Abstract
While porcine epidemic diarrhea virus (PEDV) infects and replicates in enterocytes lining villi of neonatal piglets with high efficiency, naturally isolated variants typically grow poorly in established cell lines, unless adapted by multiple passages. Cells infected with most cell-adapted PEDVs usually displayed large syncytia, a process triggered by the spike protein (S). To identify amino acids responsible for S-mediated syncytium formation, we constructed and characterized chimeric S proteins of the cell-adapted variant, YN144, in which the receptor binding domain (RBD) and S1/S2 cleavage site were replaced with those of a poorly culturable field isolate (G2). We demonstrated that the RBD, not the S1/S2 cleavage site, is critical for syncytium formation mediated by chimeric S proteins. Further mutational analyses revealed that a single mutation at the amino acid residue position 672 (V672F) could enable the chimeric S with the entire RBD derived from the G2 strain to trigger large syncytia. Moreover, recombinant PEDV viruses bearing S of the G2 strain with the single V672F substitution could induce extensive syncytium formation and replicate efficiently in VeroE6 cells stably expressing porcine aminopeptidase N (VeroE6-APN). Interestingly, we also demonstrated that while the V672F mutation is critical for the syncytium formation in VeroE6-APN cells, it exerts a minimal effect in Huh-7 cells, thereby suggesting the difference in receptor preference of PEDV among host cells.
Highlights
Porcine epidemic diarrhea virus (PEDV) causes severe enteric illness of swine (PED)
While the C-terminal motifs are believed to play a key role in its ability to trigger cell–cell fusion [14,15,16,17], it is important to note that S of PEDVYN144 (SYN144) harbored no change of amino acids at the
While it is clear that changes in the S protein accumulated during consecutive passages in Vero cells are associated with the ability of S to induce large syncytium formation, the mechanism by which these mutations in the S protein affect the cell–cell fusion and virus growth in the host cells is still not well understood
Summary
Porcine epidemic diarrhea virus (PEDV) causes severe enteric illness of swine (PED). The infection results in watery diarrhea, vomiting, and anorexia, which can lead to death in up to 100% of neonatal piglets [1,2]. Since its first appearance in the 1970s, PED outbreaks have caused enormous economic losses to the swine industry. PEDV is an enveloped, non-segmented, positive-strand RNA virus, which constitutes the genus Alphacoronavirus of the family Coronaviridae. Its RNA genome encodes replicase proteins and structural proteins including spike (S), envelope (E), membrane (M), nucleocapsid (N), and an accessory protein (ORF3). The virus replicates efficiently in the enterocytes lining the villi of the small intestine, leading to cell death and severe villous atrophy [1]
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