scholarly journals Construction of a Recombinant Porcine Epidemic Diarrhea Virus Encoding Nanoluciferase for High-Throughput Screening of Natural Antiviral Products

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1866
Author(s):  
Wan Li ◽  
Mengjia Zhang ◽  
Huijun Zheng ◽  
Peng Zhou ◽  
Zheng Liu ◽  
...  
Keyword(s):  
High Throughput ◽  
Porcine Epidemic Diarrhea Virus ◽  
High Throughput Screening ◽  
Lithocholic Acid ◽  
Rapid Screening ◽  
Plaque Morphology ◽  
Dependent Manner ◽  
Strong Positive Correlation ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.

scholarly journals In Vitro Antiviral Activities of Salinomycin on Porcine Epidemic Diarrhea Virus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 580
Author(s):  
Chen Yuan ◽  
Xintong Huang ◽  
Ruiyu Zhai ◽  
Yichao Ma ◽  
Anyuan Xu ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Mapk Pathway ◽  
Vero Cells ◽  
Dependent Manner ◽  
Diarrhea Virus ◽  
Pathway Activation ◽  
P38mapk Signaling ◽  
Porcine Epidemic Diarrhea ◽  
Post Entry

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.

scholarly journals MicroRNA-221-5p Inhibits Porcine Epidemic Diarrhea Virus Replication by Targeting Genomic Viral RNA and Activating the NF-κB Pathway

2018 ◽  
Vol 19 (11) ◽  
pp. 3381 ◽  
Author(s):  
Hongqing Zheng ◽  
Lei Xu ◽  
Yuzhong Liu ◽  
Cheng Li ◽  
Liang Zhang ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Nuclear Translocation ◽  
Regulation Of Gene Expression ◽  
Economic Loss ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Swine Industry ◽  
Regulatory Relationship ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

MicroRNAs (miRNAs) are a class of noncoding RNAs involved in posttranscriptional regulation of gene expression and many critical roles in numerous biological processes. Porcine epidemic diarrhea virus (PEDV), the etiological agent of porcine epidemic diarrhea, causes substantial economic loss in the swine industry worldwide. Previous studies reported miRNA involvement in viral infection; however, their role in regulating PEDV infection remains unknown. In this study, we investigated the regulatory relationship between miRNA-221-5p and PEDV infection, finding that miR-221-5p overexpression inhibited PEDV replication in a dose-dependent manner, and that silencing endogenous miR-221-5p enhanced viral replication. Our results showed that miR-221-5p directly targets the 3′ untranslated region (UTR) of PEDV genomic RNA to inhibit PEDV replication, and that miR-221-5p overexpression activates nuclear factor (NF)-κB signaling via p65 nuclear translocation, thereby upregulating interferon (IFN)-β, IFN-stimulated gene 15, and MX1 expression during CH/HBTS/2017 infection. We subsequently identified NF-κB-inhibitor α and suppressor of cytokine signaling 1, negative regulators of the NF-κB pathway, as miR-221-5p targets. These results demonstrated the ability of miR-221-5p to inhibit PEDV replication by targeting the 3’ UTR of the viral genome and activating the NF-κB-signaling pathway. Our findings will aid the development of preventive and therapeutic strategies for PEDV infection.

scholarly journals Detection of porcine epidemic diarrhea virus–neutralizing antibody using high-throughput imaging cytometry

2020 ◽  
Vol 32 (2) ◽  
pp. 324-328
Author(s):  
Luciana V. Sarmento ◽  
Korakrit Poonsuk ◽  
Liying Tian ◽  
Juan C. Mora-Díaz ◽  
Rodger G. Main ◽  
...  
Keyword(s):  
High Throughput ◽  
Porcine Epidemic Diarrhea Virus ◽  
Herd Immunity ◽  
Neutralizing Antibody ◽  
Serum Samples ◽  
Swine Industry ◽  
Diarrhea Virus ◽  
Long Term Storage ◽  
Imaging Cytometry ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry–based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status ( n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

scholarly journals Porcine Epidemic Diarrhea Virus Infection Inhibits Interferon Signaling by Targeted Degradation of STAT1

