We have recently developed a reverse genetic platform of PEDV and have successfully generated a recombinant virus, iPEDVPT-P96, which is phenotypically comparable to its parental attenuated Taiwan PEDV-Pintung 52 strain, PEDVPT-P96 [19]. To study the role of the S gene in the attenuation mechanism of PEDVPT-P96, we further generated the recombinant, highly virulent Taiwan PEDVPT 52 strain, iPEDVPT-P5, and two chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S) by replacing the complete sequence of the S gene reciprocally (Figure S1). In general, the approach to rescue the new recombinant viruses were virtually the same as published previously [19]. However, for the generation of iPEDVPT-P5 and iPEDVPT-P5-96S, we split the plasmid B to two fragments at nucleotide position of 9654 according to Hou et al. [23] in order to compensate for the instability caused by the toxic sequences. The in vitro and in vivo properties of iPEDVPT-P5 were confirmed similarly to those of the parental PEDVPT-P5 strain in a seven-day-old conventional piglet model.