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1.
J Virol ; 96(9): e0038022, 2022 May 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
2.
Commun Biol ; 5(1): 252, 2022 03 22.
Article in English | MEDLINE | ID: covidwho-1788322

ABSTRACT

The location of intraepithelial lymphocytes (IELs) between epithelial cells provide a first line of immune defense against enteric infection. It is assumed that IELs migrate only along the basement membrane or into the lateral intercellular space (LIS) between epithelial cells. Here, we identify a unique transepithelial migration of porcine IELs as they move to the free surface of the intestinal epithelia. The major causative agent of neonatal diarrhea in piglets, porcine epidemic diarrhea virus (PEDV), increases the number of IELs entering the LIS and free surface of the intestinal epithelia, driven by chemokine CCL2 secreted from virus-infected intestinal epithelial cells. Remarkably, only virus pre-activated IELs inhibits PEDV infection and their antiviral activity depends on the further activation by virus-infected cells. Although high levels of perforin is detected in the co-culture system, the antiviral function of activated IELs is mainly mediated by IFN-γ secretion inducing robust antiviral response in virus-infected cells. Our results uncover a unique migratory behavior of porcine IELs as well as their protective role in the defense against intestinal infection.


Subject(s)
Coronavirus Infections , Intestinal Diseases , Intraepithelial Lymphocytes , Porcine epidemic diarrhea virus , Virus Diseases , Animals , Antiviral Agents/therapeutic use , Coronavirus Infections/drug therapy , Porcine epidemic diarrhea virus/physiology , Swine
3.
Vet Microbiol ; 267: 109391, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1778497

ABSTRACT

Protein tyrosine phosphatase non-receptor type 14 (PTPN14) is a member of the protein tyrosine phosphatase (PTP) family which is a potential tumor suppressor. PTPs modulate the cellular level of tyrosine phosphorylation under normal and pathological conditions. Porcine epidemic diarrhea virus (PEDV) is one of the most important pathogens in the swine industry. Our previous membrane proteomics results showed that PTPN14 was markedly upregulated in PEDV-infected Vero cells. However, its biological roles in PEDV infection have not yet been investigated. In this study, we reported PTPN14 functions as a novel regulator of signal transducer and activator of transcription 3 (STAT3) phosphorylation during PEDV infection. Firstly, PTPN14 was markedly upregulated in PEDV-infected Vero cells with the decrease of STAT3 phosphorylation. Knockdown of PTPN14 or phosphatase inhibitor treatment promoted PEDV proliferation and increased the phosphorylation of STAT3 in Vero cells. On the contrary, overexpression of PTPN14 inhibits viral infection in Vero cells. Moreover, dephosphorylation of STAT3 by PTPN14 might occur in the cytoplasm but not in nucleus. Collectively, our results indicate that PTPN14 plays a negative role in regulating STAT3 activation in PEDV infected Vero cells and demonstrate another layer of regulation in PEDV infection.


Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Animals , Chlorocebus aethiops , Coronavirus Infections/pathology , Coronavirus Infections/veterinary , Porcine epidemic diarrhea virus/physiology , Protein Tyrosine Phosphatases/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Swine , Tyrosine/metabolism , Vero Cells
4.
Front Immunol ; 12: 826882, 2021.
Article in English | MEDLINE | ID: covidwho-1674339

