To evaluate the pathogenicity related to S gene replacement in PEDVPT 52 strain, we orally inoculated 2 mL of 5 × 102 TCID50/mL of iPEDVPT-P5, iPEDVPT-P5-96S, iPEDVPT-P96, iPEDVPT-P96-5S viruses and with PI medium in seven-day-old crossbred piglets assigned in the corresponding groups. Parameters used to assess the pathogenicity of different recombinant viruses were summarized in Table 2 and shown in Figure 2. The target sequences of the recombinant viruses at the time point of peak fecal viral shedding in each pig was confirmed identical to the original inoculum. No viral RNA shedding, diarrhea or mortality was detected in the mock-treated group during the entire experimental course. Piglets inoculated with recombinant viruses carrying the S gene derived from the highly virulent iPEDVPT-P5, namely the iPEDVPT-P5 itself and iPEDVPT-P96-5S, had an early onset of clinical symptoms including diarrhea, anorexia and decreased activity, and peak viral shedding at 1 d post-inoculation (DPI). At 3 DPI, piglets inoculated with iPEDVPT-P5 (n = 3) and iPEDVPT-P96-5S (n = 3) both exhibited extensive PEDV-induced villous blunting and atrophy in the jejunum and, to a lesser extent, in the ileum; histological changes in the duodenum were not obvious (Figure 3). Mortality rates of both groups were comparable, reaching 50% at 2 (iPEDVPT-P5 group) and 5 (iPEDVPT-P96-5S group) DPI and ultimately exceeded 80% by 10 DPI.