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1.
PLoS One ; 17(2): e0263582, 2022.
Article in English | MEDLINE | ID: covidwho-1910522

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
2.
J Virol ; 96(13): e0014322, 2022 Jul 13.
Article in English | MEDLINE | ID: covidwho-1879115

ABSTRACT

Differentiation of infected from vaccinated hosts (DIVH) is a critical step in virus eradication programs. DIVH-compatible vaccines, however, take years to develop, and are therefore unavailable for fighting the sudden outbreaks that typically drive pandemics. Here, we establish a protocol for the swift and efficient development of DIVH assays, and show that this approach is compatible with any type of vaccines. Using porcine circovirus 2 (PCV2) as the experimental model, the first step is to use Immunoglobin G (IgG) sero-dynamics (IsD) curves to aid epitope discovery (IsDAED): PCV2 Cap peptides were categorized into three types: null interaction, nonspecific interaction (NSI), and specific interaction (SI). We subsequently compared IsDAED approach and traditional approach, and demonstrated identifying SI peptides and excluding NSI peptides supports efficient diagnostic kit development, specifically using a protein-peptide hybrid microarray (PPHM). IsDAED directed the design of a DIVH protocol for three types of PCV2 vaccines (while using a single PPHM). Finally, the DIVH protocol successfully differentiated infected pigs from vaccinated pigs at five farms. This IsDAED approach is almost certainly extendable to other viruses and host species. IMPORTANCE Sudden outbreaks of pandemics caused by virus, such as SARS-CoV-2, has been determined as a public health emergency of international concern. However, the development of a DIVH-compatible vaccine is time-consuming and full of uncertainty, which is unsuitable for an emergent situation like the ongoing COVID-19 pandemic. Along with the development and public health implementation of new vaccines to prevent human diseases, e.g., human papillomavirus vaccines for cervical cancer; enterovirus 71 vaccines for hand, foot, and mouth disease; and most recently SARS-CoV-2, there is an increasing demand for DIVH. Here, we use the IsDAED approach to confirm SI peptides and to exclude NSI peptides, finally to direct the design of a DIVH protocol. It is plausible that our IsDAED approach is applicable for other infectious disease.


Subject(s)
Antibodies, Viral , Circoviridae Infections , Epitopes , Immunoglobulin G , Viral Vaccines , Animals , Antibodies, Viral/blood , COVID-19 , Circoviridae Infections/immunology , Circovirus , Disease Models, Animal , Epitopes/analysis , Epitopes/immunology , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Peptides , SARS-CoV-2 , Swine , Swine Diseases/immunology , Viral Vaccines/immunology
3.
J Virol ; 96(11): e0046922, 2022 06 08.
Article in English | MEDLINE | ID: covidwho-1854236

ABSTRACT

Coronavirus (CoV) nonstructural protein 1 (nsp1) inhibits cellular gene expression and antagonizes interferon (IFN) response. Porcine epidemic diarrhea virus (PEDV) infects pigs and causes high mortality in neonatal piglets. We hypothesized that a recombinant PEDV carrying mutations at the conserved residues N93 and N95 of nsp1 induces higher IFN responses and is more sensitive to IFN responses, leading to virus attenuation. We mutated PEDV nsp1 N93 and N95 to A93 and A95 to generate the recombinant N93/95A virus using the infectious clone of a highly virulent PEDV strain, PC22A (icPC22A), and evaluated N93/95A virus in vitro and in vivo. Compared with icPC22A, the N93/95A mutant replicated to significantly lower infectious titers, triggered stronger type I and III IFN responses, and was more sensitive to IFN treatment in vitro. To evaluate the pathogenicity and immunogenicity, 5-day-old gnotobiotic piglets were orally inoculated with the N93/95A or icPC22A strain or mock inoculated and then challenged at 22 days postinoculation (dpi) with icPC22A. icPC22A in all pigs (100% [5/5]) caused severe diarrhea and death within 6 dpi. Only one pig (25% [1/4]) died in the N93/95A group. Compared with the icPC22A group, significantly delayed and diminished fecal PEDV shedding was detected in the N93/95A group. Postchallenge, all piglets in N93/95A group were protected from severe diarrhea and death, whereas all pigs in the mock-challenged group developed severe diarrhea, and 25% (1/4) of them died. In summary, nsp1 N93A and N95A mutations attenuated PEDV but retained viral immunogenicity and can be targets for the development of live attenuated vaccines for PEDV. IMPORTANCE PEDV causes porcine epidemic diarrhea (PED) and remains a great threat to the swine industry worldwide because no effective vaccines are available yet. Safe and effective live attenuated vaccines can be designed using reverse genetics to induce lactogenic immunity in pregnant sows to protect piglets from the deadly PED. We found that an engineered PEDV mutant carrying N93A and N95A mutations of nsp1 was partially attenuated and remained immunogenic in neonatal pigs. Our study suggested that nsp1 N93 and N95 can be good targets for the rational design of live attenuated vaccines for PEDV using reverse genetics. Because CoV nsp1 is conserved among alphacoronaviruses (α-CoVs) and betacoronaviruses (ß-CoVs), it may be a good target for vaccine development for other α-CoVs or ß-CoVs.


