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1.
Viruses ; 14(5)2022 05 15.
Article in English | MEDLINE | ID: covidwho-1884377

ABSTRACT

In this study, we investigated the correlation between the mechanism involved in porcine epidemic diarrhea virus (PEDV) replication and autophagic flux. In this study, we found that as PEDV replicated, production of LC3-II was significantly induced up to 24 h post-infection (hpi). Interestingly, although there was significant production of LC3-II, greater p62 accumulation was simultaneously found. Pretreatment with rapamycin significantly induced PEDV replication, but autolysosome formation was reduced. These results were confirmed by the evaluation of ATG5/ATG12 and LAMP1/LAMP2. Taken together, we conclude that PEDV infection induces autophagosome formation but inhibits autolysosome formation during replication.


Subject(s)
Autophagosomes/metabolism , Porcine epidemic diarrhea virus , Animals , Autophagosomes/genetics , Chlorocebus aethiops , Lysosomes/genetics , Lysosomes/metabolism , Macroautophagy , Porcine epidemic diarrhea virus/immunology , Swine , Vero Cells
2.
PLoS One ; 17(2): e0263582, 2022.
Article in English | MEDLINE | ID: covidwho-1677590

ABSTRACT

The membrane protein M of the Porcine Epidemic Diarrhea Virus (PEDV) is the most abundant component of the viral envelope. The M protein plays a central role in the morphogenesis and assembly of the virus through protein interactions of the M-M, M-Spike (S) and M-nucleocapsid (N) type. The M protein is known to induce protective antibodies in pigs and to participate in the antagonistic response of the cellular antiviral system coordinated by the type I and type III interferon pathways. The 3D structure of the PEDV M protein is still unknown. The present work exposes a predicted 3D model of the M protein generated using the Robetta protocol. The M protein model is organized into a transmembrane and a globular region. The obtained 3D model of the PEDV M protein was compared with 3D models of the SARS-CoV-2 M protein created using neural networks and with initial machine learning-based models created using trRosetta. The 3D model of the present study predicted four linear B-cell epitopes (RSVNASSGTG and KHGDYSAVSNPSALT peptides are noteworthy), six discontinuous B-cell epitopes, forty weak binding and fourteen strong binding T-cell epitopes in the CV777 M protein. A high degree of conservation of the epitopes predicted in the PEDV M protein was observed among different PEDV strains isolated in different countries. The data suggest that the M protein could be a potential candidate for the development of new treatments or strategies that activate protective cellular mechanisms against viral diseases.


Subject(s)
Coronavirus Infections/virology , Coronavirus M Proteins/chemistry , Porcine epidemic diarrhea virus/chemistry , Swine Diseases/virology , Swine/virology , Amino Acid Sequence , Animals , Coronavirus Infections/immunology , Coronavirus Infections/veterinary , Coronavirus M Proteins/immunology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Models, Molecular , Porcine epidemic diarrhea virus/immunology , Protein Conformation , Swine Diseases/immunology
3.
Front Immunol ; 12: 688758, 2021.
Article in English | MEDLINE | ID: covidwho-1304592

ABSTRACT

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/metabolism , Interferons/antagonists & inhibitors , Interferons/immunology , Receptors, Immunologic/metabolism , Amino Acid Sequence/genetics , Animals , COVID-19/pathology , DEAD Box Protein 58/immunology , Deltacoronavirus/genetics , Deltacoronavirus/immunology , Humans , Infectious bronchitis virus/genetics , Infectious bronchitis virus/immunology , Interferon Regulatory Factor-3/metabolism , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Phosphorylation , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/immunology , Receptors, Immunologic/immunology , SARS Virus/genetics , SARS Virus/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Swine , Ubiquitination/physiology
4.
Front Immunol ; 12: 688758, 2021.
Article in English | MEDLINE | ID: covidwho-1295641

ABSTRACT

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/metabolism , Interferons/antagonists & inhibitors , Interferons/immunology , Receptors, Immunologic/metabolism , Amino Acid Sequence/genetics , Animals , COVID-19/pathology , DEAD Box Protein 58/immunology , Deltacoronavirus/genetics , Deltacoronavirus/immunology , Humans , Infectious bronchitis virus/genetics , Infectious bronchitis virus/immunology , Interferon Regulatory Factor-3/metabolism , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Phosphorylation , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/immunology , Receptors, Immunologic/immunology , SARS Virus/genetics , SARS Virus/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Swine , Ubiquitination/physiology
5.
Proc Natl Acad Sci U S A ; 118(18)2021 05 04.
Article in English | MEDLINE | ID: covidwho-1189344

