Porcine Epidemic Diarrhea Virus Entry Research Articles

Matrine exhibits antiviral activities against PEDV by directly targeting Spike protein of the virus and inducing apoptosis via the MAPK signaling pathway

Porcine Epidemic Diarrhea Virus (PEDV) is a highly contagious virus that causes Porcine Epidemic Diarrhea (PED). This enteric disease results in high mortality rates in piglets, leading to significant financial losses in the pig industry. However, vaccines cannot provide sufficient protection against epidemic strains. Spike (S) protein exposed on the surface of virion mediates PEDV entry into cells. Our findings imply that matrine (MT), a naturally occurring alkaloid, inhibits PEDV infection targeting S protein of virions and biological process of cells. The GLY434 residue in the autodocking site of the S protein and MT conserved based on sequence comparison. This study provides a comprehensive analysis of viral attachment, entry, and virucidal effects to investigate how that MT inhibits virus replication. MT inhibits PEDV attachment and entry by targeting S protein. MT was added to cells before, during, or after infection, it exhibits anti-PEDV activities and viricidal effects. Network pharmacology focuses on addressing causal mechanisms rather than just treating symptoms. We identified the key genes and screened the cell apoptosis involved in the inhibition of MT on PEDV infection in network pharmacology. MT significantly promotes cell apoptosis in PEDV-infected cells to inhibit PEDV infection by activating the MAPK signaling pathway. Collectively, we provide the biological foundations for the development of single components of traditional Chinese medicine to inhibit PEDV infection and spread.

ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses.

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.

Angiotensin Converting Enzyme 2 Does Not Facilitate Porcine Epidemic Diarrhea Virus Entry into Porcine Intestinal Epithelial Cells and Inhibits It-induced Inflammatory Injury by Promoting STAT1 Phosphorylation

ACE2 has been confirmed to be a functional receptor for SARS-CoV and SARS-CoV-2, but research on animal coronaviruses, especially PEDV, are still unknown. The present study investigated whether ACE2 plays a role in receptor recognition and subsequent infection during PEDV invasion of host cells. IPEC-J2 cells stably expressing porcine ACE2 did not increase the production of PEDV-N but inhibited its expression. Porcine ACE2 knockout cells was generated by CRISPR/Cas9 genome editing in IPEC-J2 cells. The expression of PEDV-N did not decrease but slightly increased. The Co-IP results showed that there was no significant association between ACE2 and PEDV-S. There were no obvious interaction between PEDV-S, PEDV-E, PEDV-M and porcine ACE2 promoters, but PEDV-N could inhibit the activity of ACE2 promoters. PEDV-N degraded STAT1 and prevented its phosphorylation, thereby inhibiting the expression of interferon-stimulated genes. Repeated infection of PEDV further confirmed the above results. PEDV activated ACE-Ang II-AT1R axis, while ACE2-Ang (1-7)-MasR axis activity was decreased and inflammatory response was intensified. However, excess ACE2 can reverse this reaction. These results reveal that ACE2 does not facilitate PEDV entry into cells, but relieves PEDV-induced inflammation by promoting STAT1 phosphorylation.

Veratramine inhibits porcine epidemic diarrhea virus entry through macropinocytosis by suppressing PI3K/Akt pathway

Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is available to prevent the disease. Therefore, research for novel antivirals is important. This study aimed to identify the antiviral mechanism of Veratramine (VAM), which actively inhibits PEDV replication with a 50 % inhibitory concentration (IC50) of ∼5 µM. Upon VAM treatment, both PEDV-nucleocapsid (N) protein level and virus titer decreased significantly. The time-of-addition assay results showed that VAM could inhibit PEDV replication by blocking viral entry. Importantly, VAM could inhibit PEDV-induced phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity and further suppress micropinocytosis, which is required for PEDV entry. In addition, PI3K inhibitor LY294002 showed anti-PEDV activity by blocking viral entry as well. Taken together, VAM possessed anti-PEDV properties against the entry stage of PEDV by inhibiting the macropinocytosis pathway by suppressing the PI3K/Akt pathway. VAM could be considered as a lead compound for the development of anti-PEDV drugs and may be used during the viral entry stage of PEDV infection.