ABSTRACT

Swine enteric coronaviruses (SECoVs) including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV), account for the majority of lethal watery diarrhea in neonatal pigs and pose significant economic and public health burdens in the world. While the three SECoVs primarily infect intestinal epithelia in vivo and cause similar clinical signs, there are evident discrepancies in their cellular tropism and pathogenicity. However, the underlying mechanisms to cause the differences remain unclear. Herein, we employed porcine enteroids that are a physiologically relevant model of the intestine to assess the host epithelial responses following infection with the three SECoVs (PEDV, TGEV, and PDCoV). Although SECoVs replicated similarly in jejunal enteroids, a parallel comparison of transcriptomics datasets uncovered that PEDV and TGEV infection induced similar transcriptional profiles and exhibited a more pronounced response with more differentially expressed genes (DEGs) in jejunal enteroids compared with PDCoV infection. Notably, TGEV and PDCoV induced high levels of type I and III IFNs and IFN-stimulated gene (ISG) responses, while PEDV displayed a delayed peak and elicited a much lesser extent of IFN responses. Furthermore, TGEV and PDCoV instead of PEDV elicited a substantial upregulation of antigen-presentation genes and T cell-recruiting chemokines in enteroids. Mechanistically, we demonstrated that IFNs treatment markedly elevated the expression of NOD-like receptor (NLR) family NLRC5 and major histocompatibility complex class I (MHC-I) molecules. Together, our results indicate unique and common viral strategies for manipulating the global IFN responses and antigen presentation utilized by SECoVs, which help us a better understanding of host-SECoVs interactions.


Subject(s)
Antigen Presentation/immunology , Coronavirus Infections/veterinary , Gene Expression Regulation , Interferons/metabolism , Porcine epidemic diarrhea virus/physiology , Swine Diseases/etiology , Swine Diseases/metabolism , Animals , Gastroenteritis, Transmissible, of Swine/etiology , Gastroenteritis, Transmissible, of Swine/metabolism , Gastroenteritis, Transmissible, of Swine/pathology , Gene Expression Profiling , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Swine , Swine Diseases/pathology , Transmissible gastroenteritis virus
5.
J Virol ; 94(16)2020 07 30.
Article in English | MEDLINE | ID: covidwho-1214962

ABSTRACT

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.


Subject(s)
Exoribonucleases/metabolism , Interferon Type I/biosynthesis , Interferons/biosynthesis , Porcine epidemic diarrhea virus/physiology , RNA Caps/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Binding Sites , Cell Line , Chlorocebus aethiops , Exoribonucleases/genetics , Gene Expression , Guanine/metabolism , Immunity, Innate , Methylation , Mutation , Porcine epidemic diarrhea virus/enzymology , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/pathogenicity , RNA, Viral/metabolism , S-Adenosylmethionine/metabolism , Swine , Vero Cells , Viral Nonstructural Proteins/genetics , Virus Replication
6.
J Virol ; 95(9)2021 04 12.
Article in English | MEDLINE | ID: covidwho-1180915

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus causing severe diarrhea and high mortality rates in suckling piglets and posing significant economic impact. PEDV replication is completed and results in a large amount of RNA in the cytoplasm. Stress granules (SGs) are dynamic cytosolic RNA granules formed under various stress conditions, including viral infections. Several previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. However, the underlying mechanisms are poorly understood. This study aimed to delineate the molecular mechanisms regulating the SG response to PEDV infection. SG formation is induced early during PEDV infection, but as infection proceeds, this ability is lost and SGs disappear at late stages of infection (>18 h postinfection). PEDV infection resulted in the cleavage of Ras-GTPase-activating protein-binding protein 1 (G3BP1) mediated by caspase-8. Using mutational analysis, the PEDV-induced cleavage site within G3BP1 was identified, which differed from the 3C protease cleavage site previously identified. Furthermore, G3BP1 cleavage by caspase-8 at D168 and D169 was confirmed in vitro as well as in vivo The overexpression of cleavage-resistant G3BP1 conferred persistent SG formation and suppression of viral replication. Additionally, the knockdown of endogenous G3BP1 abolished SG formation and potentiated viral replication. Taken together, these data provide new insights into novel strategies in which PEDV limits the host stress response and antiviral responses and indicate that caspase-8-mediated G3BP1 cleavage is important in the failure of host defense against PEDV infection.IMPORTANCE Coronaviruses (CoVs) are drawing extensive attention again since the outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. CoVs are prone to variation and own the transmission capability by crossing the species barrier resulting in reemergence. How CoVs manipulate the antiviral responses of their hosts needs to be explored. Overall, the study provides new insight into how porcine epidemic diarrhea virus (PEDV) impaired SG assembly by targeting G3BP1 via the host proteinase caspase-8. These findings enhanced the understanding of PEDV infection and might help identify new antiviral targets that could inhibit viral replication and limit the pathogenesis of PEDV.