ABSTRACT

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Porcine epidemic diarrhea virus/immunology , SARS-CoV-2/immunology , Viral Fusion Proteins/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/blood , Disease Models, Animal , Escherichia coli/genetics , Genome, Bacterial , Interferon-gamma/blood , RNA, Viral/analysis , Swine , Vaccines, Inactivated/immunology , Vaccines, Synthetic/immunology
6.
mSphere ; 5(3)2020 05 06.
Article in English | MEDLINE | ID: covidwho-1153652

ABSTRACT

Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus 1/isolation & purification , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Swine Diseases/epidemiology , Animals , Antibodies, Viral/immunology , Betacoronavirus 1/immunology , Coronavirus Infections/diagnosis , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , Immunoglobulin G/immunology , Porcine Respiratory Coronavirus/immunology , Porcine epidemic diarrhea virus/immunology , Seroepidemiologic Studies , Swine , Swine Diseases/diagnosis , Transmissible gastroenteritis virus/immunology , United States/epidemiology
7.
Vet Microbiol ; 251: 108892, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-899645

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded positive-sense RNA virus that belongs to a porcine entero-pathogenic alphacoronavirus, causing lethal watery diarrhea in piglets. Despite existing study reports the sole accessory protein ORF3 identified as NF-κB antagonist, the contribution of PEDV ORF3 to production of the pro-inflammatory cytokines mediated by NF-κB signaling remains largely unknown. Thus in this present study, we showed that PEDV ORF3 protein significantly inhibited the productions of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8. The phosphorylation of IκBα was inhibited by ORF3 protein, but no degradation of IκBα was induced in ORF3-expressing cells. Furthermore, PEDV ORF3 inhibited NF-κB activation through preventing nuclear factor p65 phosphorylation and down-regulating p65 expression level, as well as interfering nuclear translocation of p65, eventually resulting into the inhibition of IL-6 and IL-8 production. Our study definitely links PEDV ORF3 to inhibition of pro-inflammatory cytokines production, which will provide new insight into the molecular mechanisms of NF-κB activity inhibited by PEDV proteins to facilitate virus evasion of host innate immune.


Subject(s)
Interleukin-6/antagonists & inhibitors , Interleukin-8/antagonists & inhibitors , Porcine epidemic diarrhea virus/genetics , Transcription Factor RelA/genetics , Viral Proteins/genetics , Viral Proteins/immunology , Animals , Chlorocebus aethiops , HEK293 Cells , HeLa Cells , Humans , Interleukin-6/immunology , Interleukin-8/immunology , Porcine epidemic diarrhea virus/immunology , Signal Transduction , Swine , Transcription Factor RelA/immunology , Vero Cells , Virus Replication
8.
Microb Pathog ; 140: 103922, 2020 Mar.
Article in English | MEDLINE | ID: covidwho-863896

ABSTRACT

BACKGROUND: Highly virulent variants of porcine epidemic diarrhea virus (PEDV) have been closely associated with recent outbreaks of porcine epidemic diarrhea (PED) in China, which have resulted in severe economic losses to the pork industry. METHODS: In the current study, the variant PEDV strain HM2017 was isolated and purified and a viral growth curve was constructed according to the median tissue culture infective dose (TCID50). HM2017 were amplify with RT-PCR and analyzed by phylogeny analysis. Animal pathogenicity experiment was carried to evaluate the HM2017 clinical assessment. RESULTS: Genome-based phylogenetic analysis revealed that PEDV strain HM2017 was clustered into the variant subgroup GII-a that is currently circulating in pig populations in China. The highest median tissue culture infectious dose of strain HM2017 after 15 passages in Vero cells was 1.33 × 107 viral particles/mL. Strain HM2017 was highly virulent to suckling piglets, which exhibited clinical symptoms at 12 h post-infection (hpi) (i.e., weight loss at 12-84 hpi, increased body temperatures at 24-48 hpi, high viral loads in the jejunum and ileum, and 100% mortality by 84 hpi). CONCLUSION: The present study reports a variant subgroup GII-a PEDV HM2017 strain in China and characterize its pathogenicity. PEDV strain HM2017 of subgroup GII-a presents a promising vaccine candidate for the control of PED outbreaks in China.