Subject(s)
Coronavirus Infections , Interferons , Porcine epidemic diarrhea virus , Swine Diseases , Viral Nonstructural Proteins , Animals , Animals, Newborn , Coronavirus Infections/immunology , Coronavirus Infections/veterinary , Diarrhea/veterinary , Diarrhea/virology , Female , Interferons/immunology , Mutation , Swine , Swine Diseases/immunology , Swine Diseases/virology , Viral Nonstructural Proteins/genetics
4.
J Virol ; 96(9): e0038022, 2022 05 11.
Article in English | MEDLINE | ID: covidwho-1794532

ABSTRACT

Crossing the endothelium from the entry site and spreading in the bloodstream are crucial but obscure steps in the pathogenesis of many emerging viruses. Previous studies confirmed that porcine epidemic diarrhea virus (PEDV) caused intestinal infection by intranasal inoculation. However, the role of the nasal endothelial barrier in PEDV translocation remains unclear. Here, we demonstrated that PEDV infection causes nasal endothelial dysfunction to favor viral dissemination. Intranasal inoculation with PEDV compromised the integrity of endothelial cells (ECs) in nasal microvessels. The matrix metalloproteinase 7 (MMP-7) released from the PEDV-infected nasal epithelial cells (NECs) contributed to the destruction of endothelial integrity by degrading the tight junctions, rather than direct PEDV infection. Moreover, the proinflammatory cytokines released from PEDV-infected NECs activated ECs to upregulate ICAM-1 expression, which favored peripheral blood mononuclear cells (PBMCs) migration. PEDV could further exploit migrated cells to favor viral dissemination. Together, our results reveal the mechanism by which PEDV manipulates the endothelial dysfunction to favor viral dissemination and provide novel insights into how coronavirus interacts with the endothelium. IMPORTANCE The endothelial barrier is the last but vital defense against systemic viral transmission. Porcine epidemic diarrhea virus (PEDV) can cause severe atrophic enteritis and acute viremia. However, the mechanisms by which the virus crosses the endothelial barrier and causes viremia are poorly understood. In this study, we revealed the mechanisms of endothelial dysfunction in PEDV infection. The viral infection activates NECs and causes the upregulation of MMP-7 and proinflammatory cytokines. Using NECs, ECs, and PBMCs as in vitro models, we determined that the released MMP-7 contributed to the destruction of endothelial barrier, and the released proinflammatory cytokines activated ECs to facilitate PBMCs migration. Moreover, the virus further exploited the migrated cells to promote viral dissemination. Thus, our results provide new insights into the mechanisms underlying endothelial dysfunction induced by coronavirus infection.


Subject(s)
Coronavirus Infections , Endothelium , Porcine epidemic diarrhea virus , Swine Diseases , Virus Shedding , Animals , Coronavirus Infections/transmission , Coronavirus Infections/virology , Cytokines , Endothelium/virology , Intercellular Adhesion Molecule-1/genetics , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Matrix Metalloproteinase 7/metabolism , Porcine epidemic diarrhea virus/physiology , Swine , Swine Diseases/immunology , Swine Diseases/transmission , Swine Diseases/virology , Viremia
5.
Front Immunol ; 11: 596964, 2020.
Article in English | MEDLINE | ID: covidwho-1067653

