Serum samples
Ninety six serum samples collected from healthy volunteers in 2013 during an RVF surveillance study in Kenya and 93 serum samples collected from suspected-RVF patients during the 2006–2007 RVF outbreak in Kenya were used for the experiments on the detection of IgG and IgM against RVFV, respectively.
Construction of recombinant plasmids
Wild type RVFV isolate 46A-66, isolated from mosquitoes that were collected during the 2006 RVF outbreak in Kenya, was used to construct RVFV recombinant plasmids. The wild type virus was cultured at the Kenya Medical Research Institute for more than 5 passages. RNAs from RVFV infected culture fluid of Vero cells were extracted by using the QIAamp viral mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RVFV N gene was amplified by RT-PCR using the sense primer 5′- AACGGATCCACAATAATGGACAACTAT-3′ and the reverse primer 5’-CCTGGTCGACCACTTAGGCTGCTGT-3′. The sense and reverse primers contained the Bam HI and Sal I restriction sites (underlined nucleotides), respectively. The PCR amplified DNA fragment was digested with Bam HI and Sal I, purified by a QIAEX II gel extraction kit (Qiagen, Hilden, Germany), and subsequently cloned into the corresponding restriction site of the pQE30 vector (Qiagen, Hilden, Germany). The insert of recombinant plasmid was confirmed to be in frame by DNA sequencing. The expression construct, encompassing the coding region for amino acids (aa) 1–245, the full length of RVFV-N protein with a vector derived His-tag (histidine hexamer) at the N-terminus was obtained. The resultant recombinant protein was designated as recombinant RVFV-N protein.
Expression and purification of the rRVFV- N protein
The rRVFV-N protein was expressed by inserting the recombinant plasmid containing the RVFV-N sequence into E. coli strain XL-1 blue. The expression and purification was done as described previously [25]. Briefly E. coli strain XL-1 blue containing the rRVFV-N recombinant plasmid was cultured at 37 °C in 1 litre of Luria-Bertani (LB) medium containing 100 μg/ml of ampicillin and then induced for 3 hrs by 0.2 mM isopropyl β-D-thiogalactoside (IPTG) when the optical density (OD 600 nm) reached 1.0. After harvest by centrifugation at 4 °C for 30 min, the E. coli pellet was resuspended in 10 mM PBS at pH 7.5 with 500 mM NaCl and frozen at − 80 °C. After freezing and thawing three times, the cell suspension was sonicated for 2 min with an interval of 1 s between pulses and centrifuged at 30,000 g for 30 min at 4 °C. The supernatant was then applied to a Talon™ IMAC resin column (Clontech, USA) and the rRVFV-N protein was purified according to the manufacturer’s instructions. Protein concentrations were determined by the Bradford method using a Bio-Rad protein assay reagent kit (Bio-Rad, USA), and the purity of the protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Western blot analysis
Western blot analysis was performed as described previously [26]. Briefly, the recombinant protein was separated by SDS-PAGE and then transferred onto a PVDF membrane (Immobilon, Millipore, USA). After blocking with Blockace (Yokijirushi, Sapporo, Japan) overnight at 4 °C, the membrane was subjected to reaction with rabbit RVFV hyper-immune sera (1:400) or mouse anti-histidine (1:1000 dilution) for 1 hr at 37 °C, followed by incubation with horseradish peroxidase conjugated- goat anti-rabbit IgG, or rabbit anti-mouse IgG (1:1000 dilution) for 1 hr at 37 °C. Finally, the reaction was visualized by dimethyl amino benzidine (DAB) staining.
Preparation of rabbit hyper-immune sera for RVFV
Two litres of infected culture fluid (ICF) was harvested from cultures of Vero cells 5 days after inoculation with RVFV (Smithburn strain). ICF was purified by sucrose-gradient ultracentrifugation at 50,000 x g for 14 hrs at 4 °C. The purified virus (0.25 mg/mL) was inactivated with 1% (final concentration) formalin for about 2 days at 4 °C. Two 3-month-old New Zealand white rabbits were immunized once with formalin-inactivated virus mixed with an equal volume of Freund’s complete adjuvant (0.125 mg/shot/rabbit), and after 1 week boosted thrice with formailin-inactivated virus mixed with an equal volume of Freund’s incomplete adjuvant at intervals of 1 week. Serum was collected and the rabbit anti-virus IgG was purified by using MAb Trap Kit (GE Healthcare) according to the manufacturer’s instructions.
Preparation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid (rSFTSV-N) protein
The rSFTSV-N protein was expressed by inserting the recombinant plasmid containing the N gene sequence of severe with thrombocytopenia syndrome virus (a newly emerged Phlebovirus in east Asia) into E. coli strain XL-1 blue, and purified following the procedure described previously [25]. This protein was used as negative antigen in our newly developed ELISA systems.
ELISA procedures
To evaluate the possibility of applying the rRVFV-N protein for diagnosis of RVFV infection, we developed an IgG sandwich and an IgM capture ELISA system for human sera by using rRVFV-N protein as antigen. The results were compared with those of inactivated virus-based ELISA systems routinely used in the WHO RVF reference diagnostic laboratories.
