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Interferon regulatory factor 8 ( IRF8 ) is a key regulator of innate immune receptor signaling that resists pathogen invasion by regulating cell growth and differentiation. Porcine epidemic diarrhea virus (PEDV) targets the intestine and damages the mucosal barrier. However, whether IRF8 regulates PEDV replication remains unclear. We revealed that PEDV infection activated IRF8 expression. Moreover, IRF8 deletion drastically promoted PEDV replication and invasion, increasing the virus copies and titers. Hypomethylation enrichment of activating protein (AP)-2α was significantly negatively correlated with high IRF8 expression, and AP-2α directly targeted the IRF8 promoter to regulate PEDV replication. Furthermore, IRF8 overexpression decreased the cellular reactive oxygen species levels and mitochondrial membrane potential and increased the antioxidant enzyme activities to alleviate PEDV-induced oxidative damage. IRF8 overexpression suppressed apoptotic gene expression, thereby inhibiting apoptosis in response to PEDV stimulation. Taken together, this study demonstrates that AP-2α is involved in PEDV-induced epigenetic modification of IRF8 to reduce cell apoptosis and oxidative stress and facilitate host resistance to PEDV in the intestinal epithelium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zong, Qu, Zheng, Wang, Wu, Yin and Bao.)

Porcine epidemic diarrhea virus (PEDV) infection results in severe epidemic diarrhea and the death of suckling pigs. Although new knowledge about the pathogenesis of PEDV has been improved, alterations in metabolic processes and the functional regulators involved in PEDV infection with host cells remain largely unknow. To identify cellular metabolites and proteins related to PEDV pathogenesis, we synergistically investigated the metabolome and proteome profiles of PEDV-infected porcine intestinal epithelial cells by liquid chromatography tandem mass spectrometry and isobaric tags for relative and absolute quantification techniques. We identified 522 differential metabolites in positive and negative ion modes and 295 differentially expressed proteins after PEDV infection. Pathways of cysteine and methionine metabolism, glycine, serine and threonine metabolism, and mineral absorption were significantly enriched by differential metabolites and differentially expressed proteins. The betaine-homocysteine S-methyltransferase (BHMT) was indicated as a potential regulator involved in these metabolic processes. We then knocked down the BHMT gene and observed that down-expression of BHMT obviously decreased copy numbers of PEDV and virus titers ( p < 0.01). Our findings provide new insights into the metabolic and proteomic profiles in PEDV-infected host cells and contribute to our further understanding of PEDV pathogenesis.

Porcine epidemic diarrhea virus (PEDV) is an emerging coronavirus which causes acute diarrhea and destroys gastrointestinal barrier function in neonatal pigs. Trefoil factor 1 (TFF1) is a protective peptide for maintaining the integrity of gastrointestinal mucosa and reducing intestinal inflammation. However, its role in protecting intestinal epithelium against PEDV infection is still unclear. In this study, we discovered that TFF1 expression was activated in the jejunum of pigs with PEDV infection and TFF1 is required for the growth of porcine intestinal epithelial cells. For instance, inhibited cell proliferation and cell arrest were observed when TFF1 is genetically knocked-out using CRISPR-Cas9. Additionally, TFF1 depletion increased viral copy number and PEDV titer, along with the elevated genes involved in antiviral and inflammatory cytokines. The decreased TFF1 mRNA expression is in line with hypermethylation on the gene promoter. Notably, the strong interactions of protein-DNA complexes containing CCAAT motif significantly increased C/EBPα accessibility, whereas hypermethylation of mC-6 loci decreased C/EBPα binding occupancies in TFF1 promoter. Overall, our findings show that PEDV triggers the C/EBPα-mediated epigenetic regulation of TFF1 in intestine epithelium and facilitates host resistance to PEDV and other Coronavirus infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Qu, Zong, Wang, Wu, Cai and Bao.)

