Abstract
Entry of the α-coronavirus porcine epidemic diarrhea virus (PEDV) requires specific proteases to activate spike (S) protein for the membrane fusion of the virion to the host cell following receptor binding. Herein, PEDV isolate 85-7 could proliferate and induce cell–cell fusion in a trypsin independent manner on Vero cells, and eight homologous mutation strains were screened by continuous proliferation in the absence of trypsin on Vero cells. According to the whole genome sequence comparative analysis, we identified four major variations located in nonstructural protein 2, S, open reading frame 3, and envelope (E) genes, respectively. Comparative analyses of their genomic variations and proliferation characteristics identified a single mutation within the S2′ cleavage site between C30 and C40 mutants: the substitution of conserved arginine (R) by a glycine (G) (R895G). This change resulted in weaker cell–cell fusion, smaller plaque morphology, higher virus titer and serious microfilament condensation. Further analysis confirmed that this mutation was responsible for optimal cell-adaptation, but not the determinant for trypsin-dependent entry of PEDV. Otherwise, a novel variation (16–20 aa deletion and an L25P mutation) in the transmembrane domain of the E protein affected multiple infection processes, including up-regulation of the production of the ER stress indicator GRP78, improving the expression of pro-inflammatory cytokines IL-6 and IL-8, and promoting apoptosis. The results of this study provide a better understanding of the potential mechanisms of viral functional proteins in PEDV replication, infection, and fitness.
Highlights
Porcine epidemic diarrhea (PED), characterized by watery diarrhea and dehydration, results in significant economic losses in the swine industry worldwide [1,2,3]
In recent years, porcine epidemic diarrhea virus (PEDV) has reemerged in Asia and Europe, and has spread into America and Australia, demonstrating a complex virulence situation, genetic recombination, and evolution [3, 4, 35, 36]
PEDV undergoes frequent variation in the process of clinical evolution and cell culture, which contributes to its fast adaptation to the host microenvironment [5, 21, 37, 38]
Summary
Porcine epidemic diarrhea (PED), characterized by watery diarrhea and dehydration, results in significant economic losses in the swine industry worldwide [1,2,3]. The causative agent, porcine epidemic diarrhea virus (PEDV), was identified as a member of the alphacoronavirus from the family Coronaviridae [1]. In the process of cell culture and clinical spread of PEDV, several genomic sites show variation and. The PEDV spike (S) glycoprotein was recognized as a class I fusion protein, and could be divided structurally into the S1 and S2 regions, mediating the receptor binding and membrane fusion, respectively [7]. Therein S2 contains the S2′ cleavage site, fusion peptide (FP), heptad repeat region 1 (HR1), HR2, and transmembrane domain (TM), in that order [8]. Trypsin cleaves the S protein, allowing a conformational change and mediating fusion activity [8, 9]. The S2′ cleavage site, FP position, and HR1 domain have been predicted as the determinants for
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