Transmissible Gastroenteritis Virus Replication Research Articles

In vitro and in vivo evaluation of thapsigargin as an antiviral agent against transmissible gastroenteritis virus

Swine enteric coronaviruses (SeCoVs) pose a significant threat to the global pig industry, but no effective drugs are available for treatment. Previous research has demonstrated that thapsigargin (TG), an ER stress inducer, has broad-spectrum antiviral effects on human coronaviruses. In this study, we investigated the impact of TG on transmissible gastroenteritis virus (TGEV) infection using cell lines, porcine intestinal organoid models, and piglets. The results showed that TG effectively inhibited TGEV replication both in vitro and ex vivo. Furthermore, animal experiments demonstrated that oral administration of TG inhibited TGEV infection in neonatal piglets and relieved TGEV-associated tissue injury. Transcriptome analyses revealed that TG improved the expression of the ER-associated protein degradation (ERAD) component and influenced the biological processes related to secretion, nutrient responses, and epithelial cell differentiation in the intestinal epithelium. Collectively, these results suggest that TG is a potential novel oral antiviral drug for the clinical treatment of TGEV infection, even for infections caused by other SeCoVs.

Hypoxia inducible factor-1α facilitates transmissible gastroenteritis virus replication by inhibiting type I and type III interferon production

Transmissible gastroenteritis virus (TGEV) is characterized by watery diarrhea, vomiting, and dehydration and is associated with high mortality especially in newborn piglets, causing significant economic losses to the global pig industry. Hypoxia inducible factor-1α (HIF-1α) has been identified as a key regulator of TGEV-induced inflammation, but understanding of the effect of HIF-1α on TGEV infection remains limited. This study found that TGEV infection was associated with a marked increase in HIF-1α expression in ST cells and an intestinal organoid epithelial monolayer. Furthermore, HIF-1α was shown to facilitate TGEV infection by targeting viral replication, which was achieved by restraining type I and type III interferon (IFN) production. In vivo experiments in piglets demonstrated that the HIF-1α inhibitor BAY87–2243 significantly reduced HIF-1α expression and inhibited TGEV replication and pathogenesis by activating IFN production. In summary, we unveiled that HIF-1α facilitates TGEV replication by restraining type I and type III IFN production in vitro, ex vivo, and in vivo. The findings from this study suggest that HIF-1α could be a novel antiviral target and candidate drug against TGEV infection.

Integrated Metabolomic and transcriptomic analyses reveal deoxycholic acid promotes transmissible gastroenteritis virus infection by inhibiting phosphorylation of NF-κB and STAT3

BackgroundAcute diarrhea, dehydration and death in piglets are all symptoms of transmissible gastroenteritis virus (TGEV), which results in significant financial losses in the pig industry. It is important to understand the pathogenesis and identify new antiviral targets by revealing the metabolic interactions between TGEV and host cells.ResultsWe performed metabolomic and transcriptomic analyses of swine testicular cells infected with TGEV. A total of 1339 differential metabolites and 206 differentially expressed genes were detected post TEGV infection. The differentially expressed genes were significantly enriched in the HIF-1 signaling pathway and PI3K-Akt signaling. Integrated analysis of differentially expressed genes and differential metabolites indicated that they were significantly enriched in the metabolic processes such as nucleotide metabolism, biosynthesis of cofactors and purine metabolism. In addition, the results showed that most of the detected metabolites involved in the bile secretion was downregulated during TGEV infection. Furthermore, exogenous addition of key metabolite deoxycholic acid (DCA) significantly enhanced TGEV replication by NF-κB and STAT3 signal pathways.ConclusionsWe identified a significant metabolite, DCA, related to TGEV replication. It added TGEV replication in host cells by inhibiting phosphorylation of NF-κB and STAT3. This study provided novel insights into the metabolomic and transcriptomic alterations related to TGEV infection and revealed potential molecular and metabolic targets for the regulation of TGEV infection.

Effect of miR-17 on Polygonum Cillinerve polysaccharide against transmissible gastroenteritis virus.