Purpose: The expression level of aminopeptidase N (APN) is evidently correlated with porcine epidemic diarrhea virus (PEDV) infectivity. This study aims to examine the mechanisms regulating APN expression level in response to PEDV infection., Methods: Quantitative real time PCR was performed herein to detect gene expression dynamics at various timepoints after PEDV infection. Subsequently, CRISPR/Cas9 gene editing technology was used to generate a APN-knockout IPEC-J2 cell line, exploring the effects of APN on cell proliferation by propidium iodide staining and anti-PEDV activity by indirect immunofluorescence assay. Ultimately, the effects of single nucleotide polymorphisms (SNPs) and methylation in the APN promoter region on gene expression were analyzed by using bisulfite sequencing PCR and dual luciferase reporter gene assay., Results: APN expression was significantly upregulated within 4-24 h post-infection. The cytoactivity of the APN-knockout IPEC-J2 cell line was markedly suppressed at different timepoints. Further, cell cycle analyses indicated an increase in the number of G1-phase cells and a significant decrease in that of S-phase cells. Moreover, key cyclical factors regulating the G1 phase were highly expressed in APN-knockout cells. The RNA copies of viral particles and mRNA levels of antiviral genes and inflammatory cytokines in APN-knockout cells were markedly decreased within 24 h of PEDV infection. Similarly, indirect immunofluorescence assay confirmed that the number of PEDV particles was significantly decreased. Sequence analysis revealed two CpG islands in the APN promoter region. However, there was no evident correlation between the methylation status of APN promoter and mRNA levels. Dual luciferase reporter gene assay showed that the SNP rs326030589 (G/A) significantly increased the promoter activity of APN., Conclusions: These results suggested that APN knockout enhanced the resistance of IPEC-J2 cells to PEDV. Moreover, rs326030589 in the APN promoter region participated in gene transcription regulation. Our results provide a reference for studying the mechanisms regulating APN and may contribute to the application of APN gene in resistance breeding of swine epidemic diarrhea., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV) causes a highly contagious intestinal disease in neonatal pigs. Aquaporin-3 (AQP3) plays important roles in maintenance of intestinal barrier function and regulation of immune responses. However, the roles of AQP3 in mediating PEDV infection to host cells and the regulatory mechanisms of AQP3 expression remain poorly understood. Here, we identified one 16 bp (GGGCGGGGTTGCGGGC) insertion mutation in the AQP3 gene promoter in Large White pigs, with the frequencies of 49.3% of heterozygotes and 31.3% of mutant homozygotes. Functional analysis by luciferase activity assay indicated that the insertion mutation results in significant enhancement in AQP3 transcriptional activity (P < 0.01). Mechanistic analysis showed that the inserted sequence adds binding sites for transcription factor CEBPA, which promotes the expression of AQP3. Downregulation of AQP3 by shRNA silencing in porcine intestinal epithelial cells revealed obvious increases in genome copies and viral titers of PEDV. Expression of proinflammatory cytokines (IL-6, IL-8, and IL-18) and interferons (IFN-α and IFN-β) were significantly reduced (P < 0.01) in AQP3 knockdown cells upon PEDV infection. Furthermore, decreased level of ZO-1 protein was also detected in AQP3 knockdown cells in response to PEDV infection. Our findings suggested a previously unknown mechanism linking the effects of promoter genetic variants on the expression of AQP3, revealed the roles of AQP3 in response to PEDV pathogenesis, and indicated the potential associations of the 16 bp insertion mutation with resistance to PEDV infection in porcine intestinal epithelial cells. [ABSTRACT FROM AUTHOR]

Simple Summary: The porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, can cause enormous economic losses in the swine industry. There is no effective commercial vaccine against PEDV infection. In this study, we found that an aqueous leaf extract of M. oleifera (MOE) exhibited antiviral activity in response to PEDV infection at the stage of PEDV replication instead of attachment or internalization. Mechanistically, MOE suppressed the oxidative stress and the expression of inflammatory cytokines induced by PEDV infection and upregulated the expression of anti-apoptotic proteins, which further led to less cell apoptosis. This study is the first report showing that MOE has antiviral potential as a new prophylactic and therapeutic strategy against PEDV infection. Porcine epidemic diarrhea (PED), one of the serious enteric diseases caused by the porcine epidemic diarrhea virus (PEDV), is responsible for enormous economic losses in the global swine industry. However, available commercial vaccines fail to protect pigs from PEDV infection due to the appearance of PEDV variants. Hence, it is necessary to find an effective and cost-efficient natural product to protect pigs from PEDV infection. In this study, we first found that an aqueous leaf extract of M. oleifera (MOE) exhibited antiviral activity in response to PEDV infection. Furthermore, time-of-addition experiments revealed that MOE inhibited PEDV replication rather than attachment and internalization. Mechanistically, MOE significantly suppressed the production of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by PEDV infection, and restored glutathione peroxidase (GSH-Px) activity. Importantly, the addition of MOE alleviated oxidative stress and the expression of inflammatory cytokines and resulted in fewer apoptotic cells during PEDV infection. These results indicated that MOE might be an effective anti-PEDV drug used to control PED disease and may be helpful in developing a new prophylactic and therapeutic strategy against PEDV. [ABSTRACT FROM AUTHOR]

Simple Summary: Porcine epidemic diarrhea virus causes serious diarrhea in suckling piglets, resulting in huge economic losses in the pig industry. A better understanding of porcine epidemic diarrhea virus (PEDV) pathogenesis and identification of the responsive genes will contribute to controlling PEDV infection. Therefore, screening and identifying PEDV-resistance functional genes and then implementing disease resistance breeding have important scientific significance. This study explores the regulatory roles of the GLP-2 gene in regulating PEDV infection and in repairing the intestinal mucosa damage associated with PEDV infection. The findings may contribute to the identification of resistant genes or genetic markers for disease resistance breeding in pigs. Porcine epidemic diarrhea virus (PEDV) infects intestinal epithelial cells, destroys the intestinal mucosal barrier and then causes diarrhea in piglets. Glucagon-like peptide-2 (GLP-2) is a specific intestinal growth hormone that promotes the repair of damaged intestinal mucosa and improves the intestinal barrier. In this study, we investigated the functions of porcine GLP-2 gene in regulating PEDV infection. The intestinal tissues with damaged intestinal structures caused by PEDV infection were first confirmed and collected. Expression analysis indicated that the GLP-2 gene was expressed in the duodenum, jejunum and ileum tissues, and the mRNA level was significantly down-regulated in jejunum and ileum of piglets with damaged intestinal mucosa. Infection of PEDV to porcine small intestinal epithelial cells in vitro showed that GLP-2 gene was significantly decreased, which was consistent with the expression pattern in intestinal tissues. In addition, we silenced the GLP-2 gene by shRNA interfering and found that the copy numbers of PEDV were remarkably increased in the GLP-2 gene silencing cells. Our findings suggest that the GLP-2 gene was potentially involved in regulating PEDV infection and in maintaining the integrity of the intestinal mucosal barrier structure, which could contribute to our understanding of the mechanisms of PEDV pathogenesis and provide a theoretical basis for the identification and application of resistant genes in pig selective breeding for porcine epidemic diarrhea. [ABSTRACT FROM AUTHOR]