Prism software v8

Prism software v. 8.0.2 (GraphPad, USA) was used for statistical analysis and preparation of plot graphs. Statistical significance was determined using the unpaired Student’s t test.

Descriptive statistics were used to summarize the data. Continuous variables are shown as median and IQR. Baseline immuno-phenotyping data were analyzed by one-way ANOVA and post hoc test using the Tukey-Kramer test. The difference between pre- and post-treatment data was assessed using the Wilcoxon signed-rank test. The correlation coefficient was used for matrix correlation analysis. p values less than 0.05 were considered significant. All analyses were conducted using JMP version 14.0 (SAS Institute, Cary, NC, USA) or GraphPad Prism software V.8.0 (GraphPad, La Jolla, CA, USA).

Viability of mammalian Vero and insect KC cells was measured using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Boster Biological Technology, Pleasanton, CA 9, USA) following the manufacturer’s instructions. Briefly, confluent cells (96-well plate format, 5 × 10⁴ cells/well, triplicates) were treated with 100 μL of medium containing the indicated concentrations of ATA (two-fold dilutions, starting concentration of 1000 μM). Plates were incubated at 37 °C or 29 °C for 24 h. Samples were treated with 15 μL of Dye Solution and incubated at 37 °C in a 5% CO₂ atmosphere for 5 h. Next, the cells were treated with 100 μL of Formazan solubilisation solution for 16 h, and absorbance at 570 nm was read using a microplate reader. The viability of compound-treated cells was calculated as a percentage relative to values obtained with mock-treated cells. Non-linear regression curves and the median cytotoxic concentration (CC₅₀) were calculated using GraphPad Prism software v. 8.0. (GraphPad Software, San Diego, CA, USA).

Data are presented as individual data points with summary statistics (median ± IQR or mean ± SD) according to whether data are normally distributed. Parametric or nonparametric statistical tests were applied as appropriate after data were tested for normality using the Kolmogorov-Smirnov test. Tests used for comparisons are indicated in figure legends. Two-tailed P values were computed; a P value of less than 0.05 was considered statistically significant. Statistical analyses were undertaken using GraphPad Prism Software v8.0.

To assess the neutralizing efficacy of sera from immunized mice, a 50% focus reduction neutralization titer (FRNT50) assay was utilized. Sera were first inactivated by heating at 56°C for 30 min and then diluted four-fold across four points, ranging from 1:40 to 1:2560. Each serum dilution was mixed and incubated with an equal volume of SFTSV solution (containing 100 FFU) at 4°C for 1 h. This inoculated mixture was then applied to a monolayer of VeroE6 cells housed in a 24-well plate (SPL Life Sciences, Pocheon, Republic of Korea) and incubated for 2 h at 37°C. After this period, supernatants were discarded, and cells were cultured in an overlay medium (DMEM supplemented with 2% FBS, 1% penicillin/streptomycin, and 1% methylcellulose) at 37°C for 6 days. The process for visualizing viral foci is as described above. Percentage focus reduction was calculated as [(No. of plaques without antibodies) − (No. of plaques with antibodies)]/(No. of plaques without antibodies) × 100. All the experiments were duplicated and the FRNT₅₀ was calculated using nonlinear regression analysis (log[inhibitor] versus normalized response method) embedded in GraphPad Prism Software v8.0 (GraphPad Software; https://www.graphpad.com). All FRNT50 assays were conducted alongside negative control sera to establish the limit of detection under our experimental conditions.