EIPA treatment inhibits ASFV entry and infection in Vero cells.

A–B) EIPA inhibits ASFV uptake. Cells were pretreated (DMSO or EIPA) and infected (MOI 10) for 60 min. A) Infected cells were analyzed by FACS. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal. (n = 7, performed in duplicate; mean±S.D.) B) Cells were incubated with Topro3, TRITC-phalloidin and anti-p72 to stain nuclei (blue), actin filaments (red) and viral particles (green), respectively. Images were taken by CLSM and represented as a maximum z- projection of x–y plane (bottom panels) and x–z plane (upper panels). C–E) The infection is inhibited by EIPA. C) Pretreated cells (20 µM EIPA) were infected (MOI 1) for 16 h and analyzed by immunoblotting with an anti-p72 and an anti-ASFV polyclonal antibodies. D) Pretreated cells (60 µM EIPA) were infected (MOI 5) and stained with Topro3, TRITC-phalloidin and anti-p72. Images were taken by CLSM (mid z-section). Arrowheads: viral factories. E) Supernatants from pretreated (20 µM EIPA) and infected cells (MOI 1) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). F) Bypass of EIPA of ASFV infectivity. Acid mediated bypass was performed and samples of pretreated (20 µM EIPA) and infected cells (MOI 1) for 16 h were analyzed by immunoblotting with an anti-ASFV antibody. G–H) ASFV colocalizes with dextran and induces its uptake. G) Cells were pretreated (60 µM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with 647-dextran for 15 min and analyzed by FACS (n = 3; mean ±S.D.). H) After 30 mpi cells were pulsed with Texas-red-dextran for 15 min and incubated with anti-p72 antibody. Images were taken by CLSM (mid z-section) and Nomarsky. Arrowheads: dextran-virus colocalization. β-actin: load control. S.D., standard deviations. * Unspecific cellular protein detected by the antibody.