Porcine Epidemic Diarrhea Virus Infection Research Articles

Characterization of RNA G-quadruplexes in porcine epidemic diarrhea virus genome and the antiviral activity of G-quadruplex ligands

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there are still no anti-PEDV drugs with accurate targets. G-quadruplexes (G4s) are non-canonical secondary structures formed within guanine-rich regions of DNA or RNA, and have attracted great attention as potential targets for antiviral strategy. In this study, we reported two putative G4-forming sequences (PQS) in S and Nsp5 genes of PEDV genome based on bioinformatic analysis, and identified that S-PQS and Nsp5-PQS were enabled to fold into G4 structure by using circular dichroism spectroscopy and fluorescence turn-on assay. Furthermore, we verified that both S-PQS and Nsp5-PQS PQS could form G4 structure in live cells by immunofluorescence microscopy. In addition, G4-specific compounds, such as TMPyP4 and PDS, could significantly inhibit transcription, translation and proliferation of PEDV in vitro. Importantly, these compounds exert antiviral activity at the post-entry step of PEDV infection cycle, by inhibiting viral genome replication and protein expression. Lastly, we demonstrated that TMPyP4 can inhibit reporter gene expression by targeting G4 structure in Nsp5. Taken together, these findings not only reinforce the presence of viral G-quadruplex sequences in PEDV genome but also provide new insights into developing novel antiviral drugs targeting PEDV RNA G-quadruplexes.

N-Acetylcysteine Administration Improves the Redox and Functional Gene Expression Levels in Spleen, Mesenteric Lymph Node and Gastrocnemius Muscle in Piglets Infected with Porcine Epidemic Diarrhea Virus.

Our previous study reported that N-acetylcysteine (NAC) administration improved the function of intestinal absorption in piglets infected with porcine epidemic diarrhea virus (PEDV). However, the effects of NAC administration on the functions of other tissues and organs in PEDV-infected piglets have not been reported. In this study, the effects of NAC on the liver, spleen, lung, lymph node, and gastrocnemius muscle in PEDV-infected piglets were investigated. Thirty-two 7-day-old piglets with similar body weights were randomly divided into one of four groups: Control group, NAC group, PEDV group, and PEDV+NAC group (eight replicates per group and one pig per replicate). The trial had a 2 × 2 factorial design consisting of oral administration of 0 or 25 mg/kg body weight NAC and oral administration of 0 or 1.0 × 104.5 TCID50 PEDV. The trial lasted 12 days. All piglets were fed a milk replacer. On days 5-9 of the trial, piglets in the NAC and PEDV + NAC groups were orally administered NAC once a day; piglets in the control and PEDV groups were orally administered the same volume of saline. On day 9 of trial, piglets in the PEDV and PEDV+NAC groups were orally administrated 1.0 × 104.5 TCID50 PEDV, and the piglets in the control and NAC groups were orally administrated the same volume of saline. On day 12 of trial, samples, including of the liver, spleen, lung, lymph node, and gastrocnemius muscle, were collected. PEDV infection significantly increased catalase activity but significantly decreased the mRNA levels of Keap1, Nrf2, HMOX2, IFN-α, MX1, IL-10, TNF-α, S100A12, MMP3, MMP13, TGF-β, and GJA1 in the spleens of piglets. NAC administration ameliorated abnormal changes in measured variables in the spleens of PEDV-infected piglets. In addition, NAC administration also enhanced the antioxidant capacity of the mesenteric lymph nodes and gastrocnemius muscles in PEDV-infected piglets. Collectively, these novel results revealed that NAC administration improved the redox and functional gene expression levels in the spleen, mesenteric lymph nodes, and gastrocnemius muscle in PEDV-infected piglets.

