Toosendanin inhibits SFTSV internalization. (A) Effect of toosendanin on SFTSV binding. HUVEC cells were pretreated with toosendanin (1 μM) or vehicle (DMSO) at 37°C for 1 h, chilled at 4°C for 15 min, and incubated with SFTSV (MOI = 5) at 4°C for 2 h. The unbound virions were removed with pre-cooled PBS, and cells were harvested for bound virions quantification. (B,C) Effect of toosendanin on SFTSV internalization. HUVEC cells pre-treated with 1 μM toosendanin (B) or a series concentration of toosendanin (C) for 1 h were chilled at 4°C for 15 min and incubated with SFTSV (MOI = 5) for 1 h. Cells were washed with pre-cooled PBS to eliminate the unbound virions and incubated at 37°C for 3 h in the presence of ammonium chloride (20 mM), then washed and treated with trypsin. The relative level of internalized virions was determined by qRT-PCR. (D) Effect of toosendanin on SFTSV internalization was analyzed by immunofluorescence. Cell boundaries (white lines), NP staining (green), DAPI staining (blue). Bars represent 5 μm. (E) Quantification of intracellular virions captured in (D). Median value (red line), number of cells under quantification (n). TSN, toosendanin. Data shown are means ± SD (n = 3). Comparison of mean values (A,B,C) between two groups were analyzed by Student’s t test. *p < 0.05; **p < 0.01; ns, no significance. Comparison of median values (E) between two groups were analyzed by Mann-Whitney test. ****p < 0.0001.