Subject(s)
Caspase 8/metabolism , Coronavirus Infections/metabolism , Cytoplasmic Granules/metabolism , Porcine epidemic diarrhea virus/physiology , Proteolysis , RNA Recognition Motif Proteins/metabolism , Virus Replication , Animals , Caspase 8/genetics , Chlorocebus aethiops , Coronavirus Infections/genetics , Coronavirus Infections/pathology , Cytoplasmic Granules/genetics , Cytoplasmic Granules/virology , HEK293 Cells , Humans , RNA Recognition Motif Proteins/genetics , Swine , Vero Cells
7.
Vet Res ; 51(1): 136, 2020 Nov 11.
Article in English | MEDLINE | ID: covidwho-937135

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.


Subject(s)
Antiviral Agents/pharmacology , Porcine epidemic diarrhea virus/physiology , Tomatine/analogs & derivatives , Viral Proteins/metabolism , Virus Replication/drug effects , Antiviral Agents/chemistry , Peptide Hydrolases/metabolism , Plant Extracts/chemistry , Plant Extracts/pharmacology , Porcine epidemic diarrhea virus/drug effects , Porcine epidemic diarrhea virus/enzymology , Tomatine/chemistry , Tomatine/pharmacology , Virus Replication/physiology
8.
Bioconjug Chem ; 31(11): 2553-2563, 2020 11 18.
Article in English | MEDLINE | ID: covidwho-872629

ABSTRACT

As a large enveloped RNA virus, coronavirus is of considerable medical and veterinary significance, and anticoronavirus treatment is challenging due to its biodiversity and rapid variability. In this study, Au@Ag nanorods (Au@AgNRs) were successfully synthesized by coating AuNRs with silver and were shown for the first time to have activity against the replication of porcine epidemic diarrhea virus (PEDV). Viral titer analysis demonstrated that Au@AgNRs could inhibit PEDV infection by 4 orders of magnitude at 12 h post-infection, which was verified by viral protein expression analysis. The potential mechanism of action showed that Au@AgNRs could inhibit the entry of PEDV and decrease the mitochondrial membrane potential and caspase-3 activity. Additionally, we demonstrated that a large amount of virus proliferation can cause the generation of reactive oxygen species in cells, and the released Ag+ and exposed AuNRs by Au@AgNRs after the stimulation of reactive oxygen species has superior antiviral activity to ensure long-term inhibition of the PEDV replication cycle. The integrated results support that Au@AgNRs can serve as a potential therapeutic strategy to prevent the replication of coronavirus.


Subject(s)
Gold/chemistry , Gold/pharmacology , Metal Nanoparticles/chemistry , Porcine epidemic diarrhea virus/drug effects , Porcine epidemic diarrhea virus/physiology , Silver/chemistry , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/toxicity , Chlorocebus aethiops , Dose-Response Relationship, Drug , Gold/toxicity , Nanotubes/chemistry , Vero Cells
9.
Viruses ; 12(4)2020 04 01.
Article in English | MEDLINE | ID: covidwho-833482