Transient expression and purification of S2 protein from porcine epidemic diarrhea virus in plants

Porcine epidemic diarrhea (PED), caused by the Porcine epidemic diarrhea virus (PEDV), is an acute, highly contagious disease of pigs of all ages, especially piglets under one week old, with the mortality rate reaching 95–100%. Developing an effective vaccine against PEDV in Vietnam is urgent. Spike protein containing S1 and S2 subunits is considered the main target for vaccine development, and the S2 subunit contains immunodominant neutralizing epitopes of PEDV. To date, the expression of S2 protein in plants has not been reported and evaluated. In this study, the gene encoding the S2 subunit of a PEDV strain belonging to genotype 2a was amplified, sequenced, and inserted in a pRTRA vector containing the GCN4pII (pII) motif. The plant expression cassette containing S2-pII was then inserted into the pCB301 vector. The Agrobacterium tumefaciens harboring the pCB301-S2-pII vector was transformed into Nicotiana benthamiana leaves via agroinfiltration. The accumulation level of S2-pII protein in tobacco leaves was semi-quantified by Western blot, accounting for approximately 86.7 mg/kg of fresh leaves and 1.47% total soluble protein, which was 294-fold higher than the accumulation level of S1-pII protein in our previous publication. S2-pII protein was successfully purified by immobilized metal affinity chromatography (IMAC). The oligomeric state of S2-pII protein was characterized by size exclusion chromatography (SEC). The S2-pII protein was determined to be a multimer protein with a high molecular weight. These results are the basis for more extended studies to develop a plant-based S2 vaccine against PEDV infection.

Resistance of Field-Isolated Porcine Epidemic Diarrhea Virus to Interferon and Neutralizing Antibody.

Variant porcine epidemic diarrhea virus (PEDV), belonging to the genogroup G2b, has higher pathogenicity and mortality than classical PEDV, belonging to the genogroup G1a. To understand the pathogenesis of the G2b PEDV, we examined the resistance of the G2b PEDV to interferon (IFN) and neutralizing antibodies, which are important for controlling PEDV infection. We found that the G2b PEDV showed higher resistance to IFN than G1a PEDV. The G1a PEDV could replicate in IFN-deficient Vero cells, but not in IFN-releasing porcine alveolar macrophages, whereas the G2b PEDV showed similar infectivity in both types of cells. We also found that G2b PEDV was not effectively blocked by neutralizing antibodies, unlike G1a PEDV, suggesting differences in the antigenicity of the two strains. These results provide an understanding of the occurrence of variant PEDV and its pathogenesis.

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.

MiR-615 facilitates porcine epidemic diarrhea virus replication by targeting IRAK1 to inhibit type III interferon expression.

Porcine epidemic diarrhea virus (PEDV) in the Coronavirus family is a highly contagious enteric pathogen in the swine industry, which has evolved mechanisms to evade host innate immune responses. The PEDV-mediated inhibition of interferons (IFNs) has been linked to the nuclear factor-kappa B (NF-κB) pathway. MicroRNAs (miRNAs) are involved in virus-host interactions and IFN-I regulation. However, the mechanism by which the PEDV regulates IFN during PEDV infection has not yet been investigated in its natural target cells. We here report a novel mechanism of viral immune escape involving miR-615, which was screened from a high-throughput sequencing library of porcine intestinal epithelial cells (IECs) infected with PEDV. PEDV infection altered the profiles of miRNAs and the activities of several pathways involved in innate immunity. Overexpression of miR-615 increased PEDV replication, inhibited IFN expression, downregulated the NF-κB pathway, and blocked p65 nuclear translocation. In contrast, knockdown of miR-615 enhanced IFN expression, suppressed PEDV replication, and activated the NF-κB pathway. We further determined that IRAK1 is the target gene of miR-615 in IECs. Our findings show that miR-615 suppresses activation of the NF-κB pathway by suppressing the IRAK1 protein and reducing the generation of IFN-IIIs, which in turn facilitates PEDV infection in IECs. Moreover, miR-615 inhibited PEDV replication and NF-κB pathway activation in both IECs and MARC-145 cells. These findings support an important role for miR-615 in the innate immune regulation of PEDV infections and provide a novel perspective for developing new treatments.

Multifaceted Effects and Mechanisms of N-Acetylcysteine on Intestinal Injury in a Porcine Epidemic Diarrhea Virus-Infected Porcine Model.

