Sequence comparison between the autologous highly virulent PEDVPT-P5 and attenuated PEDVPT-P96 revealed several amino acid substitutions [18], especially in the S gene (see Table 1). Consistent with other studies, these mutations chiefly accumulated in the S2 domain, presumably because of adaptation to Vero cells [12,29]. In the present study, the enhanced fusogenic ability in vitro was distinctly ascribed to the S gene, more specifically the S2 domain, derived from iPEDVPT-P96 and presumably represented the evolutionary process to increase viral progeny since Vero cells are not of swine-origin but readily susceptible to PEDV infection. Animal experiments with seven-day-old piglets revealed that the exchange of the S gene from the iPEDVPT-P96 resulted in a delayed onset of peak viral shedding, diarrhea, and milder villous atrophy when examined histologically at 3 d post-inoculation. Additionally, in contrast to the findings published by Wang et al. [17], Suzuki et al. [30] reported that replacement of the entire S gene or S1 sequence from a highly virulent OKN-1/JPN/2013 American type PEDV strain enabled the attenuated rPEDVGFP-CV777 to acquire virulence in piglets. In iPEDVPT-P96 virus, however, only two amino acid substitutions were identified in the S10 (C144T) and S1B (T554C) domains compared to the iPEDVPT-P5 virus. For PEDV, S10 and S1B domains are known to contain sialic acid-binding and receptor-binding domains, respectively; thus, are crucial for viral entry [31]. Although Hou et al. [23] previously showed that deletion of 197 amino acids in S10 domain of PEDV resulted in attenuation in piglets, a more specific epitope has yet to be identified. Therefore, we are curious about whether only these two mutations in iPEDVPT-P96 can attenuate the highly virulent iPEDVPT-P5 virus or if the S2 domain must be primarily accounted for the attenuation. Future studies will be conducted to elucidate these questions.