Establishment of Replication Deficient Vesicular Stomatitis Virus for Studies of PEDV Spike-Mediated Cell Entry and Its Inhibition.

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.

Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus by Disrupting Virus Entry.

The porcine epidemic diarrhea virus (PEDV), belonging to the α-coronavirus, is the causative agent of porcine epidemic diarrhea (PED). Presently, protection from the existing PEDV vaccine is not effective. Therefore, anti-PEDV compounds should be studied. Berbamine (BBM), Fangchinoline (FAN), and (+)-Fangchinoline (+FAN), are types of bis-benzylisoquinoline alkaloids that are extracted from natural medicinal plants. These bis-benzylisoquinoline alkaloids have various biological activities, including antiviral, anticancer, and anti-inflammatory properties. In this study, we found that BBM, FAN, and +FAN suppressed PEDV activity with a 50% inhibitory concentration of 9.00 µM, 3.54 µM, and 4.68 µM, respectively. Furthermore, these alkaloids can decrease the PEDV-N protein levels and virus titers in vitro. The time-of-addition assay results showed that these alkaloids mainly inhibit PEDV entry. We also found that the inhibitory effects of BBM, FAN, and +FAN on PEDV rely on decreasing the activity of Cathepsin L (CTSL) and Cathepsin B (CTSB) by suppressing lysosome acidification. Taken together, these results indicated that BBM, FAN, and +FAN were effective anti-PEDV natural products that prevented PEDV entry and may be considered novel antiviral drugs.

Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED). In particular, the virus causes severe diarrhea, dehydration, and death in neonatal piglets. Maternal immunity effectively protects neonatal piglets from PEDV infection; however, maternal antibodies can only prevent PEDV attachment and entry into target cells, but have no effects on intracellular viruses. Intracellular antibodies targeting virus-encoded proteins are effective in preventing viral infection. We previously identified four single chain variable fragments (scFvs), ZW1-16, ZW3-21, ZW1-41, and ZW4-16, which specifically targeted the PEDV N protein and significantly inhibited PEDV replication and up-regulated interferon-λ1 (IFN-λ1) expression in host cells. In our current study, the four scFvs were subcloned into replication-defective adenovirus vectors to generate recombinant adenoviruses rAdV-ZW1-16, rAdV-ZW3-21, rAdV-ZW1-41, and rAdV-ZW4-16. ScFvs were successfully expressed in Human Embryonic Kidney 293 (HEK293) cells and intestinal porcine epithelial cell line J2 (IPEC-J2) and were biosafe for piglets as indicated by body temperature and weight, scFv excretion in feces, IFN-γ and interleukin-4 (IL-4) expression in jejunum, and pathological changes in porcine tissue after oral administration. Western blotting, immunofluorescence, and immunohistochemical analyses showed that scFvs were expressed in porcine jejunum. The prophylactic effects of rAdV-ZW, a cocktail of the four rAdV-scFvs, on piglet diarrhea caused by PEDV was investigated. Clinical symptoms in piglets orally challenged with PEDV, following a two-time treatment with rAdV-ZW, were significantly reduced when compared with PEDV-infected piglets treated with phosphate buffered saline (PBS) or rAdV-wild-type. Also, no death and jejunal lesions were observed. ScFv co-localization with the PEDV N protein in vivo was also observed. Next, the expression of pro-inflammatory serum cytokines such as tumor necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, and IFN-λ was assessed by enzyme-linked immunosorbent assay (ELISA), which showed that scFvs significantly suppressed PEDV-induced pro-inflammatory cytokine expression and restored PEDV-inhibited IFN-λ expression. Therefore, our study supported a promising role for intracellular scFvs targeting the PEDV N protein to prevent and treat diarrhea in PEDV-infected piglets.