ABSTRACT

A highly virulent porcine epidemic diarrhea virus (PEDV) appeared in China and spread rapidly to neighbor countries, which have led to great economic losses to the pig industry. In the present study, we isolated a PEDV using Vero cells and serially propagated 100 passages. PEDV SDSX16 was characterized in vitro and in vivo. The viral titers increased to 107.6 TCID50/mL (100th) by serial passages. The spike (S) gene and the whole gene of the SDSX16 virus was fully sequenced to assess the genetic stability and relatedness to previously identified PEDV. Along with successive passage in vitro, there were 18 nucleotides (nt) deletion occurred in the spike (S) gene resulting in a deletion of six amino acids when the SDSX16 strain was passaged to the 64th generation, and this deletion was stable until the P100. However, the ORF1a/b, M, N, E, and ORF3 genes had only a few point mutations in amino acids and no deletions. According to growth kinetics experiments, the SDSX16 deletion strain significantly enhanced its replication in Vero cells since it was passaged to the 64th generation. The animal studies showed that PEDV SDSX16-P10 caused more severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis than SDSX16-P75, indicating that SDSX16-P10 is enteropathogenic in the natural host, and the pathogenicity of SDSX16 decreased with successive passage in vitro. However, SDSX16-P10 was found to cause lower levels of cytokine expression than SDSX16-P75 using real-time PCR and flow cytometry, such as IL1ß, IL6, IFN-ß, TNF-α, indicating that SDSX16-P10 might inhibit the expression of cytokines. Our data indicated that successive passage in vitro resulted in virulent attenuation in vivo of the PEDV variant strain SDSX16.


Subject(s)
Coronavirus Infections/veterinary , Porcine epidemic diarrhea virus/physiology , Swine Diseases/virology , Viral Load , Animals , Biomarkers , Chlorocebus aethiops , Cytokines , Immunohistochemistry , Phylogeny , Porcine epidemic diarrhea virus/classification , Swine , Swine Diseases/metabolism , Swine Diseases/pathology , Vero Cells , Viral Proteins/chemistry , Viral Proteins/genetics , Virulence
10.
Viruses ; 12(2)2020 02 21.
Article in English | MEDLINE | ID: covidwho-833229

ABSTRACT

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.


Subject(s)
Antiviral Agents/pharmacology , Peptide Hydrolases/chemistry , Porcine epidemic diarrhea virus/drug effects , Protease Inhibitors/pharmacology , Pyrrolidines/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Catalytic Domain , Chlorocebus aethiops , Crystallography, X-Ray , Models, Molecular , Peptide Hydrolases/metabolism , Porcine epidemic diarrhea virus/enzymology , Porcine epidemic diarrhea virus/physiology , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Pyrrolidines/chemistry , Pyrrolidines/metabolism , Substrate Specificity , Sulfonic Acids , Transmissible gastroenteritis virus/enzymology , Vero Cells , Virus Replication/drug effects
11.
Viruses ; 12(2)2020 02 14.
Article in English | MEDLINE | ID: covidwho-831427

ABSTRACT

The genomes of coronaviruses carry accessory genes known to be associated with viral virulence. The single accessory gene of porcine epidemic diarrhea virus (PEDV), ORF3, is dispensable for virus replication in vitro, while viral mutants carrying ORF3 truncations exhibit an attenuated phenotype of which the underlying mechanism is unknown. Here, we studied the effect of ORF3 deletion on the proliferation of PEDV in Vero cells. To this end, four recombinant porcine epidemic diarrhea viruses (PEDVs) were rescued using targeted RNA recombination, three carrying the full-length ORF3 gene from different PEDV strains, and one from which the ORF3 gene had been deleted entirely. Our results showed that PEDVs with intact or naturally truncated ORF3 replicated to significantly higher titers than PEDV without an ORF3. Further characterization revealed that the extent of apoptosis induced by PEDV infection was significantly lower with the viruses carrying an intact or C-terminally truncated ORF3 than with the virus lacking ORF3, indicating that the ORF3 protein as well as its truncated form interfered with the apoptosis process. Collectively, we conclude that PEDV ORF3 protein promotes virus proliferation by inhibiting cell apoptosis caused by virus infection. Our findings provide important insight into the role of ORF3 protein in the pathogenicity of PEDV.


