Protein a g conjugated with hrp

A virus-neutralizing (VN) test for SFTSV was conducted using a 50% focus-reduction neutralizing test (FRNT₅₀). Approximately 2,000 focus-forming units/ml of
SFTSV HB29 strain were equally mixed with inactivated serum, which were diluted two-fold from 1:5 with DMEM containing 2% FCS and 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo
Fisher Scientific) and incubated at 37°C for 1 hr. Then, 100 µl of the mixture was inoculated onto monolayered Vero cells in a 12-well plate (SUMITOMO BAKELITE, Tokyo, Japan). After 1 hr of
adsorption, cells were washed with DMEM and overlaid with DMEM containing 2% FCS and 1% methylcellulose and cultured at 37°C in 5% CO₂ for seven days. Cells were fixed by 10%
buffered formalin, exposed to UV irradiation and permeabilized by PBS containing 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Infected cells were stained by rabbit antibodies
against SFTSV N protein [26 (link)] and Protein A/G conjugated with HRP (Thermo Fisher Scientific) using 3,3′-diaminobenzidine tetrahydrochloride hydrate
(DAB; FUJIFILM Wako Pure Chemical Corp.). Serum samples that reduced the number of focuses to ≤50% of the number in control wells were considered to be positive.

To determine the specific antibody against SFTSV in animal sera, enzyme-linked immunosorbent assay (ELISA) was performed using extracts from SFTSV HB29- or mock-infected HuH-7 cells [5 (link), 9 (link)]. In brief, antigens were coated with coating buffer (0.05 M carbonate-bicarbonate buffer, pH9.6) in an ELISA
plate (MaxiSorp; NUNC, Roskilde, Denmark), incubated at 37°C for 2 hr, and blocked by 1% Block Ace (KAC, Kyoto, Japan) in PBS at 37°C for 30 min. As the 1st antibody, serum was diluted to
1:100 in 0.4% Block Ace in PBS containing 0.05% of tween 20 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) (PBS-T) and 100 µl was added to duplicate wells. After incubation at 37°C for 30
min, Protein A/G conjugated with HRP (Thermo Fisher Scientific) diluted in 0.4% Block Ace in PBS-T was used as the secondary antibody. These reactions were visualized by ABTS Peroxidase
Substrate (SeraCare Life Science, Milford, MA, USA) and the optical density (OD) was measured by a microplate reader (Bio-Rad, Hercules, CA, USA) using a 405 nm filter. For raccoons, an
ELISA cut-off value of 0.564 was applied after comparison with a virus-neutralizing test.