This study investigates the potential effects of N-acetylcysteine (NAC) on intestinal injury in a porcine epidemic diarrhea virus (PEDV)-infected porcine model. Thirty-two piglets are randomly assigned to one of four groups: the control, PEDV, NAC, and NAC+PEDV. Piglets in the NAC+PEDV group are orally administrated with NAC (100mg(kg·BW)-1 day-1 ) for 4 consecutive days after 2 days of PEDV infection. The results show that NAC administration decreases the diarrhea rate and improves intestinal morphology. The concentration of diamine oxidase and intestinal fatty-acid binding protein, as well as IL-1β, IL-8, and TNF-α in the plasma, is decreased by NAC. Intriguingly, NAC administration significantly increases the viral load in the jejunum and ileum and down-regulates the expression of interferon-related genes. Microarray and proteomic analyses show that the differentially expressed genes/proteins between NAC+PEDV and PEDV groups are highly enriched in substance transport. Furthermore, aquaporin 8/10 expression is significantly increased by NAC upon PEDV infection. NAC administration alleviates PEDV-induced intestinal injury by inhibiting inflammatory responses and improving substance transport, but promotes viral replication by inhibiting interferon signaling. These results suggest NAC exhibits multifaceted effects upon PEDV infection, and thus caution is required when using NAC as a dietary supplement to prevent viral infection.

Differences in cytokines expression between Vero cells and IPEC-J2 cells infected with porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) primarily infects suckling piglets and causes severe economic losses to the swine industry. Cytokines, as part of the innate immune response, are important in PEDV infection. The cytokines secreted by cell infection models in vitro might reflect true response to viral infection of target cells in vivo. Vero cells and IPEC-J2 are commonly used as an in vitro model to investigate PEDV infection. However, it is not clear which type of cells is more beneficial to the study of PEDV. In our study, firstly, Vero cells and IPEC-J2 were successfully infected with PEDV virulent strains (HBQY2016) and attenuated vaccine strains (CV777) and were capable of supporting virus replication and progeny release. Moreover, cytokine differences expression by Vero cells and IPEC-J2 cells infected with two PEDV strains were analyzed. Compared with IPEC-J2 cells, only the mRNA levels of TGF-β, MIP-1β and MCP-1 were detected in Vero cells. ELISA assay indicated that compared to the control group, the PEDV-infected group had significantly induced expression levels of IL-1β, MIP-1β, MCP-1, IL-8, and CXCL10 in IPEC-J2 cells, while only secretion level of IL-1β, MIP-1β and IL-8 in Vero cells were higher in PEDV infected group. Finally, cytokines change of piglets infected PEDV-HBQY2016 strains were detected by cDNA microarray, and similar to those of IPEC-J2 cells infected PEDV. Collectively, these data determined that the IPEC-J2 could be more suitable used as a cell model for studying PEDV infection in vitro compared with Vero cells, based on the close approximation of cytokine expression profile to in vivo target cells.

Development of a multiplex RT-PCR method for the detection of four porcine enteric coronaviruses.

Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV M gene, PDCoV N gene, TGEV S gene and SADS-CoV RdRp gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 105 copies/μL, 6.48 × 105 copies/μL, 8.54 × 105 copies/μL and 7.79 × 106 copies/μL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.

Chemokines induced by PEDV infection and chemotactic effects on monocyte, T and B cells

