3.1. Recovery of recombinant PEDVs
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.
At 24–48 h post-electroporation, typical PEDV cytopathic effects of giant syncytia were observed for all recombinant viruses. Viral stocks were prepared by passaging viruses in Vero cells one additional time (P1), and the presence of PEDV was confirmed by detection of PEDV S protein by immunofluorescence assay (Figure 1A) and sequence analyses.