For all the ELISA procedures, the plates used were 96-well Nunc immunoplates (Thermo Scientific, Denmark), and all the reagents were in 100 μL volumes. Wash buffer was 0.01 M PBS with 0.1% (vol/vol) Tween 20 (PBS-T). Plates were washed thrice with PBS-T between reagent steps. The coating buffer was 0.01 M PBS, pH 7.4, and coated plate was left at 4 °C overnight. All subsequent serum samples and reagent dilutions were done in PBS-T with 5% nonfat milk (Difco, Detroit, USA). Incubations, except for substrate, were done for 1 hr at 37 °C. Plates added with H2O2-ABTS substrate (Kirkegaard & Perry, Gaithersburg, MD) were incubated for 30 min at 37 °C before reading spectrophotometrically. On each plate, 3 control serum samples namely positive control, weak positive control and negative control were added. Optical density (OD) values at 410 nm were recorded. The adjusted OD was calculated by subtracting the OD of the negative antigen reacted wells from the OD of the positive antigen reacted wells. The OD cut-off value was calculated as the mean of the adjusted OD of the negative control sera plus three times the standard deviation, and generally this would give an OD of ≤0.2 at a 1:100 sample dilution. A serum sample was considered positive if the adjusted OD value was greater than or equal to the assay cut-off or 0.2, which ever value was higher.
IgG sandwich ELISA using inactivated RVFV
The IgG sandwich ELISA system using inactivated RVFV was routinely used in the WHO RVF diagnosis reference laboratory in the Kenya Medical Research Institute. The virus strain used in the inactivated virus-based assay systems was ZH501 strain (GenBank: DQ380149.1). The virus was propagated in Vero cells and the infected culture fluid (supernatants) from these cells provided the RVFV antigens used in the assay. The supernatants were inactivated by using 0.3% beta-propiolactone and cobalt irradiated using 3 million rads and were tested for safety using established protocols [27, 28]. Briefly, for each sample to be tested, 4 wells were subjected to the following reaction steps: coating with anti-RVFV hyper-immune mouse ascetic fluid (HMAF) diluted at 1:8000; then, addition of 1:18 dilution of inactivated RVFV culture fluid (positive antigen) to two of these wells and to the other two wells with 1:18 dilution of mock culture fluid (negative antigen); followed by the addition of test serum samples (diluted 1:100) to all the 4 wells; and then, detection of bound IgG by 1:8000 diluted peroxidase-conjugated mouse anti-human IgG (Fc specific) (Accurate Chemical & Scientific Corp, Westbury, NY) after which the ABTS substrate was added to produce a color reaction for OD detection.
In our initial experiments, the procedure above was applied to 12 serum samples and the results obtained were compared with the results when only the inactivated virus antigen in the inacivated virus based sandwich ELISA system was replaced with the rRVF-N (50 ng/well) we produced. This comparison was done to determine the reactivity of rRVF-N in the inactivated virus-based ELISA system used in the WHO reference laboratory in Kenya.
IgG sandwich ELISA using recombinant RVFV-N
The procedure that we developed for IgG sandwich ELISA utilizing rRVFV-N followed the same steps of inactivated virus-based ELISA procedure with some modifications.
Briefly, for each serum sample, 4 wells were subjected to the following reaction steps: coating with 1:10,000 dilution of the purified rabbit RVFV hyper-immune serum IgG prepared as mentioned above, addition of 50 ng rRVFV-N protein (positive antigen) to two wells and of 50 ng rSFTSV-N protein (negative antigen) to the other two wells, followed by the addition of 1:100 diluted human serum sample to the four wells, then addition of 1:30,000 diluted horseradish-peroxidase-conjugated goat anti-human IgG (American Qualex, Califonia, USA); and lastly, color development was done after the addition of ABTS substrate.
The optimal concentration of capture antibody (purified rabbit RVFV hyper-immune serum IgG) used to coat the plate and the rRVFV-N protein was determined by checkerboard titration with two positive and two negative reference serum samples.
IgM capture ELISA using inactivated RVFV
The IgM capture ELISA system using inactivated RVFV was routinely used for diagnosis in the WHO RVF diagnosis reference laboratory in the Kenya Medical Research Institute. Briefly, for each sample to be tested, 4 wells were coated with 1:500 dilution of anti-human IgM (Kirkegaard & Perry Laboratories) after which 1:100 diluted human serum sample was added to all four wells. Two wells were added with positive antigen (inactivated RVFV culture fluid, which was also the same source of antigen in our IgG sandwich ELISA) and the other two wells with negative antigen (mock culture fluid). The wells were then reacted with 1:2000 anti-RVFV HMAF, which were then reacted with 1:16,000 diluted HRP conjugated anti-mouse IgG (Kirkegaard & Perry Laboratories) and color development was done by adding ABTS substrate.
Similar to the IgG sandwich ELISA, to determine the reactivity of rRVFV-N in the inactivated virus-based IgM capture ELISA system, 12 serum samples were tested using the described procedure above with the inactivated RVFV antigen and with our alternative rRVF-N (50 ng/well).
IgM capture ELISA using rRVFV-N protein
The rRVFV-N-based IgM capture ELISA followed the same steps as with the inactivated virus-based ELISA system with some modifications. For each serum sample to be tested, 4 wells were coated with 1:500 dilution of anti-human IgM (Cappel, MP Biochemicals). Then 1:100 diluted human serum was added to the 4 wells. Two wells were added with 50 ng rRVFV-N protein (positive antigen) and the other two wells were added with 50 ng of rSFTSV-N protein (negative antigen). A 1:10,000 dilution of anti-RVFV rabbit hyperimmune serum was added to all four wells, after which a 1:10,000 diluted peroxidase conjugated anti-rabbit IgG (American Qualex, Califonia, USA) was then added. Color development was done by adding ABTS substrate.