Subject(s)
Coronavirus Infections/veterinary , Porcine epidemic diarrhea virus/isolation & purification , Animals , China/epidemiology , Chlorocebus aethiops , Disease Outbreaks/prevention & control , Genome, Viral , Phylogeny , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/immunology , Porcine epidemic diarrhea virus/pathogenicity , Swine , Swine Diseases/virology , Vero Cells , Viral Vaccines/immunology
9.
J Virol Methods ; 279: 113855, 2020 05.
Article in English | MEDLINE | ID: covidwho-827847

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) causes very high mortality in newborn piglets. The mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. Antibodies derived from the secretory immunoglobulin A (SIgA) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. Therefore, the measurement of SIgA levels is an important index in evaluating PEDV infections and immune status. A simple and rapid method for the detection of PEDV-specific SIgA using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-SIgA secretory component (SC) mAb probe for the detection of anti-PEDV-specific SIgA in swine. On the strip, a gold-labeled anti-SIgA SC mAb was applied to a conjugate pad; purified PEDV particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. Results showed that the immunochromatographic test strip had high sensitivity and specificity. When compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect PEDV specific SIgA in colostrum samples. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. We found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of PEDV specific SIgA, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying PEDV.


Subject(s)
Antibodies, Viral/analysis , Colostrum/immunology , Immunoassay/methods , Immunoglobulin A, Secretory/isolation & purification , Porcine epidemic diarrhea virus/immunology , Animals , Female , Gold Colloid , Reagent Strips , Sensitivity and Specificity , Specific Pathogen-Free Organisms , Swine , Swine Diseases/diagnosis , Swine Diseases/immunology , Swine Diseases/virology
10.
PLoS One ; 15(4): e0231723, 2020.
Article in English | MEDLINE | ID: covidwho-827309

ABSTRACT

Understanding the immune responses against Porcine epidemic diarrhea virus (PEDV) is important to prevent infection and to design control strategies. We evaluated both systemic and mucosal immune responses to PEDV in pigs and assessed if prior exposure to virus protects against re-infection. Three-week-old pigs were infected with PEDV and immune response in blood, intestine, and mesenteric lymph node (MLN) was evaluated. At 30 dpi, virus exposed pigs were challenged with a field isolate of PEDV and immune response at 5 d post challenge was evaluated. We found that PEDV RNA persists in the intestine even after fecal shedding of the virus was stopped at 28 dpi and pigs previously exposed to PEDV are protected from virus shedding after re-infection. PEDV infection induced both humoral and cell mediated immune response with an increase in PEDV specific IgA and IgG antibodies in intestine and serum. Flow cytometry analysis showed a significantly higher frequency of B cells and lower frequency of T cells at 4 dpi. The frequency of CD4/CD8 double positive (DP) memory T cells was significantly increased in the MLN of challenged animals. These studies may provide further insights into understanding the mucosal immune response to PEDV and its role in protection against disease.


Subject(s)
Antibodies, Viral/analysis , Coronavirus Infections/immunology , Diarrhea/immunology , Porcine epidemic diarrhea virus/immunology , Animals , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , B-Lymphocytes/immunology , Coronavirus Infections/blood , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Diarrhea/blood , Diarrhea/veterinary , Diarrhea/virology , Disease Resistance/immunology , Feces/microbiology , Immunity, Cellular , Immunity, Humoral , Immunity, Mucosal , Immunoglobulin A/analysis , Immunoglobulin A/immunology , Immunoglobulin A/metabolism , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/isolation & purification , RNA, Viral/isolation & purification , Swine , T-Lymphocytes/immunology , Virus Shedding
11.
Virol J ; 17(1): 46, 2020 04 03.
Article in English | MEDLINE | ID: covidwho-827237

