BASV pseudotyped virus was generated according to a previously described method19. Briefly, to prepare BASV pseudotyped virus expressing GFP (BASVpv/GFP), BASV-G/pCAGGS was transfected into HEK 293 T cells with X-Treme GENE 9 (Sigma-Aldrich), and VSV-G-complemented (*G) VSVΔG/GFP (*G-VSVΔG/GFP) was inoculated at an moi of 0.1 per cell in 2 days post-transfection. The virus was adsorbed and then washed 4 times with serum-free EMEM. After 24 h of incubation, the supernatants containing BASVpv were centrifuged and stored at −80℃. For the titration of infectious BASVpv, Huh7 cells were infected with a serial 10-fold dilution of harvested BASVpv in the 96-well plate. As a control, VSVpv pseudotyped with VSV-G protein was used. At 1 day post-inoculation, GFP-positive cells were counted under a fluorescence microscope, and the virus titer was expressed as transduction unit (TU) per mL. BASVpv and VSVpv were inactivated with 3% (final concentration) H2O2 for 6 h at room temperature (RT) and H2O2 was removed by dialyzation with phosphate buffer solution (PBS). Finally, dialyzed BASVpv and VSVpv was concentrated by ultracentrifugation (Beckman Coulter Optima L-100 K, Beckman Coulter Life Sciences, California, U.S.A) and the pellet was resuspended with PBS. To confirm the infectivity of H2O2-treated BASVpv and VSVpv, Huh7 was inoculated with the PBS-resuspended BASVpv and VSVpv, and then, GFP-positive cells were measured under a fluorescence microscope.
Immunofluorescence staining
Immunofluorescence (IF) staining was performed to determine the titer of the antibody against the BASV-G protein. To perform IF staining, BASV-G protein-expressing HeLa cells were prepared according to the method of our previous study18. The full-length BASV-G cNDA was amplified by PCR using primers, BamHI-BG-F (5’-AGGATCCAAGATGACCCGCCTGTCCCA-3’) and BG-BamHI-R (5’-TGGATCCTCATTTAGAATTTACAGAGA-3’), and cloned into BamHI sites of the pKS336 expression vector. The resultant vector, BASV-G/pKS336, was transfected into HeLa cells and selected by DMEM supplemented with 5% FBS and blasticidin S hydrochloride (2 µg/mL, Kaken Pharmaceutical. Co, Tokyo, Japan). The generated BASV-G protein-expressing HeLa cells were mixed with mock HeLa cells at 1:1, spotted on 14-well HT-coated slide glasses (AR Brown, Tokyo, Japan), air dried, and fixed with acetone at room temperature (RT) for 5 min and stored at −80℃ until use. The antigen slides were used for IF staining.
To perform IF staining, rabbit sera were serially 2-fold diluted in phosphate-buffered saline (PBS(-)) at a dilution of 1:40 to 1:20,480. They were applied to the wells of the antigen slides and reacted for 1 h at 37℃ in a humidified chamber. After washing in PBS(-), FITC-goat anti-rabbit (Invitrogen, MD, USA) was applied and the slides were incubated for 40 min at 37℃. Pre-sera from the same rabbits were used as negative controls. After washing in PBS(-), the slides were examined under a fluorescence microscope (Olympus BX51 and Olympus DP Controller 1. 1. 1.65; Olympus, Tokyo, Japan). For some IF staining, counterstaining of nuclei was done with DAPI (NucBlue Fixed Cell ReadyProbes Reagent (DAPI), Invitrogen). The antibody titer was recorded as the reciprocals of the highest dilutions producing positive staining.
Immunoblot
Recombinant BASV-G protein, BASVpv, H2O2-treated BASVpv and negative controls were separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad, California, U.S.A.). The membrane was blocked with Blocking One (Nacali tesque, Kyoto, Japan) for 1 h at RT. Then, the membrane was incubated with the primary antibody, anti BASV-G rabbit sera (1:500), overnight at 4℃, followed by wash three times with TBS containing 0.05% Tween 20 (v/v). Then, HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:2,000, Invitrogen, Massachusetts, U.S.A.) was applied to the membrane, and incubated for 1 h at RT. After wash, protein was visualized using Ez West Blue reagent (ATTO, Tokyo, Japan) and the images were analyzed by CanoScan 5200 F scanner (Canon, Tokyo, Japan). In some experiments, chemiluminescence detection (ECL Select Western Blotting Detection Reagent, GE Healthcare, U.S.A., Illinois) was used and the images were analyzed by Amersham ImageQuant 500 (Cytiva, Tokyo, Japan).
50% reduction neutralization titer (RNT50)
An RNT50 test was performed to determine the neutralizing antibody titer using BASVpv/Luc. BASVpv/Luc was generated according to the above protocol using *G-VSVΔG/Luc. To perform RNT50, 2 × 104 cells/well of Huh7 cells were cultured on a 96-well plate (black/clear, FALCON, NY, USA). The next day, rabbit sera were serially diluted in DMEM with 2% FBS at a dilution of 1:100 to 1:204,800. Then, they were mixed with BASVpv/Luc diluted to 1:100 with DMEM with 2% FBS at the rate of 1:1 and incubated for 1 h at 37℃. They were inoculated on the Huh7 cells and incubated for 24 h after the medium on the 96-well plate of Huh7 cells was removed. BASVpv/Luc infectivity was assessed by luciferase activity. The relative light unit (RLU) value of luciferase was determined using a Bright-Glo luciferase assay system (Promega Corporation, Madison, WI). RNT50 was determined as the reciprocal of the highest dilution at which the RLU value of luciferase was < 50% of the RLU value of luciferase measured without rabbit sera.
Animal experiments
Female Japanese white rabbits (Kb1:JW, Kitayama Labes Inc., Ina, Japan) of 8–9 weeks of age at the time of primary inoculation were used in all animal experiments. The rabbits were subcutaneously inoculated with the recombinant BASV-G protein (100 µg) mixed with Inject-Alum (Thermo Fisher Scientific, MA, U.S.A.), BASV-G/pCAGGS (400 µg) mixed with X-tremeGENE™ 9 DNA transfection solution (Roche Life Science, Darmstadt, Germany), and H2O2-BASVpv (108 and 109 TU) mixed with Inject-Alum (Thermo Fisher Scientific) at 2-week intervals. The recBASV-G/LC16 m8 (107 plaque-forming units, pfu) were inoculated subcutaneously at a 1-month interval. The rabbits were monitored weekly regarding condition and body weight during the experiments. Two milliliters of blood samples were collected from the marginal ear vein from the first inoculation, and the antibody or neutralizing antibody titer was determined by IF and the neutralizing test. When the antibody titer reached a plateau value, the rabbits were euthanized using pentobarbital sodium (Somnopentyl, Kyoritsu Seiyaku, Tokyo, Japan) and blood was collected by cardiac puncture. All animal experiments were approved (approved numbers 116014-III, 116076-II, and 118036-II) by the Institutional Animal Care and Use Committee of the NIID and performed under the strict regulations of the animal experimentation guidelines of the National Institute of Infectious Diseases in Japan, and in compliance with the ARRIVE guidelines (https://arriveguidelines.org/about).
Statistical analysis
Data was analyzed using a two-way ANOVA with Sidak’s multiple comparisons test. All statistical analyses were performed using Graph Pad Prism 9.2 for Windows (GraphPad Software, San Diego, California, U.S.A.).