ABSTRACT

We designed the killed swine influenza A virus (SwIAV) H1N2 antigen (KAg) with polyriboinosinic:polyribocytidylic acid [(Poly(I:C)] adsorbed corn-derived Nano-11 particle based nanovaccine called Nano-11-KAg+Poly(I:C), and evaluated its immune correlates in maternally derived antibody (MDA)-positive pigs against a heterologous H1N1 SwIAV infection. Immunologically, in tracheobronchial lymph nodes (TBLN) detected enhanced H1N2-specific cytotoxic T-lymphocytes (CTLs) in Nano-11-KAg+Poly(I:C) vaccinates, and in commercial vaccinates detected CTLs with mainly IL-17A+ and early effector phenotypes specific to both H1N2 and H1N1 SwAIV. In commercial vaccinates, activated H1N2- and H1N1-specific IFNγ+&TNFα+, IL-17A+ and central memory T-helper/Memory cells, and in Nano-11-KAg+Poly(I:C) vaccinates H1N2-specific central memory, IFNγ+ and IFNγ+&TNFα+, and H1N1-specific IL-17A+ T-helper/Memory cells were observed. Systemically, Nano-11-KAg+Poly(I:C) vaccine augmented H1N2-specific IFNγ+ CTLs and H1N1-specific IFNγ+ T-helper/Memory cells, and commercial vaccine boosted H1N2- specific early effector CTLs and H1N1-specific IFNγ+&TNFα+ CTLs, as well as H1N2- and H1N1-specific T-helper/Memory cells with central memory, IFNγ+&TNFα+, and IL-17A+ phenotypes. Remarkably, commercial vaccine induced an increase in H1N1-specific T-helper cells in TBLN and naive T-helper cells in both TBLN and peripheral blood mononuclear cells (PBMCs), while H1N1- and H1N2-specific only T-helper cells were augmented in Nano-11-KAg+Poly(I:C) vaccinates in both TBLN and PBMCs. Furthermore, the Nano-11-KAg+Poly(I:C) vaccine stimulated robust cross-reactive IgG and secretory IgA (SIgA) responses in lungs, while the commercial vaccine elicited high levels of serum and lung IgG and serum hemagglutination inhibition (HI) titers. In conclusion, despite vast genetic difference (77% in HA gene identity) between the vaccine H1N2 and H1N1 challenge viruses in Nano-11-KAg+Poly(I:C) vaccinates, compared to over 95% identity between H1N1 of commercial vaccine and challenge viruses, the virus load and macroscopic lesions in the lungs of both types of vaccinates were comparable, but the Nano-11-KAg+Poly(I:C) vaccine cleared the virus from the nasal passage better. These data suggested the important role played by Nano-11 and Poly(I:C) in the induction of polyfunctional, cross-protective cell-mediated immunity against SwIAV in MDA-positive pigs.


Subject(s)
Influenza A virus/immunology , Influenza Vaccines/administration & dosage , Influenza Vaccines/immunology , Nanoparticles , Orthomyxoviridae Infections/veterinary , Poly I-C , Swine Diseases/prevention & control , Vaccines, Inactivated , Animals , Antibodies, Viral/immunology , Antigens, Viral/immunology , Cross Reactions , Cytokines/metabolism , Immunity, Cellular , Immunologic Memory , Influenza Vaccines/chemistry , Nanoparticles/chemistry , Poly I-C/chemistry , Swine , Swine Diseases/immunology , Swine Diseases/virology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Viral Load
6.
Viruses ; 12(1)2020 01 09.
Article in English | MEDLINE | ID: covidwho-830212

ABSTRACT

Porcine deltacoronavirus (PDCoV) is a porcine enteropathogenic coronavirus that causes watery diarrhea, vomiting, and frequently death in piglets, causing serious economic losses to the pig industry. The strain CHN-JS-2017 was isolated and identified by cytopathology, immunofluorescence assays, transmission electron microscopy, and sequence analysis. A nucleotide sequence alignment showed that the whole genome of CHN-JS-2017 is 97.4%-99.6% identical to other PDCoV strains. The pathogenicity of the CHN-JS-2017 strain was investigated in orally inoculated five-day-old piglets; the piglets developed acute, watery diarrhea, but all recovered and survived. CHN-JS-2017 infection-induced microscopic lesions were observed, and viral antigens were detected mainly by immunohistochemical staining in the small intestine. The neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) are crucial immunoglobulin (Ig) receptors for the transcytosis ofimmunoglobulin G (IgG), IgA, or IgM. Importantly, CHN-JS-2017 infected five-day-old piglets could significantly down-regulate the expression of FcRn, pIgR, and nuclear factor-kappa B (NF-κB)in the intestinal mucosa. Note that the level of FcRn mRNA in the intestinal mucosa of normal piglets is positively correlated with pIgR and NF-κB. At the same time, the expressions of FcRn, pIgR, and NF-κB mRNA are also positively correlated in infected piglets. These results may help explain the immunological and pathological changes associated with porcine deltacorononirus infection.


