scholarly journals Porcine Epidemic Diarrhea Virus nsp15 Antagonizes Interferon Signaling by RNA Degradation of TBK1 and IRF3

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 599 ◽  
Author(s):  
Yang Wu ◽  
Hongling Zhang ◽  
Zhaorong Shi ◽  
Jianfei Chen ◽  
Mingwei Li ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Rna Degradation ◽  
Respiratory Syndrome Virus ◽  
Type I ◽  
Diarrhea Virus ◽  
Itraq Analysis ◽  
Porcine Epidemic Diarrhea ◽  
Innate Antiviral Response ◽  
Immune Pathway ◽  
Innate Immune Pathway

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. The type I interferon (IFN-I or IFN α/β) is a key mediator of innate antiviral response during virus infection. Different antagonistic strategies have been identified and determined as to how PEDV infection inhibits the host’s IFN responses to escape the host innate immune pathway, but the pathogenic mechanisms of PEDV infection are not fully elucidated. Our preliminary results revealed that endogenous TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), the key components in the IFN signaling pathway were downregulated in PEDV infected IPEC-J2 cells by iTRAQ analysis. In this study, we screened nsp15 as the most important viral encoded protein involved in TBK1 and IRF3 reduction. Endoribonuclease (EndoU) activity has been well determined for coronavirus nsp15. Three residues (H226, H241, and K282) of PEDV nsp15 were identified as critical amino acids for PEDV EndoU but not D265, which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (SARS-CoV). Moreover, PEDV nsp15 can directly degrade the RNA levels of TBK1 and IRF3 dependent on its EndoU activity to suppress IFN production and constrain the induction of IFN stimulated genes (ISGs), by which PEDV antagonizes the host innate response to facilitate its replication. Collectively, these results have confirmed that PEDV nsp15 was capable of subverting the IFN response by the RNA degradation of TBK1 and IRF3.

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 < 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 Engineering a Live Attenuated Porcine Epidemic Diarrhea Virus Vaccine Candidate via Inactivation of the Viral 2'-O-Methyltransferase and the Endocytosis Signal of the Spike Protein

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Yixuan Hou ◽  
Hanzhong Ke ◽  
Jineui Kim ◽  
Dongwan Yoo ◽  
Yunfang Su ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Spike Protein ◽  
Economic Losses ◽  
High Dose ◽  
Type I ◽  
Viral Pathogen ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2′-O-methyltransferase (2′-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2′-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A. Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2′-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2′-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

scholarly journals Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Pengcheng Wang ◽  
Juan Bai ◽  
Xuewei Liu ◽  
Mi Wang ◽  
Xianwei Wang ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Economic Losses ◽  
Titration Calorimetry ◽  
Respiratory Syndrome Virus ◽  
Transmissible Gastroenteritis Virus ◽  
Diarrhea Virus ◽  
Infected Cells ◽  
Porcine Epidemic Diarrhea ◽  
3Cl Protease

Abstract Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. There is an urgent need to find new therapeutic methods to prevent and control PEDV. Not only is there a shortage of commercial anti-PEDV drugs, but available commercial vaccines fail to protect against highly virulent PEDV variants. We screened an FDA-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of PEDV replication in Vero and IPEC-J2 cells in vitro. Molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of PEDV 3CL protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). The inhibiting effect of tomatidine on 3CL protease was determined using cleavage visualization and FRET assay. Tomatidine-mediated blocking of 3CL protease activity in PEDV-infected cells was examined by western blot detection of the viral polyprotein in PEDV-infected cells. It indicates that tomatidine inhibits PEDV replication mainly by targeting 3CL protease. In addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalo myocarditis virus (EMCV) and seneca virus A (SVA) in vitro. These results may be helpful in developing a new prophylactic and therapeutic strategy against PEDV and other swine disease infections.

scholarly journals The papain-like protease of porcine epidemic diarrhea virus negatively regulates type I interferon pathway by acting as a viral deubiquitinase

2013 ◽  
Vol 94 (7) ◽  
pp. 1554-1567 ◽  
Author(s):  
Yaling Xing ◽  
Jianfei Chen ◽  
Jian Tu ◽  
Bailing Zhang ◽  
Xiaojuan Chen ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Innate Immune ◽  
Signalling Pathway ◽  
Type I ◽  
Polyubiquitin Chains ◽  
Diarrhea Virus ◽  
Ubiquitinated Proteins ◽  
Interferon Pathway ◽  
Porcine Epidemic Diarrhea ◽  
Swine Disease

