Cytoxicity and anti-PRRSV activity of Toosendanin (TSN) in Marc-145 cells. (A) Chemical structure of TSN. (B) TSN Cytoxicity in Marc-145 cells was examined using MTT assay and was presented as relative cell viability of the viable cells in the absence of the compound (set up as 100%). (C, D) TSN antiviral activity against PRRSV (NADC30-like, VR2332 and CH-1a) infection of Marc-145 cells was examined by immunofluorescence assays (IFA). Cells grown in 96-well plates were infected with PRRSV (0.05 MOI) for 2 h at 37 ℃ and then cultured in fresh media containing various concentrations of TSN. IFA for the N protein of PRRSV was performed at 48 hpi using Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (red). Nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI) (blue). Results shown in C are the mean values of percentages of PRRSV-infected cell ratio in TSN-treated groups compared to the DMSO-treated control (0 µM TSN, set as 100%) from three independent experiments; and D is one representative IFA data from C. Scale bar: 100 µm. Statistical significances are denoted by *p < 0.05, **p < 0.01, and ***p < 0.001.