Subject(s)
Coronavirus Infections/veterinary , Coronavirus/classification , Histocompatibility Antigens Class I/immunology , Intestinal Mucosa/immunology , Receptors, Fc/immunology , Receptors, Polymeric Immunoglobulin/immunology , Swine Diseases/virology , Animals , Antigens, Viral/analysis , Coronavirus/isolation & purification , Coronavirus Infections/immunology , Diarrhea/veterinary , Diarrhea/virology , Gene Expression Regulation , Intestinal Mucosa/virology , Intestine, Small/immunology , Intestine, Small/virology , NF-kappa B/immunology , Phylogeny , RNA, Viral/analysis , Sequence Alignment , Sequence Analysis, DNA , Swine , Swine Diseases/immunology , Virus Shedding
7.
Vet Microbiol ; 247: 108785, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-827867

ABSTRACT

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.


Subject(s)
Coronavirus Infections/veterinary , Epithelial Cells/immunology , Epithelial Cells/virology , Host-Pathogen Interactions/immunology , Interferons/antagonists & inhibitors , Animals , Cell Line , Coronavirus , Coronavirus Infections/immunology , Coronavirus Infections/virology , Interferon Regulatory Factor-1/antagonists & inhibitors , Interferon Regulatory Factor-1/immunology , Interferons/immunology , Intestines/cytology , Intestines/virology , Kidney/cytology , Kidney/virology , NF-kappa B/antagonists & inhibitors , NF-kappa B/immunology , Sendai virus/immunology , Signal Transduction/immunology , Swine/virology , Swine Diseases/immunology , Swine Diseases/virology
8.
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
9.
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
10.
Emerg Microbes Infect ; 9(1): 2278-2288, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-811383

ABSTRACT

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naïve sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/veterinary , Pandemics/veterinary , Pneumonia, Viral/veterinary , Swine Diseases/virology , Animals , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19 , Cell Line , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Coronavirus Infections/transmission , Disease Models, Animal , Disease Reservoirs , Disease Susceptibility , Female , Male , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Pneumonia, Viral/transmission , RNA, Viral/blood , Reverse Transcriptase Polymerase Chain Reaction/veterinary , SARS-CoV-2 , Swine , Swine Diseases/immunology , Swine Diseases/pathology , Swine Diseases/transmission , Virus Cultivation , Virus Replication , Whole Exome Sequencing
11.
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
12.
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
13.
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
14.
J Microbiol Biotechnol ; 30(4): 515-525, 2020 04 28.
Article in English | MEDLINE | ID: covidwho-325674

ABSTRACT

Interferon (IFN)-λ plays an essential role in mucosal cells which exhibit strong antiviral activity. Lactobacillus plantarum (L. plantarum) has substantial application potential in the food and medical industries because of its probiotic properties. Alphacoronaviruses, especially porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), cause high morbidity and mortality in piglets resulting in economic loss. Co-infection by these two viruses is becoming increasingly frequent. Therefore, it is particularly important to develop a new drug to prevent diarrhea infected with mixed viruses in piglets. In this study, we first constructed an anchored expression vector with CWA (C-terminal cell wall anchor) on L. plantarum. Second, we constructed two recombinant L. plantarum strains that anchored IFN-λ3 via pgsA (N-terminal transmembrane anchor) and CWA. Third, we demonstrated that both recombinant strains possess strong antiviral effects against coronavirus infection in the intestinal porcine epithelial cell line J2 (IPEC-J2). However, recombinant L. plantarum with the CWA anchor exhibited a more powerful antiviral effect than recombinant L. plantarum with pgsA. Consistent with this finding, Lb.plantarum-pSIP-409-IFN-λ3-CWA enhanced the expression levels of IFN-stimulated genes (ISGs) (ISG15, OASL, and Mx1) in IPEC-J2 cells more than did recombinant Lb.plantarum-pSIP-409-pgsA'-IFN-λ3. Our study verifies that recombinant L. plantarum inhibits PEDV and TGEV infection in IPEC-J2 cells, which may offer great potential for use as a novel oral antiviral agent in therapeutic applications for combating porcine epidemic diarrhea and transmissible gastroenteritis. This study is the first to show that recombinant L. plantarum suppresses PEDV and TGEV infection of IPEC-J2 cells.