Subject(s)
Apoptosis , Open Reading Frames/genetics , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/pathogenicity , Viral Proteins/genetics , Virus Replication , Animals , Cell Proliferation , Chlorocebus aethiops , Porcine epidemic diarrhea virus/physiology , Vero Cells , Virulence
12.
Prev Vet Med ; 175: 104848, 2020 Feb.
Article in English | MEDLINE | ID: covidwho-831031

ABSTRACT

It is well known that infectious diseases such as porcine reproductive and respiratory syndrome (PRRS) and porcine epidemic diarrhea (PED) decrease herd productivity and lead to economic loss. It is believed that biosecurity practices are effective for the prevention and control of such infectious diseases. Therefore, the objective of the present study was to investigate whether or not an association between biosecurity level and herd productivity, as well as disease status exists on Japanese commercial swine farms. The present study was conducted on 141 farms. Biosecurity in each farm was assessed by a biosecurity assessment tool named BioAsseT. BioAsseT has a full score of 100 and consists of three sections (external biosecurity, internal biosecurity and diagnostic monitoring). Production data for number of pigs weaned per sow per year (PWSY) and post-weaning mortality per year (PWM) were collected for data analysis. Regarding PRRS status, the farms were categorized into two groups: unknown or unstable and stable or negative. In addition, these farms were categorized based on their PED status, either positive or negative. The total BioAsseT score was associated with herd productivity: as total score increased by 1, PWSY increased by 0.104 pigs and PWM decreased by 0.051 % (P < 0.05). Herd productivity was associated with the score of external and internal biosecurity (P < 0.05), but did not correlate with the score of diagnostic monitoring. Regarding PRRS status, farms with an unknown or unstable status had lower total score than those with stable or negative status (P < 0.05). Similarly, PED positive farms had a lower total score compared to PED negative farms (P < 0.05). In conclusion, the present study provides evidence for the association between high biosecurity levels and increased herd productivity as well as a decreased risk for novel introductions of infectious diseases such as PED.


Subject(s)
Animal Husbandry/methods , Porcine epidemic diarrhea virus/physiology , Porcine respiratory and reproductive syndrome virus/physiology , Swine Diseases/mortality , Animals , Coronavirus Infections/mortality , Coronavirus Infections/veterinary , Female , Japan/epidemiology , Porcine Reproductive and Respiratory Syndrome/mortality , Reproduction , Sus scrofa/physiology , Swine
13.
Vet Microbiol ; 244: 108684, 2020 May.
Article in English | MEDLINE | ID: covidwho-827805

ABSTRACT

Porcine epidemic diarrhea virus (PEDV) has caused, and continues to cause, severe economic losses to the swine industry worldwide. The pathogenic mechanism and immune regulatory interactions between PEDV and the host remain largely unknown. In this study, the interaction between autophagy and PEDV replication in intestinal porcine epithelial (IPEC-J2) cells was investigated. The effects of the structural and nonstructural proteins of PEDV on the autophagy process and the autophagy-related signaling pathways were also examined. The results shown that PEDV replication increased the autophagy flux in IPEC-J2 cells, and that autophagy was beneficial to PEDV replication, which may be one of the reasons for the rapid damage to intestinal epithelial cells and the enhanced virulence of PEDV in both newborn piglets and finishing pigs. When autophagy was pharmacologically induced by rapamycin, PEDV replication increased from 8.5 × 105 TCID50/mL to 8.8 × 106 TCID50/mL in IPEC-J2 cells. When autophagy was pharmacologically suppressed by hydroxychloroquine, PEDV replication decreased from 8.5 × 105 TCID50/mL to 7.9 × 104 TCID50/mL. To identify which PEDV proteins were the key inducers of autophagy, all 4 structural proteins and 17 nonstructural proteins of PEDV were eukaryotic expressed. It was found that the nonstructural protein 6 (nsp6) and ORF3 of PEDV were able to induce significant autophagy in IPEC-J2 cells, but the other proteins were unable to induce autophagy. It was indicated that nsp6-induced autophagy mainly occurred via the PI3K/Akt/mTOR signaling pathway. The results accelerate the understanding of the biology and pathogenesis of PEDV infection and provide new insights into the development of effective therapeutic strategies.


