Vero cells (ATCC Number: CCL-81) were resuscitated and propagated to passage 8, cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), and incubated at 37°C with 5% CO₂ until reaching 90% confluence.

DBV virus (Zhejiang/01/2011(SFTSV), GenBank accession: KJ597824.1), isolated from a Zhejiang SFTS patient and stored in our laboratory, was inoculated into monolayer Vero cells in MEM maintenance medium (2% FBS) and incubated at 37°C with 5% CO₂. Cells were monitored daily for cytopathic effects (CPE). Upon observation of 80–100% CPE, viral supernatant were collected and concentrated by ultracentrifugation over a 20% sucrose cushion at 32,000 rpm for 4 h at 4°C. Purified DBV particles were stored at −70°C.

Sequence analysis of multiple DBV strains from GenBank revealed >99% amino acid sequence similarity in envelope glycoproteins Gn and Gc across different strains, indicating remarkable evolutionary conservation of these structural proteins. This high conservation supports the utility of any representative strain for Gn/Gc protein characterization. Based on these findings, two pairs of specific primers targeting the complete Gn and Gc gene sequences of the Zhejiang/01/2011 (SFTSV) reference strain were designed.

Gn-F (5′-GACACGACACCATATGGACTCAGGCCC-3′), Gn-R (5′-GTGTCCTCGAGTCATTATCCATAG-3′), amplifying 1–900 bp of the Gn gene; Gc-F (5′-GACACGACACCATATGTGTGACGA-3′) and Gc-R (5′-GTGTCCTCGAGTCATTAGCGCACGATATACGA-3′), amplifying 1–1,200 bp of the Gc gene. Restriction enzyme sites for NdeI and XhoI were incorporated into the primers (underlined), with flanking sequences including GACACGACAC, GTGTC, and TCATTA.

Total RNA was extracted from DBV using an RNA extraction kit, and cDNA was synthesized using a reverse transcription kit. The DBV Gn and Gc genes were amplified from this cDNA via PCR under the following conditions: 95°C for 2 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min; followed by a final extension at 68°C for 10 min, according to the PCR kit manufacturer’s instructions. The resulting PCR products were analysed by 1.5% agarose gel electrophoresis and purified using a gel extraction kit.

The purified PCR products and the expression vector pET15b were double-digested with NdeI and XhoI. The digested products were purified using a gel extraction kit and ligated with T4 DNA ligase. The ligation products were transformed into competent BL21(DE3) cells, and single colonies were selected for plasmid extraction and PCR verification. Positive recombinant plasmids were sequenced and named pET15b-Gn and pET15b-Gc.

Recombinant plasmids pET15b-Gn and pET15b-Gc were expressed in E. coli. Colonies were cultured in ampicillin-containing LB broth at 37°C until optical density (OD) reached 0.6, IPTG was added, and the culture was transferred to 16°C for induction of expression. Cells were harvested, sonicated, and centrifuged. The pellet containing inclusion bodies was solubilized, and His-tagged rGn and rGc were purified by Ni-NTA affinity chromatography. Proteins concentration were quantified using a BCA kit. For SDS-PAGE, samples (1 μg/lane) were resolved on 12% gels (5% stacking gel) with Tris-Glycine buffer at 80 V for 30 min and 120 V for 60 min, followed by Coomassie Blue staining for 20 min and destaining. Western blot analysis used the same electrophoretic conditions. Proteins were wet-transferred to PVDF membranes at 250 mA for 90 min. After blocking with 5% skim milk at 37°C for 2 h, the membranes were incubated with rabbit anti-His tag antibody and HRP-conjugated goat anti-rabbit IgG at 37°C for 1 h. Bands were visualized using a TMB substrate.

