90 (Genbank: KJ682814.1) was synthesized commercially (GenScript), the strain was isolated from an abattoir worker in the northwest province in South Africa, who tragically succumbed to a fatal CCHF disease [28]. Primers were designed to amplify the Gc ectodomain (from amino acid 1050 to 1594 of SPU 187/90), and a truncated version (from amino acid 1050 to 1577). The signal peptide of the CCHFV glycoprotein (GPC: MHISLMYAVLCLQLCSLG) or the human insulin leader sequence (INS: MALWMRLLPLLALLALWGPDPAAA) were inserted in the forward primers together with a Kozak sequence and the EcoRI restriction site, while an Avi-tag (GLNDIFEAQKIEWHE), a hexahistidine tag and the XhoI restriction site were inserted in the reverse primers (Table 1). The Gc gene was amplified from a plasmid containing a codon optimized SPU187/90 M-segment, using Phusion PCR polymerase following conditions described in table 1 and cloned into the mammalian expression vector pCDNA3.1(+) using EcoRI and XhoI restriction sites. Ligation reactions were used to transform beta-10 competent cells (New England Biolabs) and positive transformants were identified using Sanger sequencing.
Table 1: Primers used to generate expression constructs and PCR conditions.
2.4. Immunofluorescence assay
To confirm the expression of Gc from construct 1, an IFA was carried out on transfected HEK293. Transfected cells were detached using trypsin (Gibco) and centrifuged for 5 min at 500g, the supernatant was discarded, and cells were re-suspended in PBS (Merck). 10μl aliquot of the cells was added to each well of the 10 well slides and left to air-dry for an hour, then fixed with methanol and acetone in a 1:1 ratio for 30 mins at minus 20°C. An IgG CCHF positive serum sample diluted 1:100 in blocking buffer was applied to each well and incubated for 30 mins at 37°C. A CCHF negative serum was used as control. The IFA slides were washed three times for one minute with PBS to remove unbound antibodies. The secondary antibody, goat anti-human IgG conjugated to fluorescein isothiocyanate (SeraCare), diluted 1:20 in 0.1% Evans blue (Merck) was then added and incubated at 37°C for 30 mins. The slides were washed again three times with PBS for one minute and were left to air dry. The slides were then mounted by inverting the slides onto mounting media on a cover slip. Cells were observed for fluorescence using a Nikon Eclipse Ni fluorescent microscope (Nikon), and the image of fluorescent cells was captured with the mounted Camera at 40x magnification.
2.5. Expression and purification of recombinant CCHFV Gc
Recombinant proteins were produced by transfecting 293F cells with purified plasmid DNA using PEI-max (Polysciences). Briefly, 293F cells were cultivated until concentration reached 2x106 cells/ml. DNA was mixed with PEI-max at a 1:3 mass ratio (w/w) and incubated a room temperature for 20 min. The mixture was then added to cells and incubated at room temperature for 20 min, then transferred in the shaking incubator and incubated at 37⁰C, 8%CO2. After 72 hours incubation the supernatant from transfected cells was harvested and centrifuged at 4,000g for 30 min. The supernatant was then passed through a Nickel-NTA Agarose (ThermoFisher Scientific) column and allowed to flow by gravity. The column was then washed with a washing buffer (50 mM Tris-HCl, pH8.0, 20 mM imidazole, 300 mM NaCl) and the recombinant CCHFV Gc, bound to the column was then eluted with Elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM Imidazole, 150 mM NaCl). The protein was concentrated using a 30 KDa cut-off Amicon centrifugal units (Merck). Size-exclusion chromatography (SEC) was further performed with a Superdex 200 (10/300) GL column (Cytiva) equilibrated and eluted with PBS, pH7.4 at a flow rate of 0.5 mL/min.
2.6. SDS-PAGE and Western Blotting
The purified recombinant CCHFV Gc was analysed by SDS-PAGE and Western blotting. To determine protein yield and purity, samples were mixed with 2x Laemmli sample buffer (non-reducing) or 5x lane marker reducing sample buffer (ThermoFisher Scientific) and loaded onto a NuPAGETM 4–12% Bis-Tris gel (Invitrogen) and proteins were visualized with GelCodeTM Blue Safe protein. Protein concentrations were estimated using NanodropTM at 280 nm. For the western blot analysis, the eluted was first separated on a SDS-PAGE, then transferred onto a nitrocellulose membrane and incubated with anti-CCHFV serum sample from a positive sample as primary antibody (dilution 1:200), and detected using goat anti-human IgG alkaline phosphatase (AP)-labelled secondary antibody (Merck, dilution 1:10000). The expression and the target protein were then visualized by BCIP/NBT substrate (ThermoFisher Scientific) reaction.
2.7. Native PAGE
The purified recombinant CCHFV Gc protein was further analyzed using a NativePAGETM 4-16% Bis-Tris gel (Invitrogen). The CCHFV Gc was prepared by mixing with NativePAGETM 4x sample buffer (Invitrogen) and 1%Triton X-100 (BDH Chemicals). The NativeMarkTM (Invitrogen) unstained protein standard was resolved alongside CCHFV Gc to estimate the size. After electrophoresis, the protein and markers were stained using GelCodeTM Blue Safe protein (ThermoFisher Scientific) and de-stained using a de-staining solution (10% Acetic acid and 50% methanol diluted in distilled water).
2.8. Anti-CCHFV Gc-specific ELISA
Recombinant CCHFV Gc protein was used for the development of an indirect IgG ELISA. Unless stated otherwise, all volumes were 100 µl/well, plates were incubated at 37ºC for an hour, and washed three times for 15 seconds with PBS containing 0.1% tween 20 (PBST). Briefly, 96-well PolySorp microtiter plates (Nalgene) were coated with 0.5 µg/ml of purified CCHFV Gc antigen and incubated overnight at 4ºC. The following day, plates were washed and blocked with 200 µl of 5% skim milk/PBS and incubated. Post-incubation plates were washed and incubated with 10-fold serially diluted serum in duplicates, serial dilutions were prepared in 2% skimmed milk/PBS. Samples were diluted 10-fold from 1x 101 to 1x107. On each plate the bottom row was reacted with PBS and used as a control to calculate the net OD. Post-incubation, plates were washed and incubated with goat anti-human IgG conjugated to horseradish peroxidase (SeraCare), diluted 1:10000. Post-incubation plates were washed and visualized by adding 2,2′ -Azino di-ethyl-benzothiazoline-sulfonic acid peroxidase substrate (ABTS) (SeraCare). Plates were incubated for 10 min at room temperature in the dark and the OD values were read at 405 nm using the 800TM TS microplate reader (Biotek). Net OD values were determined for each sample as follows: net OD =OD values from serum samples minus OD values from 2% skimmed milk/PBS. The same protocol was used to compare the reactivity of a CCHF-positive and CCHF-negative samples on CCHFV Gc aggregates and the monomers with serum dilution starting at 1:100.
2.9. Statistical analysis
Statistical differences between CCHF negative and CCHF positive samples were analysed using an unpaired t-test using GraphPad Prism (version 9.5.1). Statistical difference between the reactivity of a CCHF positive sample against Gc aggregates and Gc monomers was analyzed using a paired t-test. P-value of less than 0.05 was considered statistically significant.