Identification and Characterization of Cell Lines HepG2, Hep3B217 and SNU387 as Models for Porcine Epidemic Diarrhea Coronavirus Infection.

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.

Death Receptor DR5 as a Proviral Factor for Viral Entry and Replication of Coronavirus PEDV.

Porcine epidemic diarrhea virus (PEDV), a member of Coronaviridae, causes high mortality in newborn piglets, and has caused significant economic losses in the pig industry. PEDV infection can induce apoptosis, both caspase-dependent and caspase-independent, but the details of apoptosis remain clarified. This study investigated the effect of death receptor DR5 on PEDV infection and its relationship with PEDV-induced apoptosis. We found that DR5 knockdown reduced viral mRNA and protein levels of PEDV, and the viral titer decreased from 104.5 TCID50 to 103.4 TCID50 at 12 hpi. Overexpression of DR5 significantly increased the viral titer. Further studies showed that DR5 facilitates viral replication by regulating caspase-8-dependent apoptosis, and the knockdown of DR5 significantly reduced PEDV-induced apoptosis. Interestingly, we detected a biphasic upregulation expression of DR5 in both Vero cells and piglets in response to PEDV infection. We found that DR5 also facilitates viral entry of PEDV, especially, incubation with DR5 antibody can reduce the PEDV binding to Vero cells. Our study improves the understanding of the mechanism by which PEDV induces apoptosis and provides new insights into the biological function of DR5 in PEDV infection.

CCR4-NOT Complex 2-A Cofactor in Host Cell for Porcine Epidemic Diarrhea Virus Infection.

The porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. However, there is no consensus on the primary receptor associated with the PEDV invasion of host cells. An increasing number of studies have reported that PEDV invading host cells may require collaboration between multiple receptors and to better understand the virus-host interaction during PEDV entry, surface plasmon resonance (SPR) assays are performed to investigate relevant host factors interacting with PEDV spike-1 protein (S1) in Vero and IPEC-J2 cell membranes. Subsequently, the rabbit anti-PEDV S1 polyclonal antibody is used as bait to recognize the complexes of IPEC-J2 membrane proteins with or without PEDV infection, followed by detection using liquid chromatography with tandem mass spectrometry (LC-MS-MS). Our results show that 13 and 10 proteins interacting between the S1 protein and plasma membrane protein of Vero or IPEC-J2 can be identified. More specifically, a total of 11 differentially expressed interacting proteins were identified in IPEC-J2 membrane proteins after PEDV infection, compared to the uninfected group. Furthermore, we found that the differentially interacting protein CCR4-NOT complex 2 (CNOT2), identified in PEDV S1 with plasma membrane proteins of Vero cells, is involved in viral infection. The results show that the knockout of CNOT2 significantly inhibits PEDV replication in vitro. These data provide novel insights into the entry mechanism of PEDV.

PEDV: Insights and Advances into Types, Function, Structure, and Receptor Recognition.

Porcine epidemic diarrhea virus (PEDV) has been endemic in most parts of the world since its emergence in the 1970s. It infects the small intestine and intestinal villous cells, spreads rapidly, and causes infectious intestinal disease characterized by vomiting, diarrhea, and dehydration, leading to high mortality in newborn piglets and causing massive economic losses to the pig industry. The entry of PEDV into cells is mediated by the binding of its spike protein (S protein) to a host cell receptor. Here, we review the structure of PEDV, its strains, and the structure and function of the S protein shared by coronaviruses, and summarize the progress of research on possible host cell receptors since the discovery of PEDV.