Transmissible gastroenteritis virus (TGEV) could cause diarrhea, vomiting, dehydration and even death in piglets, miRNA played an important role in the interaction between virus and cell. The study aimed to investigate the impact of miR-17 on the polysaccharide of Polygonum Cillinerve (PCP) in combating TGEV. miR-17 was screened and transfection validation was performed by Real-time PCR. The function of miR-17 on PK15 cells infected with TGEV and treated with PCP was investigated by DCFH-DA loading probe, JC-1 staining and Hoechst fluorescence staining. Furthermore, the effect of miR-17 on PCP inhibiting TGEV replication and apoptosis signaling pathways during PCP against TGEV infection was measured through Real-time PCR and Western blot. The results showed that miR-17 mimic and inhibitor could be transferred into PK15 cells and the expression of miR-17 significantly increased and decreased respectively compared with miR-17 mimic and inhibitor (P < 0.05). A total 250 μg/mL of PCP could inhibit cells apoptosis after transfection with miR-17. PCP (250 μg/mL and 125 μg/mL) significantly inhibited the decrease in mitochondrial membrane potential induced by TGEV after transfection with miR-17 (P < 0.05). After transfection of miR-17 mimic, PCP at concentrations of 250 μg/mL and 125 μg/mL significantly promoted the mRNA expression of P53, cyt C and caspase 9 (P < 0.05). Compared with the control group, the replication of TGEV gRNA and gene N was significantly inhibited by PCP at concentrations of 250 μg/mL and 125 μg/mL after transfection of both miR-17 mimic and inhibitor (P < 0.05). PCP at 62.5 μg/mL significantly inhibited the replication of gene S following transfection with miR-17 inhibitor (P < 0.05). These results suggested that PCP could inhibit the replication of TGEV and apoptosis induced by TGEV by regulating miR-17.

Different Infectivity of Swine Enteric Coronaviruses in Cells of Various Species.

Swine enteric coronaviruses (SECoVs), including porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), have caused high mortality in piglets and, therefore, pose serious threats to the pork industry. Coronaviruses exhibit a trend of interspecies transmission, and understanding the host range of SECoVs is crucial for improving our ability to predict and control future epidemics. Here, the replication of PDCoV, TGEV, and PEDV in cells from different host species was compared by measuring viral genomic RNA transcription and protein synthesis. We demonstrated that PDCoV had a higher efficiency in infecting human lung adenocarcinoma cells (A549), Madin-Darby bovine kidney cells (MDBK), Madin-Darby canine kidney cells (MDCK), and chicken embryonic fibroblast cells (DF-1) than PEDV and TGEV. Moreover, trypsin can enhance the infectivity of PDCoV to MDCK cells that are nonsusceptible to TGEV. Additionally, structural analyses of the receptor ectodomain indicate that PDCoV S1 engages Aminopeptidase N (APN) via domain II, which is highly conserved among animal species of different vertebrates. Our findings provide a basis for understanding the interspecies transmission potential of these three porcine coronaviruses.

Integrated metabolomics and transcriptomics analyses reveal metabolic responses to TGEV infection in porcine intestinal epithelial cells.

Transmissible gastroenteritis virus (TGEV) is a coronavirus that infects piglets with severe diarrhoea, vomiting, dehydration, and even death, causing huge economic losses to the pig industry. The underlying pathogenesis of TGEV infection and the effects of TGEV infection on host metabolites remain poorly understood. To investigate the critical metabolites and regulatory factors during TGEV infection in intestinal porcine epithelial cells (IPEC-J2), we performed metabolomic and transcriptomic analyses of TGEV-infected IPEC-J2 cells by LC/MS and RNA-seq techniques. A total of 87 differential metabolites and 489 differentially expressed genes were detected. A series of metabolites and candidate genes from glutathione metabolism and AMPK signalling pathway were examined through combined analysis of metabolome and transcriptome. We found glutathione peroxidase 3 (GPX3) is markedly reduced after TGEV infection, and a significant negative correlation between AMPK signalling pathway and TGEV infection. Exogenous addition of the AMPK activator COH-SR4 significantly downregulates stearoyl coenzyme A (SCD1) mRNA and inhibits TGEV replication; while exogenous GSK-690693 significantly promotes TGEV infection by inhibiting AMPK signalling pathway. In summary, our study provides insights into the key metabolites and regulators for TGEV infection from the metabolome and transcriptome perspective, which will offer promising antiviral metabolic and molecular targets and enrich the understanding of the existence of a similar mechanism in the host.

DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner.

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.

IFITM3 is a host restriction factor that inhibits porcine transmissible gastroenteritis virus infection

Interferon-induced transmembrane proteins (IFITMs) play an important role in the innate immune response triggered by viral infection. Transmissible gastroenteritis virus (TGEV) causes severe diarrhea, vomiting and dehydration in piglets, resulting in huge economic losses to the swine industry. In this study, we showed that IFITM3 inhibits the replication of TGEV and interferes with the binding of TGEV to PK15 cells. Moreover, the inhibitory effect of IFITM3 on TGEV circumvents the upregulation of inflammatory cytokines. Subsequently, we found that the M22A mutant loses part of the antiviral effect of IFITM3 on TGEV; in contrast, the K24A mutant enhances the antiviral effect of IFITM3. Notably, our data shows a synergistic effect between IFITM3 and CQ, which further amplifies the antiviral effect against TGEV.