Porcine epidemic diarrhea virus (PEDV) is a re-emerging pathogen that causes severe economic loss in the pig industry. The host’s innate immune system is the first line of defense on virus invasion of the small intestinal epithelial cells. Chemokines, as a part of the innate immune system, play an important role in host immunity against infection, however, and their expression and chemotactic effect on key immune cells in PEDV infection remains unclear. In this study, cDNA microarray was firstly performed to analyzed ileum tissue of piglets on the third day after PEDV infection. The differentially expressed genes mainly involved in multiple biological processes, chemokine signaling pathway and cytokine receptor interaction signaling pathway had the highest enrichment according to GO and KEGG enrichment analysis. The expression levels of chemokines MCP-1, MIP-1β, IL-8, CXCL9, CXCL10 and CXCL13 in ileum of PEDV- infected piglets were significantly higher than those in the control group. The expression of chemokines in vivo experiment was further verified by RT-qPCR and ELISA using PEDV-infected IPEC-J2 cells. The results showed that the PEDV-infected IPEC-J2 cells had significantly induced protein expression of MCP-1, MIP-1β, IL-8, CXCL9, CXCL-10 and CXCL13. These results indicated that the changes of chemokines expressed in the ileum of piglets (in vivo) were consistent with those in IPEC-J2 cells (in vitro) after PEDV infection. Finally, the role of chemokines in immune cell migration during PEDV infection was illustrated by siRNA-mediated knock down method and the co-culture model of IPEC-J2 cells with peripheral blood leukocyte cells (PBLCs). The FACS analysis showed that MCP-1 induced by PEDV infection played a chemotactic effect on CD14+ cells, CXCL9 on CD3+CD4-CD8-γδ T, CD3+CD4-CD8+ Tc, CD3+CD4+CD8- Th and CD3+CD4+CD8+ Tm subsets, and CXCL13 on CD19+ B cells. Collectively, our findings first indicate that PEDV-induced chemokines MCP-1, CXCL-9 and CXCL-13 attracted CD14+ cells, T cells and B cells, respectively. These results provide a theoretical basis for studying the mechanism of anti-PEDV infection in piglets.

Porcine Epidemic Diarrhea Virus: An Updated Overview of Virus Epidemiology, Virulence Variation Patterns and Virus-Host Interactions.

The porcine epidemic diarrhea virus (PEDV) is a member of the coronavirus family, causing deadly watery diarrhea in newborn piglets. The global pandemic of PEDV, with significant morbidity and mortality, poses a huge threat to the swine industry. The currently developed vaccines and drugs are only effective against the classic GI strains that were prevalent before 2010, while there is no effective control against the GII variant strains that are currently a global pandemic. In this review, we summarize the latest progress in the biology of PEDV, including its transmission and origin, structure and function, evolution, and virus-host interaction, in an attempt to find the potential virulence factors influencing PEDV pathogenesis. We conclude with the mechanism by which PEDV components antagonize the immune responses of the virus, and the role of host factors in virus infection. Essentially, this review serves as a valuable reference for the development of attenuated virus vaccines and the potential of host factors as antiviral targets for the prevention and control of PEDV infection.

Difference analysis of intestinal microbiota and metabolites in piglets of different breeds exposed to porcine epidemic diarrhea virus infection.

The gut microbial composition of the Luchuan (LC) piglet, one of China's native breeds, has rarely been studied, especially when compared to other breeds. This study developed a porcine epidemic diarrhea virus (PEDV) infection model in LC and Largewhite (LW) piglets, and analyzed the patterns and differences of intestinal microbial communities and metabolites in piglets of these two breeds after infection. The diarrhea score, survival time, and distribution of viral antigens in the intestine of piglets infected with PEDV differed among breeds, with the jejunal immunohistochemistry score of LW piglets being significantly higher than that of LC piglets (P < 0.001). The results of 16S rRNA sequencing showed differences in microbial diversity and community composition in the intestine of piglets with different breeds between PEDV infection piglets and the healthy controls. There were differences in the species and number of dominant phyla and dominant genera in the same intestinal segment. The relative abundance of Shigella in the jejunum of LC piglets after PEDV infection was significantly lower than that of LW piglets (P < 0.05). The key microorganisms differed in the microbiota were Streptococcus alactolyticus, Roseburia faecis, Lactobacillus iners, Streptococcus equi, and Lactobacillus mucosae (P < 0.05). The non-targeted metabolite analysis revealed that intestinal metabolites showed great differences among the different breeds related to infection. Spearman correlation analysis was conducted to examine any links between the microbiota and metabolites. The metabolites in the intestine of different breeds related to infection were mainly involved in arginine biosynthesis, synaptic vesicle cycle, nicotinic acid and nicotinamide metabolism and mTOR signaling pathway, with significantly positive or negative correlations (P < 0.05) between the various microorganisms. This study provides a theoretical foundation for investigating the application of core microorganisms in the gut of piglets of different breeds in the digestive tracts of those infected with PEDV, and helps to tackle the antimicrobial resistance problem further.