Enhancing epitope of PEDV spike protein.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of a highly contagious enteric disease of pigs characterized by diarrhea, vomiting, and severe dehydration. PEDV infects pigs of all ages, but neonatal pigs during the first week of life are highly susceptible; the mortality rates among newborn piglets may reach 80–100%. Thus, PEDV is regarded as one of the most devastating pig viruses that cause huge economic damage to pig industries worldwide. Vaccination of sows and gilts at the pre-fertilization or pre-farrowing stage is a good strategy for the protection of suckling piglets against PEDV through the acquisition of the lactating immunity. However, vaccination of the mother pigs for inducing a high level of virus-neutralizing antibodies is complicated with unstandardized immunization protocol and unreliable outcomes. Besides, the vaccine may also induce enhancing antibodies that promote virus entry and replication, so-called antibody-dependent enhancement (ADE), which aggravates the disease upon new virus exposure. Recognition of the virus epitope that induces the production of the enhancing antibodies is an existential necessity for safe and effective PEDV vaccine design. In this study, the enhancing epitope of the PEDV spike (S) protein was revealed for the first time, by using phage display technology and mouse monoclonal antibody (mAbG3) that bound to the PEDV S1 subunit of the S protein and enhanced PEDV entry into permissive Vero cells that lack Fc receptor. The phages displaying mAbG3-bound peptides derived from the phage library by panning with the mAbG3 matched with several regions in the S1-0 sub-domain of the PEDV S1 subunit, indicating that the epitope is discontinuous (conformational). The mAbG3-bound phage sequence also matched with a linear sequence of the S1-BCD sub-domains. Immunological assays verified the phage mimotope results. Although the molecular mechanism of ADE caused by the mAbG3 via binding to the newly identified S1 enhancing epitope awaits investigation, the data obtained from this study are helpful and useful in designing a safe and effective PEDV protein subunit/DNA vaccine devoid of the enhancing epitope.

Expression of the human or porcine C-type lectins DC-SIGN/L-SIGN confers susceptibility to porcine epidemic diarrhea virus entry and infection in otherwise refractory cell lines

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that causes great economic losses in the porcine industry. Although the functional receptor for the virus has not been identified, multiple isolates are able to infect different cell lines. Recently, it has been shown that the human C-type lectin DC-SIGN/L-SIGN (hDC-SIGN/L-SIGN) can promote entry of several coronaviruses. Here we examined whether hDC-SIGN/L-SIGN and its porcine homolog (pDC-SIGN) are entry determinants for PEDV. Expression of hDC-SIGN/L-SIGN or pDC-SIGN in refractory cells dramatically increased infection by a recombinant PEDV expressing green fluorescent protein. In both cases, lectin-mediated infection was inhibited by mannan or anti-hDC-SIGN/L-SIGN or pDC-SIGN antibodies; however, d-galactose had no effect on the virus-infected cells. Our results demonstrate that hDC-SIGN/L-SIGN or pDC-SIGN can mediate the cellular entry and propagation of PEDV, which provides a new theoretical basis for further understanding the infection mechanism of PEDV, and will be helpful for the development of novel therapeutic agents.

In Vitro Antiviral Activities of Salinomycin on Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. Owing to the lack of effective vaccines and specific therapeutic options for PEDV, it is pertinent to develop new and available antivirals. This study identified, for the first time, a salinomycin that actively inhibited PEDV replication in Vero cells in a dose-dependent manner. Furthermore, salinomycin significantly inhibited PEDV infection by suppressing the entry and post-entry of PEDV in Vero cells. It did not directly interact with or inactivate PEDV particles, but it significantly ameliorated the activation of Erk1/2, JNK and p38MAPK signaling pathways that are associated with PEDV infection. This implied that salinomycin inhibits PEDV replication by altering MAPK pathway activation. Notably, the PEDV induced increase in reactive oxidative species (ROS) was not decreased, indicating that salinomycin suppresses PEDV replication through a pathway that is an independent pathway of viral-induced ROS. Therefore, salinomycin is a potential drug that can be used for treating PEDV infection.

