Porcine coronavirus infections have known as they are specific to pigs with predominantly enteric or respiratory diseases. No laboratory animal model is yet been developed in porcine coronaviruses study. Here, we report that development of a transgenic mouse model expressing porcine APN which is susceptible to porcine coronavirus infection. The porcine APN transgene was constructed by fusing with mouse proximal APN promoter at 5' terminus and bovine growth hormone polyadenylation site at its 3' terminus. After screen on pubs from the microinjected mice, we confirmed two transgenic lines expressing porcine APN in various organs. We confirmed the susceptibility to porcine epidemic diarrhea virus, one of the porcine coronaviruses. These transgenic mice will be an important tool for research into the porcine coronaviruses.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was reported in China in 2017 and is a causative agent of porcine enteric disease. Recent studies indicate that cells from various hosts are susceptible to SADS-CoV, suggesting the zoonotic potential of this virus. However, little is known about the mechanisms through which this virus enters cells. In this study, we investigated the role of furin in SADS-CoV spike (S)-mediated cell – cell fusion and entry. We found that the SADS-CoV S protein induced the fusion of various cells. Cell – cell fusion was inhibited by the proprotein convertase inhibitor dec-RVKR-cmk, and between cells transfected with mutant S proteins resistant to furin cleavage. These findings revealed that furin-induced cleavage of the SADS-CoV S protein is required for cell – cell fusion. Using mutagenesis analysis, we demonstrated that furin cleaves the SADS-CoV S protein near the S1/S2 cleavage site, 446 RYVR 449 and 543 AVRR 546 . We used pseudotyped viruses to determine whether furin-induced S cleavage is also required for viral entry. Pseudotyped viruses expressing S proteins with a mutated furin cleavage site could be transduced into target cells, indicating that furin-induced cleavage is not required for pseudotyped virus entry. Our data indicate that S cleavage is critical for SADS-CoV S-mediated cell – cell fusion and suggest that furin might be a host target for SADS-CoV antivirals.

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In this study, we investigated the role of heat shock protein 70 (HSP70) in porcine epidemic diarrhoea virus (PEDV) replication. We found that PEDV infection induced strong HSP70 overexpression in the very early stage of infection. We also confirmed that HSP70 overexpression increased the speed of PEDV replication, resulting in the generation of more virions. In contrast, knockout of HSP70 in cells significantly downregulated PEDV protein expression, resulting in a significant reduction in PEDV replication. Most importantly, we confirmed that among the structural proteins of PEDV, membrane (M) proteins have this important role. We found that membrane proteins control cellular HSP70 expression in PEDV-infected cells. We confirmed HSP70/M complex formation by both immunoprecipitation and immunofluorescence assays. Additionally, PEDV M overexpression induced strong HSP70 expression. All our results clearly confirmed that in PEDV-infected cells, the M protein plays a very important role in PEDV replication in collaboration with HSP70.

Porcine epidemic diarrhea (PED), a highly contagious and lethal enteric disease in piglets, is characterized by diarrhea, vomiting, and dehydration, with high mortality in neonatal piglets. Despite the nationwide use of attenuated and inactivated vaccines, the outbreak of PED is still a major problem in the swine industry. Virus-like particles (VLPs) are artificial nanoparticles similar to viruses that are devoid of genetic material and are unable to replicate. VLPs have good safety profiles and elicit robust cellular and humoral immune responses. Here, we generated PED VLPs in eukaryotic cells and examined their immune responses in mice. We found that the M protein is essential for the formation of PED VLPs. Interestingly, PED VLP formation was decreased in the presence of E proteins and increased in the presence of N proteins. Both IgG and IgA antibodies were induced in mice immunized with PED VLPs. Moreover, these antibodies protected against PED virus infection in Vero cells. PED VLPs immunization induced Th2-dominant immune responses in mice. Our results indicate that PED VLPs induce strong immune responses in mice, suggesting that the VLP-based vaccine is a promising vaccine candidate.