Deletion of pentad residues in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets

Our previous study revealed that tissue culture-adapted porcine epidemic diarrhea virus (PEDV) strains, namely KNU-141112-S DEL2/ORF3 and -S DEL5/ORF3, were attenuated to different extents in vivo, suggesting that their independent deletion (DEL) signatures, including 2-amino acid (aa; residues 56–57) or 5-aa (residues 56–60) DEL in the N-terminal domain (NTD) of the spike (S) protein, may contribute to the reduced virulence of each strain. To investigate whether each DEL in the NTD of the S1 subunit is a determinant for the virulence of PEDV, we generated two mutant viruses, named icS DEL2 and icS DEL5, by introducing the identical double or quintuple aa DEL into S1 using reverse genetics with an infectious cDNA clone of KNU-141112 (icKNU-141112). We then orally inoculated conventional suckling piglets with icKNU-141112, icS DEL2, or icS DEL5 to compare their pathogenicities. The virulence of both DEL mutant viruses was significantly diminished compared to that of icKNU-141112, which causes severe clinical signs and 100 % mortality. Interestingly, the degree of attenuation differed between the two mutant viruses: icS DEL5 caused neither diarrhea nor mortality, whereas icS DEL2 caused mild to moderate diarrhea, higher viral titers in feces and intestinal tissues, and 25 % mortality. Furthermore, the icS DEL5-infected piglets displayed no remarkable macroscopic and microscopic intestinal lesions, while the icS DEL2-infected piglets showed histopathological changes in small intestine tissues, including moderate-to-severe villous atrophy. Our data indicate that the loss of the pentad (56GENQG60) residues in S alone can be sufficient to give rise to an attenuated phenotype of PEDV. •The 56GE57 or 56GENQG60 deletion in the NTD of S1 did not affect PEDV growth traits.•The 56GE57 or 56GENQG60 deletion in S1 NTD influenced sialic acid binding of PEDV.•The deletion of 56GE57 or 56GENQG60 in S1 NTD reduced enteropathogenicity of PEDV.•The loss of 56GENQG60 residues in S1 NTD resulted in full attenuation in vivo.•The pentad 56GENQG60 motif in S1 NTD is a virulence determinant of PEDV.