Epigallocatechin-3-Gallate, the Main Polyphenol in Green Tea, Inhibits Porcine Epidemic Diarrhea Virus In Vitro.

There are currently no licensed drugs against porcine epidemic diarrhea virus (PEDV), but vaccines are available. We identified a natural molecule, epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, which is effective against infection with PEDV. We used a variety of methods to test its effects on PEDV in Vero cells. Our experiments show that EGCG can effectively inhibit PEDV infections (with HLJBY and CV777 strains) at different time points in the infection using western blot analysis. We found that EGCG inhibited PEDV infection in a dose-dependent manner 24 h after the infection commenced using western blotting, plaque formation assays, immunofluorescence assays (IFAs), and quantitative reverse-transcriptase PCR (qRT-PCR). We discovered that EGCG treatment of Vero cells decreased PEDV attachment and entry into them by the same method analysis. Western blotting also showed that PEDV replication was inhibited by EGCG treatment. Whereas EGCG treatment was found to inhibit PEDV assembly, it had no effect on PEDV release. In summary, EGCG acts against PEDV infection by inhibiting PEDV attachment, entry, replication, and assembly.

Gold/Silver Hybrid Nanoparticles with Enduring Inhibition of Coronavirus Multiplication through Multisite Mechanisms.

As a large enveloped RNA virus, coronavirus is of considerable medical and veterinary significance, and anticoronavirus treatment is challenging due to its biodiversity and rapid variability. In this study, Au@Ag nanorods (Au@AgNRs) were successfully synthesized by coating AuNRs with silver and were shown for the first time to have activity against the replication of porcine epidemic diarrhea virus (PEDV). Viral titer analysis demonstrated that Au@AgNRs could inhibit PEDV infection by 4 orders of magnitude at 12 h post-infection, which was verified by viral protein expression analysis. The potential mechanism of action showed that Au@AgNRs could inhibit the entry of PEDV and decrease the mitochondrial membrane potential and caspase-3 activity. Additionally, we demonstrated that a large amount of virus proliferation can cause the generation of reactive oxygen species in cells, and the released Ag+ and exposed AuNRs by Au@AgNRs after the stimulation of reactive oxygen species has superior antiviral activity to ensure long-term inhibition of the PEDV replication cycle. The integrated results support that Au@AgNRs can serve as a potential therapeutic strategy to prevent the replication of coronavirus.

Transferrin receptor 1 levels at the cell surface influence the susceptibility of newborn piglets to PEDV infection.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of newborn piglets causing acute, severe atrophic enteritis. The underlying mechanisms of PEDV infection and the reasons why newborn piglets are more susceptible than older pigs remain incompletely understood. Iron deficiency is common in newborn piglets. Here we found that high levels of transferrin receptor 1 (TfR1) distributed in the apical tissue of the intestinal villi of newborns, and intracellular iron levels influence the susceptibility of newborn piglets to PEDV. We show that iron deficiency induced by deferoxamine (DFO, an iron chelating agent) promotes PEDV infection while iron accumulation induced by ferric ammonium citrate (FAC, an iron supplement) impairs PEDV infection in vitro and in vivo. Besides, PEDV infection was inhibited by occluding TfR1 with antibodies or decreasing TfR1 expression. Additionally, PEDV infection was increased in PEDV-resistant Caco-2 and HEK 293T cells over-expressed porcine TfR1. Mechanistically, the PEDV S1 protein interacts with the extracellular region of TfR1 during PEDV entry, promotes TfR1 re-localization and clustering, then activates TfR1 tyrosine phosphorylation mediated by Src kinase, and heightens the internalization of TfR1, thereby promoting PEDV entry. Taken together, these data suggest that the higher expression of TfR1 in the apical tissue of the intestinal villi caused by iron deficiency, accounts for newborn piglets being acutely susceptible to PEDV.

