Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

The porcine epidemic diarrhea virus (PEDV) is a highly contagious and virulent enteric coronavirus that causes severe enteric disease in pigs worldwide. PEDV infection causes profound diarrhea, vomiting, and dehydration in pigs of all ages, resulting in high mortality rates, particularly among neonatal piglets. The spike glycoprotein (S) of PEDV plays a crucial role in binding to the host cell receptor and facilitating fusion between the viral and host membranes. Pseudotyped viral particles featuring the PEDV S protein are valuable tools for investigating virus entry, identifying neutralizing antibodies, and developing small molecules to impede virus replication. In this study, we used a codon-optimized PEDV S protein to generate recombinant pseudotyped vesicular stomatitis virus (VSV) particles (rVSV-ΔG-EGFP-S). The full-length S protein was efficiently incorporated into VSV particles. The S protein pseudotyped VSV exhibited infectivity towards permissive cell lines of PEDV. Moreover, we identified a new permissive cell line, JHH7, which showed robust support for PEDV replication. In contrast to the SARS-CoV-2 spike protein, the removal of amino acids from the cytoplasmic tail resulted in reduced efficiency of viral pseudotyping. Furthermore, we demonstrated that 25-hydroxycholesterol inhibited rVSV-ΔG-EGFP-S entry, while human APN facilitated rVSV-ΔG-EGFP-S entry through the use of ANPEP knockout Huh7 cells. Finally, by transducing swine intestinal organoids with the rVSV-ΔG-EGFP-S virus, we observed efficient infection of the swine intestinal organoids by the PEDV spike-pseudotyped VSV. Our work offers valuable tools for studying the cellular entry of PEDV and developing interventions to curb its transmission.

Porcine epidemic diarrhea virus (PEDV) is a major enteric pathogen of swine, and the spike (S) protein determines how the virus enters cells. Coronaviruses enter either at the plasma membrane or through endosomes, but whether variation in S switches the route itself and how this relates to cell and tissue tropism is unclear. In this study, we generated isogenic recombinant PEDVs differing only in S by introducing the S genes of the cell culture-adapted G1b strain SD and the virulent G2b strain FJzz1 into the G2b strain HM backbone. rPEDV-HM-S_HM and rPEDV-HM-S_FJzz1 required exogenous trypsin for infection, whereas rPEDV-HM-S_SD infected Vero cells efficiently without trypsin, and the three viruses differed in cell tropism. rPEDV-HM-S_SD infected BHK-21, Huh7, JHH7, and Vero cells most efficiently but failed to infect IPEC-J2 cells and porcine intestinal organoids, which the two trypsin-dependent chimeras infected. The cathepsin inhibitor E64d blocked rPEDV-HM-S_SD but not the trypsin-dependent viruses, whereas the serine protease inhibitor camostat blocked the trypsin-dependent chimeras but not rPEDV-HM-S_SD. Furthermore, WDR81 was required preferentially by rPEDV-HM-S_SD. Collectively, PEDV S protein determines protease dependence, cell tropism, and entry route, and cell culture adaptation redirects entry to an endosomal pathway that no longer supports infection of the natural target tissue.