Spike Gene Variability in Porcine Epidemic Diarrhea Virus Determines Cell Tropism and the Route of Cell Entry

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-SHM and rPEDV-HM-SFJzz1 required exogenous trypsin for infection, whereas rPEDV-HM-SSD infected Vero cells efficiently without trypsin, and the three viruses differed in cell tropism. rPEDV-HM-SSD 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-SSD but not the trypsin-dependent viruses, whereas the serine protease inhibitor camostat blocked the trypsin-dependent chimeras but not rPEDV-HM-SSD. Furthermore, WDR81 was required preferentially by rPEDV-HM-SSD. 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.