DNAJA3 Interacts with PEDV S1 Protein and Inhibits Virus Replication by Affecting Virus Adsorption to Host Cells.

Porcine epidemic diarrhea virus (PEDV) infection causes huge economic losses to the pig industry worldwide. DNAJA3, a member of the Hsp40 family proteins, is known to play an important role in the replication of several viruses. However, it remains unknown if it interacts with PEDV. We found that DNAJA3 interacted with PEDV S1, initially with yeast two-hybrid screening and later with Co-IP, GST pull-down, and confocal imaging. Further experiments showed the functional relationship between DNAJA3 and PEDV in the infected IPEC-J2 cells. DNAJA3 overexpression significantly inhibited PEDV replication while its knockdown had the opposite effect, suggesting that it is a negative regulator of PEDV replication. In addition, DNAJA3 expression could be downregulated by PEDV infection possibly as the viral strategy to evade the suppressive role of DNAJA3. By gene silencing and overexpression, we were able to show that DNAJA3 inhibited PEDV adsorption to IPEC-J2 cells but did not affect virus invasion. In conclusion, our study provides clear evidence that DNAJA3 mediates PEDV adsorption to host cells and plays an antiviral role in IPEC-J2 cells.

A discrete-time survival model for porcine epidemic diarrhoea virus.

Since the arrival of porcine epidemic diarrhea virus (PEDV) in the United States in 2013, elimination and control programmes have had partial success. The dynamics of its spread are hard to quantify, though previous work has shown that local transmission and the transfer of pigs within production systems are most associated with the spread of PEDV. Our work relies on the history of PEDV infections in a region of the southeastern United States. This infection data is complemented by farm-level features and extensive industry data on the movement of both pigs and vehicles. We implement a discrete-time survival model and evaluate different approaches to modelling the local-transmission and network effects. We find strong evidence in that the local-transmission and pig-movement effects are associated with the spread of PEDV, even while controlling for seasonality, farm-level features and the possible spread of disease by vehicles. Our fully Bayesian model permits full uncertainty quantification of these effects. Our farm-level out-of-sample predictions have a receiver-operating characteristic area under the curve (AUC) of 0.779 and a precision-recall AUC of 0.097. The quantification of these effects in a comprehensive model allows stakeholders to make more informed decisions about disease prevention efforts.

The Network of Interactions between the Porcine Epidemic Diarrhea Virus Nucleocapsid and Host Cellular Proteins.

Host–virus protein interactions are critical for intracellular viral propagation. Understanding the interactions between cellular and viral proteins may help us develop new antiviral strategies. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe damage to the global swine industry. Here, we employed co-immunoprecipitation and liquid chromatography-mass spectrometry to characterize 426 unique PEDV nucleocapsid (N) protein-binding proteins in infected Vero cells. A protein–protein interaction network (PPI) was created, and gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses revealed that the PEDV N-bound proteins belong to different cellular pathways, such as nucleic acid binding, ribonucleoprotein complex binding, RNA methyltransferase, and polymerase activities. Interactions of the PEDV N protein with 11 putative proteins: tripartite motif containing 21, DEAD-box RNA helicase 24, G3BP stress granule assembly factor 1, heat shock protein family A member 8, heat shock protein 90 alpha family class B member 1, YTH domain containing 1, nucleolin, Y-box binding protein 1, vimentin, heterogeneous nuclear ribonucleoprotein A2/B1, and karyopherin subunit alpha 1, were further confirmed by in vitro co-immunoprecipitation assay. In summary, studying an interaction network can facilitate the identification of antiviral therapeutic strategies and novel targets for PEDV infection.