Subject(s)
Coronavirus Infections/veterinary , Gastroenteritis, Transmissible, of Swine/prevention & control , Interferons/administration & dosage , Lactobacillus plantarum/genetics , Swine Diseases/prevention & control , Animals , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Epithelial Cells/immunology , Epithelial Cells/virology , Female , Gastroenteritis, Transmissible, of Swine/genetics , Gastroenteritis, Transmissible, of Swine/immunology , Gastroenteritis, Transmissible, of Swine/virology , Gene Expression , Interferons/genetics , Interferons/immunology , Lactobacillus plantarum/metabolism , Male , Porcine epidemic diarrhea virus/physiology , Swine , Swine Diseases/genetics , Swine Diseases/immunology , Swine Diseases/virology , Transmissible gastroenteritis virus/physiology
15.
Arch Virol ; 165(7): 1653-1658, 2020 Jul.
Article in English | MEDLINE | ID: covidwho-245259

ABSTRACT

Although porcine deltacoronavirus (PDCoV) is a significant pandemic threat in the swine population and has caused significant economic losses, information regarding the immune response in conventionally weaned pigs infected with PDCoV is scarce. Hence, the immune response in conventionally weaned pigs infected with PDCoV was assessed after challenge and rechallenge. After the first challenge, obvious diarrhea and viral shedding developed successively in all pigs in the four inoculation dose groups from 3 to 14 days postinfection (dpi), and all pigs recovered (no clinical symptoms or viral shedding) by 21 dpi. All pigs in the four groups exhibited significantly increased PDCoV-specific IgG, IgA and virus-neutralizing (VN) antibody (Ab) titers and IFN-γ levels in the serum after the first challenge. All pigs were completely protected against rechallenge at 21 dpi. The serum levels of PDCoV-specific IgG, IgA, and VN Abs increased further after rechallenge. Notably, the IFN-γ level declined continuously after 7 dpi. In addition, the levels of PDCoV-specific IgG, IgA and VN Abs in saliva increased significantly after rechallenge and correlated well with the serum Ab titers. Furthermore, the appearance of clinical symptoms of PDCoV infection in conventionally weaned pigs was delayed with reduced inoculation doses. In summary, the data presented here offer important reference information for future PDCoV animal infection and vaccine-induced immunoprotection experiments.


Subject(s)
Coronavirus Infections/veterinary , Coronavirus/physiology , Swine Diseases/immunology , Animals , Antibodies, Viral/immunology , Coronavirus/genetics , Coronavirus/isolation & purification , Coronavirus Infections/immunology , Coronavirus Infections/virology , Diarrhea/immunology , Diarrhea/virology , Interferon-gamma/immunology , Swine , Swine Diseases/virology , Virus Shedding
16.
J Vet Sci ; 21(1): e12, 2020 Jan.
Article in English | MEDLINE | ID: covidwho-124741

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in piglets. However, the biological characteristics of PDCoV are unclear. In this study, the hemagglutination (HA) abilities of two PDCoV strains (CH-01 and HNZK-04) were investigated. Our results showed that PDCoV has the ability to agglutinate rabbit erythrocytes after virion pretreatment with trypsin or neuraminidase. Additionally, the HA assay results showed a significant positive correlation with the infectious viral titer. Our results suggest that assessing the HA activity of PDCoV may be a useful diagnostic method for investigating and surveilling PDCoV infections.


Subject(s)
Coronavirus Infections/veterinary , Coronavirus/physiology , Hemagglutination , Swine Diseases/immunology , Animals , Coronavirus Infections/immunology , Coronavirus Infections/virology , Diarrhea/immunology , Diarrhea/veterinary , Diarrhea/virology , Erythrocytes/immunology , Neuraminidase/administration & dosage , Rabbits , Swine , Swine Diseases/virology , Trypsin/administration & dosage , Virion/drug effects
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