ABSTRACT

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. During PEDV infection, the spike protein (S) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. However, the neutralizing B-cell epitopes within PEDV S protein have not been well studied. METHODS: To accurately identify the important immunodominant region of S1, the purified truncated S1 proteins (SA, SB, SC, SD and SE) were used to immunize BALB/c mice to prepare polyclonal antibodies. The antisera titers were determined by indirect ELISA, western blot and IFA after four immunizations to find the important immunodominant region of S1, and then purified the immunodominant region of S1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the B-cell epitopes of monoclonal antibodies. RESULTS: Five antisera of recombinant proteins of the spike protein region of PEDV were generated and we found that only the polyclonal antibody against part of the S1 region (signed as SE protein, residues 666-789) could recognize the native PEDV. Purified SE protein was used to immunize BALB/c mice and generate mAb 2E10. Pepscan of the SE protein demonstrated that SE16 (722SSTFNSTREL731) is the minimal linear epitope required for reactivity with the mAb 2E10. Further investigation indicated that the epitope SE16 was localized on the surface of PEDV S protein in the 3D structure. CONCLUSIONS: A mAb 2E10 that is specifically bound to PEDV was generated and identified a specific linear B-cell epitope (SE16, 722SSTFNSTREL731) of the mAb. The epitope region of PEDV S1 localized in the different regions in comparison with the earlier identified epitopes. These findings enhance the understanding of the PEDV spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect PEDV.


Subject(s)
Epitopes, B-Lymphocyte/immunology , Immunodominant Epitopes/immunology , Porcine epidemic diarrhea virus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Chlorocebus aethiops , Female , Mice , Mice, Inbred BALB C , Porcine epidemic diarrhea virus/chemistry , Vero Cells
12.
Arch Virol ; 165(3): 609-618, 2020 Mar.
Article in English | MEDLINE | ID: covidwho-824459

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) targets the intestinal mucosa in pigs. To protect against PEDV invasion, a mucosal vaccine is utilized effectively. In this study, we generated a recombinant adenovirus vaccine encoding the heat-labile enterotoxin B (LTB) and the core neutralizing epitope (COE) of PEDV (rAd-LTB-COE). The fusion protein LTB-COE was successfully expressed by the recombinant adenovirus in HEK293 cells, and the immunogenicity of the vaccine candidate was assessed in BALB/c mice and piglets. Three intramuscular or oral vaccinations with rAd-LTB-COE at two-week intervals induced robust humoral and mucosal immune responses. Moreover, a cell-mediated immune response was promoted in immunized mice, and the neutralizing antibody inhibited both the vaccine strain and the emerging PEDV isolate. Immunization experiments in piglets revealed that rAd-LTB-COE was immunogenic and induced good immune responses in piglets. Further studies are required to evaluate the efficacy of rAd-LTB-COE against a highly virulent PEDV challenge.


Subject(s)
Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Porcine epidemic diarrhea virus/immunology , Swine Diseases/prevention & control , Viral Vaccines/immunology , Adenoviridae/genetics , Adenoviridae/immunology , Animals , Cell Line , Coronavirus Infections/immunology , Enterotoxins/genetics , Enterotoxins/immunology , Epitopes/genetics , Epitopes/immunology , Escherichia coli/immunology , Escherichia coli/pathogenicity , Female , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Porcine epidemic diarrhea virus/genetics , Recombinant Fusion Proteins/immunology , Swine , Swine Diseases/immunology , Swine Diseases/virology , Viral Vaccines/administration & dosage , Viral Vaccines/therapeutic use
13.
Microb Pathog ; 149: 104553, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-808667

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) causes an emerging and re-emerging coronavirus disease characterized by vomiting, acute diarrhea, dehydration, and up to 100% mortality in neonatal suckling piglets, leading to huge economic losses in the global swine industry. Vaccination remains the most promising and effective way to prevent and control PEDV. However, effective vaccines for PEDV are still under development. Understanding the genomic structure and function of PEDV and the influence of the viral components on innate immunity is essential for developing effective vaccines. In the current review, we systematically describe the recent developments in vaccine against PEDV and the roles of structural proteins, non-structural proteins and accessory proteins of PEDV in affecting viral virulence and regulating innate immunity, which will provide insight into the rational design of effective and safe vaccines for PEDV or other coronaviruses.


Subject(s)
Coronavirus Infections/veterinary , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/immunology , Swine Diseases/immunology , Swine Diseases/virology , Viral Vaccines/immunology , Animals , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Immunity, Innate , Porcine epidemic diarrhea virus/pathogenicity , Swine , Swine Diseases/prevention & control , Vaccination/veterinary , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Viral Proteins/genetics , Viral Vaccines/administration & dosage , Virulence
14.
Viruses ; 12(6)2020 05 31.
Article in English | MEDLINE | ID: covidwho-645542

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. The type I interferon (IFN-I or IFN α/ß) 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.