Porcine epidemic diarrhea virus (PEDV) is the cause of an economically important swine disease. Previous studies suggested that PEDV does not elicit a robust IFN response, but the mechanism(s) used to evade or block this innate immune response was not known. In this study, we found that PEDV infection blocked synthetic dsRNA-induced IFN-β production by interfering with the activation of interferon regulatory factor 3 (IRF3). We identified PEDV replicase encoded papain-like protease 2 (PLP2) as an IFN antagonist that depends on catalytic activity for its function. We show that levels of ubiquitinated proteins are reduced during PEDV infection and that PEDV PLP2 has deubiquitinase (DUB) activity that recognizes and processes both K-48 and K-63 linked polyubiquitin chains. Furthermore, we found that PEDV PLP2 strongly inhibits RIG-I- and STING-activated IFN expression and that PEDV PLP2 can be co-immunoprecipitated with and deubiquitinates RIG-I and STING, the key components of the signalling pathway for IFN expression. These results show that PEDV infection suppresses production of IFN-β and provides evidence indicating that the PEDV papain-like protease 2 acts as a viral DUB to interfere with the RIG-I- and STING-mediated signalling pathway.

scholarly journals Porcine Epidemic Diarrhea Virus 3C-Like Protease Regulates Its Interferon Antagonism by Cleaving NEMO

2015 ◽  
Vol 90 (4) ◽  
pp. 2090-2101 ◽  
Author(s):  
Dang Wang ◽  
Liurong Fang ◽  
Yanling Shi ◽  
Huan Zhang ◽  
Li Gao ◽  
...  
Keyword(s):  
Immune Responses ◽  
Cysteine Protease ◽  
Porcine Epidemic Diarrhea Virus ◽  
Protease Activity ◽  
The United States ◽  
Watery Diarrhea ◽  
Type I ◽  
Diarrhea Virus ◽  
Cysteine Protease Activity ◽  
Porcine Epidemic Diarrhea

ABSTRACTPorcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-κB activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and this cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses.IMPORTANCEThe continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN) antagonists, papain-like protease (PLP) and N protein. Here, we report that the PEDV nsp5 gene, which encodes the 3C-like protease of PEDV, is another IFN antagonist. Mechanistically, the cysteine protease activity of PEDV nsp5 mediates proteolysis of NEMO, the key adaptor for IFN synthesis, and NEMO is cleaved at glutamine 231 (Q231). The new molecular details and determinants impacting NEMO scission by PEDV nsp5 delineated in this study are fundamental to our understanding of critical virus-host interactions that determine PEDV pathogenesis.

PSVI-7 Evaluation of Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus When Inoculated Either Alone or Together

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 217-218
Author(s):  
Catherine Elijah ◽  
G E Nichols ◽  
J T Gebhardt ◽  
Cassandra K Jones ◽  
Jason C Woodworth ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Feed Additives ◽  
Respiratory Syndrome Virus ◽  
Diarrhea Virus ◽  
Additional Information ◽  
Qrt Pcr ◽  
Ct Value ◽  
Des Moines ◽  
Porcine Epidemic Diarrhea ◽  
Single Reaction

Abstract Research has demonstrated that swine feed can be a fomite for viral transmission and certain feed additives can effectively reduce viral contamination. However, additional information is needed to evaluate the efficacy of additives when feed is inoculated with more than one virus. The objective of this study was to evaluate the efficacy of two feed additives for mitigation of porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) when inoculated individually or together. Feed additives included: 1) no treatment, 2) 0.33% commercial formaldehyde-based product (Sal Curb, Kemin Industries, Des Moines, IA), and 3) 0.50% medium chain fatty acids blend (MCFA; 1:1:1 ratio of C6:C8:C10, Sigma Aldrich, St. Louis, MO). Samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for all viruses. Once inoculated, feed was stored at ambient temperature for 24-h before analyzed via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a qRT-PCR assay was used which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to independently detect both PEDV and PRRSV within a single reaction. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P < 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P < 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both mitigants increased Ct value in feed when co-inoculated with PRRSV and PEDV.

Sign in / Sign up

Export Citation Format

Share Document