2016 ◽  
Vol 90 (18) ◽  
pp. 8281-8292 ◽  
Author(s):  
Longjun Guo ◽  
Xiaolei Luo ◽  
Ren Li ◽  
Yunfei Xu ◽  
Jian Zhang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Porcine Epidemic Diarrhea Virus ◽  
Molecular Mechanisms ◽  
Type I Interferon ◽  
Infection Type ◽  
Interferon Response ◽  
Type I ◽  
Dependent Manner ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACTPorcine epidemic diarrhea virus (PEDV) is a worldwide-distributed alphacoronavirus, but the pathogenesis of PEDV infection is not fully characterized. During virus infection, type I interferon (IFN) is a key mediator of innate antiviral responses. Most coronaviruses develop some strategy for at least partially circumventing the IFN response by limiting the production of IFN and by delaying the activation of the IFN response. However, the molecular mechanisms by which PEDV antagonizes the antiviral effects of interferon have not been fully characterized. Especially, how PEDV impacts IFN signaling components has yet to be elucidated. In this study, we observed that PEDV was relatively resistant to treatment with type I IFN. Western blot analysis showed that STAT1 expression was markedly reduced in PEDV-infected cells and that this reduction was not due to inhibition of STAT1 transcription. STAT1 downregulation was blocked by a proteasome inhibitor but not by an autophagy inhibitor, strongly implicating the ubiquitin-proteasome targeting degradation system. Since PEDV infection-induced STAT1 degradation was evident in cells pretreated with the general tyrosine kinase inhibitor, we conclude that STAT1 degradation is independent of the IFN signaling pathway. Furthermore, we report that PEDV-induced STAT1 degradation inhibits IFN-α signal transduction pathways. Pharmacological inhibition of STAT1 degradation rescued the ability of the host to suppress virus replication. Collectively, these data show that PEDV is capable of subverting the type I interferon response by inducing STAT1 degradation.IMPORTANCEIn this study, we show that PEDV is resistant to the antiviral effect of IFN. The molecular mechanism is the degradation of STAT1 by PEDV infection in a proteasome-dependent manner. This PEDV infection-induced STAT1 degradation contributes to PEDV replication. Our findings reveal a new mechanism evolved by PEDV to circumvent the host antiviral response.

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

2021 ◽  
Vol 12 ◽  
Author(s):  
Changchao Huan ◽  
Weiyin Xu ◽  
Bo Ni ◽  
Tingting Guo ◽  
Haochun Pan ◽  
...  
Keyword(s):  
Green Tea ◽  
Western Blotting ◽  
Porcine Epidemic Diarrhea Virus ◽  
Vero Cells ◽  
Dependent Manner ◽  
Egcg Treatment ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea ◽  
Dose Dependent

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.

scholarly journals Identifying outbreaks of Porcine Epidemic Diarrhea virus through animal movements and spatial neighborhoods

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gustavo Machado ◽  
Carles Vilalta ◽  
Mariana Recamonde-Mendoza ◽  
Cesar Corzo ◽  
Montserrat Torremorell ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Animal Movements ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

PSVI-4 Efficacy of Medium Chain Fatty Acids and Fatty Acid-based Feed Additives as a Mitigation Strategy Against Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 216-217
Author(s):  
O L Harrison ◽  
G E Nichols ◽  
J T Gebhardt ◽  
Cassandra K Jones ◽  
Jason C Woodworth ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Porcine Epidemic Diarrhea Virus ◽  
Positive Control ◽  
Feed Additives ◽  
Feed Additive ◽  
Respiratory Syndrome Virus ◽  
Post Inoculation ◽  
Diarrhea Virus ◽  
Medium Chain ◽  
Porcine Epidemic Diarrhea

