*2.3. Expression and Purification of SUDV GP and VP40 Antigens and Preparation of Polyclonal Antibodies*

The SUDV GP (158~368 aa) and VP40 proteins were generated by a prokaryotic expression system. Briefly, the synthesized genes encoding the SUDV GP (158–368 aa) and VP40 proteins were separately inserted into the prokaryotic expression vector PET 30a (+) by using BamHI and NotI, in-frame with the 6×His-tag on the C- and N-terminus to construct PET 30a (+) -GP-his-C/N and PET 30a (+) -VP40-his-C/N. After the recombinant plasmids were confirmed by restriction digest, the two recombinant plasmids were transformed into *E. coli* BL21(DE3) (Transgen Biotech, Beijing, China) and the transformants selected on Luria-Bertani (LB) agar plates with 100 μg/mL kanamycin. A single clone was picked and inoculated into 4 mL of LB medium with 100 μg/mL kanamycin at 37 ◦C for overnight growth. Expression was induced with the addition of 0.4 mM IPTG when the optical density at 600 nm (OD600) reached 0.6. The proteins were purified using a HisPurTM Ni-NTA Spin Purification kit (Thermo Scientific, Carlsbad, CA, USA) according to the manufacturer's instructions. To visualize the expression of the purified proteins, samples were resolved on a 12% SDS-polyacrylamide gel (SDS-PAGE) and stained with Coomassie blue, and protein concentrations were determined using a bicinchoninic acid (BCA) assay (Thermo Scientific, Carlsbad, CA, USA) followed by analysis at an absorbance of 570 nm; bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) was used as the protein standard.

Polyclonal antisera against SUDV GP (158~368 aa) or VP40 were prepared by immunizing BALB/c mice with 10 μg purified GP or VP40 recombinant proteins twice at 2-week intervals, and harvesting the mouse serum, which were mouse polyclonal antisera against SUDV GP (158~368 aa) or VP40.

#### *2.4. Immunofluorescence Testing of the Recombinant Baculoviruses*

The Sf9 cells were infected with the fourth generation recombinant baculovirus rBac-GP-GP or rBac-VP40-VP40 for approximately 48 h, infected Sf9 cells were fixed with 4% paraformaldehyde for 15 min at the room temperature. Infected-Sf9 cells were washed with PBS and blocked with PBS containing 5% BSA (Sigma-Aldrich, St. Louis, MO, USA). Then, the cells were incubated with a 1:100 dilution of a mouse polyclonal antisera against SUDV GP or a mouse polyclonal antisera against SUDV VP40 (generated in our lab) for 1 h at 37 ◦C, and then washed with PBS and incubated with FITC-conjugated goat anti-mouse IgG (Bioss antibodies, Beijing, China) containing 1% diluted Evans blue for 1 h at 37 ◦C; then the infected Sf9 cells were washed with PBST (containing 0.05% Tween-20) three times and were observed with a fluorescence microscope. The control cells were incubated with the two primary antisera at the same time. Baculovirus titers were determined using a BacPAKTM Baculovirus Rapid Titer kit (TaKaRa, Dalian, China).
