In vitro characterization of the recombinant iPEDVPT-P5, iPEDVPT-P5-96S, iPEDVPT-P96-5S and iPEDVPT-P96 viruses. (A) Representative immunofluorescence micrographs of porcine epidemic diarrhea virus (PEDV) syncytia in Vero cells stained with anti-PEDV spike antibody at 18 h post-infection (HPI). Note the recombinant viruses carrying spike gene derived from the attenuated iPEDVPT-P96 showed larger syncytia. Scale Bar = 50 μm. (B) The number of nuclei per syncytium was quantified and assessed statistically using Kruskal–Wallis test after immunofluorescence staining. Statistical differences (p < 0.05) among groups were indicated by different letters. (C) Growth kinetics of recombinant viruses in Vero cells after infection at multiplicity of infection (MOI) of 1 and 0.001. Supernatant were collected at indicated time points and the titers were quantified in Vero cells by standard 50% tissue-culture infectious dose (TCID50) assay. (D) Plaque morphologies in Vero cells of the recombinant viruses. The plates were stained with 1% crystal violet solution after 72 HPI. Note that comparing to the iPEDVPT-P96 and iPEDVPT-P5-96S, the plaques produced by iPEDVPT-P5 virus and iPEDVPT-P96-5S were barely visible macroscopically. iP5: iPEDVPT-P5; 96S: iPEDVPT-P5-96S; 5S, iPEDVPT-P96-5S; iP96, iPEDVPT-P96.