Small-Molecule RAF265 as an Antiviral Therapy Acts against PEDV Infection

Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, causes acute diarrhea, vomiting, dehydration, and high mortality in newborn piglets, and has caused significant economic losses in the pig industry. There are currently no specific drugs available to treat PEDV. Viruses depend exclusively on the cellular machinery to ensure an efficient replication cycle. In the present study, we found that small-molecule RAF265, an anticancer drug that has been shown to be a potent inhibitor of RAF, reduced viral loads of PEDV by 4 orders of magnitude in Vero cells, and protected piglets from virus challenge. RAF265 reduced PEDV production by mediating cytoskeleton arrangement and targeting the host cell’s translation machinery. Treatment with RAF265 inhibited viral entry of PEDV S-glycoprotein pseudotyped viral vector particle (PEDV-pp), at half maximal effective concentrations (EC50) of 79.1 nM. RAF265 also presented potent inhibitory activity against viral infection by SARS-CoV-2-pp and SARS-CoV-pp. The present work may provide a starting point for further progress toward the development of antiviral strategies effective against coronavirus PEDV.

Tannic acid-chelated zinc supplementation alleviates intestinal injury in piglets challenged by porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) has become a challenging problem in pig industry all over the world, causing significant profit losses. Tannins and organic zinc have been shown to exert protective effects on the intestinal dysfunction caused by endotoxins. However, there is little information on tannic acid-chelated zinc (TAZ) supplementation in the diet of newborn piglets. This study was conducted to determine the effects of TAZ on the intestinal function of piglets infected with PEDV. Thirty-two 7-day-old piglets were randomly allocated to 1 of 4 treatments in a 2 × 2 factorial design consisting of 2 diets (0 or 50 mg/kg BW TAZ) and challenge (saline or PEDV). On day 9 of the trial, 8 pigs per treatment received either sterile saline or PEDV solution at 106 TCID50 (50% tissue culture infectious dose) per pig. Pigs infected with PEDV had greater diarrhea rate and lower average daily gain (ADG) (P < 0.05). PEDV infection decreased plasma D-xylose concentration, most antioxidative enzyme activities in plasma and intestine, as well as the small intestinal villus height (P < 0.05). Plasma diamine oxidase and blood parameters were also affected by PEDV infection. Dietary supplementation with TAZ could ameliorate the PEDV-induced changes in all measured variables (P < 0.05). Moreover, TAZ decreased the concentration of malondialdehyde in plasma, duodenum, jejunum, and colon (P < 0.05). Collectively, our results indicated that dietary TAZ could alleviate PEDV induced damage on intestinal mucosa and antioxidative capacity, and improve the absorptive function and growth in piglets. Therefore, our novel findings also suggest that TAZ, as a new feed additive for neonatal and weaning piglets, has the potential to be an alternative to ZnO.

A therapeutic chimeric IgG/IgA expressed by CHO cells for oral treatment of PED in piglets.

Immunoglobulin A (IgA) of sows is critically important for assessing piglets’ protective capacity against porcine epidemic diarrhea virus (PEDV). Here, we report a therapeutic chimeric anti-PEDV IgG/IgA expressed by Chinese hamster ovary (CHO) cells for oral treatment of PED. The chimeric anti-PEDV IgG/IgA was produced by the CHO cell lines, in which the heavy chain was constructed by combining the VH, Cγ1 and hinge regions of PEDV IgG mAb 8A3, and the Cα2 and Cα3 domains of a Mus musculus immunoglobulin alpha chain. The chimeric anti-PEDV IgG/IgA could neutralize the strains of CV777 (G1), P014 (G2) and HN1303 (G2) in vitro effectively, showing broad-spectrum neutralization activity. The in vivo challenge experiments demonstrated that chimeric anti-PEDV IgG/IgA (9C4) produced in the CHO cell supernatant could alleviate clinical diarrhea symptoms of the PEDV infection in piglets. In general, our study showed that chimeric anti-PEDV IgG/IgA produced from CHO cell line supernatants effectively alleviates PEDV infection in piglets, which also gives the foundation for the construction of fully functional secretory IgA by the J chain introduction to maximize the antibody therapeutic effect.

Porcine epidemic diarrhoea virus (PEDV) infection activates AMPK and JNK through TAK1 to induce autophagy and enhance virus replication

ABSTRACT Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.