Low-level contamination of deoxynivalenol: A threat from environmental toxins to porcine epidemic diarrhea virus infection

Mycotoxins are toxic metabolites produced by fungal species that commonly present in the global environment, especially in cereals and animal forages. The changing global environment may further increase the exposure to these toxins, posing a serious threat to humans and animals. Recently, coronavirus has become one of the most important pathogens threatening human and animal health. It is not clear whether environmental toxins, such as mycotoxins, will affect coronavirus infection. Given that pigs are among the animals most affected by coronavirus and highly homologous to humans, weaned piglets and IPEC-J2 cells were respectively chosen as in vivo and in vitro model to explore the impacts of deoxynivalenol (DON), the most abundant trichothecene mycotoxin in feed, on porcine epidemic diarrhea virus (PEDV) infection and the mechanisms involved. In vivo, twenty-seven piglets infected naturally with PEDV were randomly divided into three groups, receiving the basal diet containing 0, 750 and 1500 μg/kg DON, respectively. Significant increases in the diarrhea rates, gut barrier injury and PEDV proliferation of piglets’ small intestine were observed in experimental groups compared with the control. Additionally, the autophagosome-like vesicles and the autophagy-related proteins expression were also increased in experimental groups. In vitro, we observed that 0.1, 0.5 and 1.0 μM DON significantly promoted the entry and replication of PEDV in IPEC-J2 cells, along with the induction of a complete autophagy. CRISPR‐Cas9‐mediated knockout of LC3B indicated a vital role of autophagy in the promotion. Pretreatment with p38 signaling inhibitor could significantly block the induction of autophagy, indicating that DON could promote the PEDV infection by triggering p38-mediated autophagy. Our findings suggest that mycotoxin could influence the prevalence of coronavirus and provide new ideas for the prevention and control of coronavirus.

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

With the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (PEDV) has led to significant economic loss in the global swine industry. Many studies have described how coronaviruses enter cells, but information on PEDV invasion strategies remains insufficient. Given that the differences in gene sequences and pathogenicity between classical and mutant strains of PEDV may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the PEDV GI and GII subtype strains in Vero cells and IPEC-J2 cells. We first characterized the kinetics of PEDV entry into cells and found that the highest invasion rate of PEDV was approximately 33% in the IPEC-J2 cells and approximately 100% in the Vero cells. To clarify the specific endocytic pathways, systematic research methods were used and showed that PEDV enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin II, clathrin heavy chain, Eps15, cholesterol, and caveolin-1 were indispensably involved. In addition, lipid raft extraction assay showed that PEDV can also enter cells through lipid raft-mediated endocytosis. To investigate the trafficking of internalized PEDV, we found that PEDV entry into cells relied on low pH and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. The results concretely revealed the entry mechanisms of PEDV and provided an insightful theoretical basis for the further understanding of PEDV pathogenesis and guidance for new targets of antiviral drugs.

Mortalin restricts porcine epidemic diarrhea virus entry by downregulating clathrin-mediated endocytosis

Clathrin-mediated endocytosis is a mechanism used for the invasion of cells by a variety of viruses. Mortalin protein is involved in a variety of cellular functions and plays a role in viral infection. In this study, we found that mortalin significantly inhibited the replication of porcine epidemic diarrhea virus (PEDV) through restricting virus entry. Mechanistically, a biochemical interaction between the carboxyl terminus of mortalin and clathrin heavy chain (CLTC) was been found, and mortalin could induce CLTC degradation through the proteasomal pathway, thereby inhibiting the clathrin-mediated endocytosis of PEDV into host cells. In addition, artificial changes in mortalin expression affected the cell entry of transferrin, further confirming the above results. Finally, we confirmed that this host-mounted antiviral mechanism was broadly applicable to other viruses, such as vesicular stomatitis virus (VSV), rotavirus (RV), and transmissible gastroenteritis virus (TGEV), which use the same clathrin-mediated endocytic to entry. These results reveal a new function of mortalin in inhibiting endocytosis, and provide a novel strategy for treating PEDV infections.