Effect of EB on CPXV and MPXV attachment and entry. CPXV (A, B, C, D) or MPXV (E, F) in the presence (B, D, F) or absence (A, C, E) of 100 μM EB was allowed to attach to BSC-1 cells for 1 hr at room temperature. Samples for attachment staining (A, B, E, F) were fixed with 4% paraformaldehyde, quenched for 5 minutes with 100 mM glycine, blocked with 10% FBS in PBS, and stained with 1:200 anti-VACV antibody (V0500-11D, US Biologial). Samples for entry staining (C, D) were incubated 1 hr at 37°C prior to PFA fixation, glycine quenching, permeabilization with 0.1% saponin, and staining with anti-core antibody (R236). Nuclei were counterstained with DAPI (blue). Arrows indicate selected virions (green) or virus cores (red).