Subject(s)
Autophagy , Porcine epidemic diarrhea virus/pathogenicity , Signal Transduction , Viral Nonstructural Proteins/genetics , Virus Replication , Animals , Cell Line , Epithelial Cells/pathology , Epithelial Cells/virology , Host-Pathogen Interactions , Intestines/cytology , Intestines/virology , Oncogene Protein v-akt/genetics , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Porcine epidemic diarrhea virus/physiology , Swine , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Viral Nonstructural Proteins/metabolism
14.
Vet Microbiol ; 244: 108660, 2020 May.
Article in English | MEDLINE | ID: covidwho-826788

ABSTRACT

Unlike porcine epidemic diarrhea virus (PEDV) that infects only pigs, porcine deltacoronavirus (PDCoV) has the capacity to infect different animal species. In vivo gnotobiotic calves were previously confirmed to be susceptible to infection with PDCoV, but not with PEDV. We next investigated in vitro whether primary bovine cells are susceptible to PDCoV or PEDV infection. We conducted quantification of viral RNA in cell culture supernatants and immunofluorescent staining for the detection of PDCoV or PEDV antigen in two primary bovine cell types inoculated with the PDCoV strain OH-FD22 or PEDV strain PC22-P40 grown in LLC-PK or Vero cells, respectively, and supplemented with 1.25∼5 µg/mL of trypsin in the cell culture medium. The primary cells were isolated from the kidney or heart of a gnotobiotic calf, and both cell types were vimentin-positive, but E-Cadherin-negative, resembling mesenchymal cells. Similar to the previous in vivo observation, cytopathic effects (CPE) that consisted of enlarged and rounded cells, followed by cell shrinkage and detachment, were identified in the two primary cell types inoculated with PDCoV. Unexpectedly, similar CPE was also identified in the two cell types inoculated with PEDV. High PDCoV or PEDV RNA titers and PDCoV or PEDV antigens were detected in the cell culture supernatants and CPE-positive cells, respectively. Our study revealed that primary bovine mesenchymal cells are susceptible to infection with PDCoV and PEDV. The in vitro observation partially coincided with the corresponding in vivo data from gnotobiotic calves.


Subject(s)
Deltacoronavirus/physiology , Porcine epidemic diarrhea virus/physiology , Virus Replication , Animals , Cattle , Cells, Cultured , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Germ-Free Life , Myocardium/cytology , Primary Cell Culture , Swine , Vero Cells
15.
Int J Mol Sci ; 21(8)2020 Apr 23.
Article in English | MEDLINE | ID: covidwho-825269

ABSTRACT

Our previous study showed that glycyrrhizin (GLY) inhibited porcine epidemic diarrhea virus (PEDV) infection, but the mechanisms of GLY anti-PEDV action remain unclear. In this study, we focused on the anti-PEDV and anti-proinflammatory cytokine secretion mechanisms of GLY. We found that PEDV infection had no effect on toll-like receptor 4 (TLR4) protein and mRNA levels, but that TLR4 regulated PEDV infection and the mRNA levels of proinflammatory cytokines. In addition, we demonstrated that TLR4 regulated p38 phosphorylation but not extracellular regulated protein kinases1/2 (Erk1/2) and c-Jun N-terminal kinases (JNK) phosphorylation, and that GLY inhibited p38 phosphorylation but not Erk1/2 and JNK phosphorylation. Therefore, we further explored the relationship between high mobility group box-1 (HMGB1) and p38. We demonstrated that inhibition of HMGB1 using an antibody, mutation, or knockdown decreased p38 phosphorylation. Thus, HMGB1 participated in activation of p38 through TLR4. Collectively, our data indicated that GLY inhibited PEDV infection and decreased proinflammatory cytokine secretion via the HMGB1/TLR4-mitogen-activated protein kinase (MAPK) p38 pathway.


Subject(s)
Glycyrrhizic Acid/pharmacology , HMGB1 Protein/metabolism , Porcine epidemic diarrhea virus/drug effects , Porcine epidemic diarrhea virus/physiology , Signal Transduction/drug effects , Toll-Like Receptor 4/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cells, Cultured , Chlorocebus aethiops , Coronavirus Infections/veterinary , Swine , Swine Diseases/metabolism , Swine Diseases/virology , Vero Cells
16.
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
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