Abstract Recent research has demonstrated that swine viruses can be transmitted via feed. Chemical feed additives have been suggested for the mitigation of these viruses in complete feed. Therefore, the objective of this study was to evaluate the efficacy of a commercially available formaldehyde-based feed additive, medium chain fatty acid blend (MCFA), and commercially available fatty acid-based products for mitigation of porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) in a feed matrix. Treatments consisted of: 1) non-treated positive control, 2) 0.33% commercial formaldehyde-based product (Sal Curb; Kemin Industries, Inc.; Des Moines, IA), 3) 0.5% MCFA blend (1:1:1 ratio of C6:0, C8:0, and C10:0, Sigma Aldrich, St. Louis, MO), 4) 0.25%, 5) 0.5%, or 6) 1% of commercial dry mono and diglyceride-based product (Furst Strike; Furst-McNess Company, Freeport, IL), 7) 0.25%, 8) 0.5%, or 9) 1% of commercial dry mono and diglyceride-based product (Furst Protect; Furst-McNess Company, Freeport, IL), 10) 0.25%, 11) 0.5%, or 12) 1% dry mono and diglyceride-based experimental product (Furst-McNess Company, Freeport, IL) with 3 replications/treatment. Treatments were applied to complete swine feed before inoculation with 106 TCID50/g of feed with PEDV or PRRSV. Post inoculation feed was held at ambient temperature for 24 h before being analyzed via qRT-PCR. The analyzed values represent the cycle threshold. Formaldehyde and MCFA decreased (P &lt; 0.05) the detectable RNA of PEDV and PRRSV compared to all other treatments. Furst Strike, Furst Protect, and the experimental product did not significantly impact detectability of PEDV or PRRSV RNA. In conclusion, MCFA and formaldehyde treatments are effective at reducing detection of RNA from PEDV and PRRSV in feed.

scholarly journals Differential expression and correlation analysis of miRNA–mRNA profiles in swine testicular cells infected with porcine epidemic diarrhea virus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaoqian Zhang ◽  
Chang Li ◽  
Bingzhou Zhang ◽  
Zhonghua Li ◽  
Wei Zeng ◽  
...  
Keyword(s):  
Differential Expression ◽  
Porcine Epidemic Diarrhea Virus ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Bacterial Invasion ◽  
Sequencing Data ◽  
Diarrhea Virus ◽  
Comparison Groups ◽  
Porcine Epidemic Diarrhea

AbstractThe variant virulent porcine epidemic diarrhea virus (PEDV) strain (YN15) can cause severe porcine epidemic diarrhea (PED); however, the attenuated vaccine-like PEDV strain (YN144) can induce immunity in piglets. To investigate the differences in pathogenesis and epigenetic mechanisms between the two strains, differential expression and correlation analyses of the microRNA (miRNA) and mRNA in swine testicular (ST) cells infected with YN15, YN144, and mock were performed on three comparison groups (YN15 vs Control, YN144 vs Control, and YN15 vs YN144). The mRNA and miRNA expression profiles were obtained using next-generation sequencing (NGS), and the differentially expressed (DE) (p-value < 0.05) mRNA and miRNA were obtained using DESeq R package. mRNAs targeted by DE miRNAs were predicted using the miRanda algortithm. 8039, 8631 and 3310 DE mRNAs, and 36, 36, and 22 DE miRNAs were identified in the three comparison groups, respectively. 14,140, 15,367 and 3771 DE miRNA–mRNA (targeted by DE miRNAs) interaction pairs with negatively correlated expression patterns were identified, and interaction networks were constructed using Cytoscape. Six DE miRNAs and six DE mRNAs were randomly selected to verify the sequencing data by real-time relative quantitative reverse transcription polymerase chain reaction (qRT-PCR). Based on bioinformatics analysis, we discovered the differences were mostly involved in host immune responses and viral pathogenicity, including NF-κB signaling pathway and bacterial invasion of epithelial cells, etc. This is the first comprehensive comparison of DE miRNA–mRNA pairs in YN15 and YN144 infection in vitro, which could provide novel strategies for the prevention and control of PED.

scholarly journals Sequence analysis of new variants of porcine epidemic diarrhea virus in Luzon, Philippines, in 2017

2021 ◽  
Author(s):  
Saubel Ezrael A. Salamat ◽  
Therese Marie A. Collantes ◽  
Wenchie Marie L. Lumbera ◽  
Francis A. Tablizo ◽  
Christian Thomas M. Mutia ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Porcine Epidemic Diarrhea Virus ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea
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