Subject(s)
Endoribonucleases/immunology , Interferon Regulatory Factor-3/metabolism , Interferon Type I/immunology , Porcine epidemic diarrhea virus/immunology , Viral Nonstructural Proteins/immunology , Animals , Cell Line , Chlorocebus aethiops , Coronavirus Infections/pathology , Coronavirus Infections/veterinary , Down-Regulation , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Type I/antagonists & inhibitors , RNA Stability/genetics , RNA, Messenger/metabolism , Signal Transduction/immunology , Swine , Swine Diseases/immunology , Swine Diseases/pathology , Vero Cells
15.
J Immunol Methods ; 483: 112808, 2020 08.
Article in English | MEDLINE | ID: covidwho-602160

ABSTRACT

Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV.


Subject(s)
Alphacoronavirus/immunology , Antibodies, Viral/blood , Coronavirus Infections/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Immunoglobulin G/blood , Serologic Tests/veterinary , Animals , Biomarkers/blood , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Coronavirus Infections/virology , Deltacoronavirus/immunology , Diagnosis, Differential , Gastroenteritis, Transmissible, of Swine/diagnosis , Gastroenteritis, Transmissible, of Swine/immunology , Gastroenteritis, Transmissible, of Swine/virology , Porcine epidemic diarrhea virus/immunology , Predictive Value of Tests , Reproducibility of Results , Swine , Transmissible gastroenteritis virus/immunology
16.
J Virol ; 94(17)2020 08 17.
Article in English | MEDLINE | ID: covidwho-601769

ABSTRACT

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.


Subject(s)
Coronavirus Infections/immunology , Coronavirus/immunology , Interferons/immunology , Porcine epidemic diarrhea virus/immunology , Vaccines, Attenuated/immunology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Betacoronavirus/immunology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/prevention & control , Diarrhea/pathology , Diarrhea/virology , Disease Models, Animal , Endoribonucleases/antagonists & inhibitors , Feces/virology , Ileum/pathology , Immunity, Innate , Jejunum/pathology , Pandemics , Pneumonia, Viral/immunology , Porcine epidemic diarrhea virus/genetics , RNA, Viral , RNA-Dependent RNA Polymerase , SARS-CoV-2 , Swine , Swine Diseases/virology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology
17.
Vet Q ; 40(1): 183-189, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-361230

ABSTRACT

Background: Outbreaks of porcine epidemic diarrhea virus (PEDV) infection have re-emerged and spread rapidly worldwide, resulting in significant economic losses. Vaccination is the best way to prevent PEDV infection in young piglets.Objective: To enhance the efficacy of an inactivated vaccine against PEDV, we evaluated the adjuvant properties of Fc domain of IgG.Methods: Fifteen crossbred gilts (180 ∼ 210 days old) were used. Five pigs in group 1 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 106 TCID50 of inactivated PEDV. Five pigs in group 2 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 106 TCID50 of inactivated PEDV-sFc. Five pigs in group 3 were not vaccinated and served as negative controls. Serum samples were collected at farrowing and subjected to ELISA, a serum neutralizing (SN) test, and a cytokine assay. Statistical analysis was performed by a two-tailed unpaired t-test.Results: Vero cells expressing swine IgG Fc on its surface was established. When PEDV was propagated in the cells expressing the swine Fc, PEDV virion incorporated the Fc. Immunization of pigs with inactivated PEDV harbouring Fc induced significantly higher antibody production against PEDV, comparing to the immunization with normal inactivated PEDV. In addition, we observed significantly increased IFN-γ levels in sera.Conclusion: Our results indicate that Fc molecule facilitate immune responses and PEDV harbouring Fc molecule could be a possible vaccine candidate. However, a challenge experiment would be needed to investigate the protective efficacy of PEDV harbouring Fc.


Subject(s)
Coronavirus Infections/veterinary , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Porcine epidemic diarrhea virus/immunology , Swine Diseases/immunology , Adjuvants, Immunologic , Animals , Chlorocebus aethiops , Coronavirus Infections/immunology , Female , Immunization , Neutralization Tests , Sus scrofa , Swine , Swine Diseases/virology , Vaccines, Inactivated/immunology , Vero Cells , Viral Vaccines/immunology
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