Binding of PEDV to RBCs
RBCs were infected with PEDV (MOI=0.1) for 1 h at 37 °C and then wash repeatedly to remove unbound virus. For survival assays, RBCs were cultured from 1 h to 24 h after infection with PEDV and analyzed by plaque assay and Western blot. For Transmission electron microscopy (TEM) observation, RBCs were centrifuged at 300×g for 5 min and fixed in glutaraldehyde/cacodylate buffer.
For observation the effect of oxygen concentration on PEDV binding RBCs, RBCs were pre-cultured for 2 h and then infected with PEDV (MOI=0.1) for 1 h in a fully humidified incubator supplied with pure nitrogen gas to reduce oxygen as well as with 5% CO2 at 37 °C. After incubation with PEDV, cells were still cultured in the incubator as described above for 1 h and 6 h. For observation the effect of temperature on PEDV binding RBCs, RBCs were pre-cultured for 2 h and then infected with PEDV for 1 h in normoxic condition at 33 °C. After infection with PEDV, cells were still cultured in normoxic condition at 33 °C for 1 h and 6 h.