Healthy female New Zealand white rabbits (4 months old, 2 kg) were used, with 4 rabbits per group. The initial immunization dose was 0.3 mg per rabbit, and the subsequent immunizations (second, third, and fourth) were 0.15 mg per rabbit. The antigen for the initial immunization was emulsified with an equal volume of Freund’s complete adjuvant, while the antigens for the subsequent immunizations were emulsified with an equal volume of Freund’s incomplete adjuvant. The rabbits were immunized once a week for a total of 4 weeks. After the final immunization, blood was collected from the rabbits, and serum was separated. Pre-immune sera were collected as a negative control before the initial immunization. IgG was purified from the serum using ammonium sulfate precipitation and DEAE-52 ion exchange chromatography (1.6 × 20 cm), which was conducted by Shanghai Sangong Biological Co., Ltd.

The proteins were diluted to a final concentration of 10 μg/mL in 0.05 mol/L carbonate buffer (pH 9.6) and coated onto 96-well plates at 100 μL per well, incubated overnight at 4°C. The next day, the plates were washed three times with PBST (0.01 M PBS with 0.05% Tween-20) for 3 min each, and then the wells were patted dry. The wells were blocked with 100 μL of PBST containing 10% fetal bovine serum at 37°C for 60 min. After three 3-min washes with PBST, serially diluted antibodies (initial dilution 1:1000) were added and incubated at 37°C for 1 h. Following three 3-min PBST washes, HRP-conjugated goat anti-rabbit IgG (1:8000) was incubated at 37°C for 40 min. After five 3-min PBST washes, 100 μL of substrate solution (TMB) was added and incubated in the dark for 15 min. The reaction was terminated with 50 μL of 2 mol/L H₂SO₄, and the OD₄₅₀ was measured. A result was designated as positive if the mean OD₄₅₀ value of the diluted serum exceeds the mean value of the negative control by a factor of 2.5; otherwise, it was designated as negative.

The supernatant of DBV-infected cell cultures was sonicated, and the proteins were separated by SDS-PAGE and then transferred to a PVDF membrane using a semi-dry blotting apparatus. The membrane was blocked with 5% skim milk for 2 h, washed, and then incubated with primary antibodies (rabbit anti-rGn-IgG and anti-rGc-IgG diluted 1:500) for 2 h with gentle shaking. After washing, the membrane was incubated with a secondary antibody (HRP-conjugated goat anti-rabbit antibody diluted 1:8000) for 2 h with gentle shaking. The membrane was then washed and developed with TMB substrate in a darkroom.

The recombinant proteins rGn, rGc, and BSA (negative control) were diluted to 10 μg/mL in 0.05 mol/L carbonate buffer (pH 9.6), coated onto 96-well plates (100 μL/ well), and incubated overnight at 4°C. The next day, the plates were washed three times with PBST for 3 min each and patted dry. Each well was blocked with 100 μl PBST containing 10% fetal bovine serum and incubated at 37°C for 60 min. After washing three times with PBST (0.05% Tween-20) for 3 min each and patted dry, the plates were stored at 4°C.

Fifteen clinical serum samples (five groups) were selected from the infectious disease recheck samples of Zhejiang Provincial Centers for Disease Control and Prevention, including three DBV IgM antibody-positive samples, three dengue virus IgM antibody-positive samples, three Chikungunya virus antibody-positive samples, three Japanese encephalitis virus IgM antibody-positive samples, and three normal human serum samples. Each experiment was performed in thriplicate. All serum samples were diluted 1:10 and used as primary antibodies, incubated at 37°C for 1 h. The plates were then washed five times with PBST for 3 min each. HRP-conjugated goat anti-rabbit IgG was diluted 1:8000 and incubated at 37°C for 40 min. After washing five times with PBST for 3 min each, 100 μL of substrate solution (TMB) was added to each well and reacted in the dark for 15 min. The reaction was terminated by adding 50 μL of 2 mol/L sulfuric acid, and the OD values were measured at a wavelength of 450 nm using a microplate reader.

Nineteen case samples for DBV IgM antibody-positive were selected and tested using microplates coated with rGn and rGc proteins to calculate the positive detection rates of rGn and rGc. The detected results were analysed by GraphPad Prism 7.0.