Different Mechanisms Are Utilized by Coronavirus Transmissible Gastroenteritis Virus To Regulate Interferon Lambda 1 and Interferon Lambda 3 Production.

Type III interferons (IFN-λ) are shown to be preferentially produced by epithelial cells, which provide front-line protection at barrier surfaces. Transmissible gastroenteritis virus (TGEV), belonging to the genus Alphacoronavirus of the family Coronaviridae, can cause severe intestinal injuries in porcine, resulting in enormous economic losses for the swine industry, worldwide. Here, we demonstrated that although IFN-λ1 had a higher basal expression, TGEV infection induced more intense IFN-λ3 production in vitro and in vivo than did IFN-λ1. We explored the underlying mechanism of IFN-λ induction by TGEV and found a distinct regulation mechanism of IFN-λ1 and IFN-λ3. The classical RIG-I-like receptor (RLR) pathway is involved in IFN-λ3 but not IFN-λ1 production. Except for the signaling pathways mediated by RIG-I and MDA5, TGEV nsp1 induces IFN-λ1 and IFN-λ3 by activating NF-κB via the unfolded protein responses (UPR) PERK-eIF2α pathway. Furthermore, functional domain analysis indicated that the induction of IFN-λ by the TGEV nsp1 protein was located at amino acids 85 to 102 and was dependent on the phosphorylation of eIF2α and the nuclear translocation of NF-κB. Moreover, the recombinant TGEV with the altered amino acid motif of nsp1 85-102 was constructed, and the nsp1 (85-102sg) mutant virus significantly reduced the production of IFN-λ, compared with the wild strain. Compared to the antiviral activities of IFN-λ1, the administration of IFN-λ3 showed greater antiviral activity against TGEV infections in IPEC-J2 cells. In summary, our data point to the significant role of IFN-λ in the host innate antiviral responses to coronavirus infections within mucosal organs and in the distinct mechanisms of IFN-λ1 and IFN-λ3 regulation. IMPORTANCE Coronaviruses cause infectious diseases in various mammals and birds and exhibit an epithelial cell tropism in enteric and respiratory tracts. It is critical to explore how coronavirus infections modulate IFN-λ, a key innate cytokine against mucosal viral infection. Our results uncovered the different processes of IFN-λ1 and IFN-λ3 production that are involved in the classical RLR pathway and determined that TGEV nsp1 induces IFN-λ1 and IFN-λ3 production by activating NF-κB via the PERK-eIF2α pathway in UPR. These studies highlight the unique regulation of antiviral defense in the intestine during TGEV infection. We also demonstrated that IFN-λ3 induced greater antiviral activity against TGEV replication than did IFN-λ1 in IPEC-J2 cells, which is helpful in finding a novel strategy for the treatment of coronavirus infections.

Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication.

Emerging coronaviruses (CoVs) pose a severe threat to human and animal health worldwide. To identify host factors required for CoV infection, we used α-CoV transmissible gastroenteritis virus (TGEV) as a model for genome-scale CRISPR knockout (KO) screening. Transmembrane protein 41B (TMEM41B) was found to be a bona fide host factor involved in infection by CoV and three additional virus families. We found that TMEM41B is critical for the internalization and early-stage replication of TGEV. Notably, our results also showed that cells lacking TMEM41B are unable to form the double-membrane vesicles necessary for TGEV replication, indicating that TMEM41B contributes to the formation of CoV replication organelles. Lastly, our data from a mouse infection model showed that the KO of this factor can strongly inhibit viral infection and delay the progression of a CoV disease. Our study revealed that targeting TMEM41B is a highly promising approach for the development of broad-spectrum anti-viral therapeutics.

Coronavirus transmissible gastroenteritis virus antagonizes the antiviral effect of the microRNA miR-27b via the IRE1 pathway.

Although the functional parameters of microRNAs (miRNAs) have been explored to some extent, the roles of these molecules in coronavirus infection and the regulatory mechanism of miRNAs in virus infection are still unclear. Transmissible gastroenteritis virus (TGEV) is an enteropathgenic coronavirus and causes high morbidity and mortality in suckling piglets. Here, we demonstrated that microRNA-27b-3p (miR-27b-3p) suppressed TGEV replication by directly targeting porcine suppressor of cytokine signaling 6 (SOCS6), while TGEV infection downregulated miR-27b-3p expression in swine testicular (ST) cells and in piglets. Mechanistically, the decrease of miR-27b-3p expression during TGEV infection was mediated by the activated inositol-requiring enzyme 1 (IRE1) pathway of the endoplasmic reticulum (ER) stress. Further studies showed that when ER stress was induced by TGEV, IRE1 acted as an RNase activated by autophosphorylation and unconventionally spliced mRNA encoding a potent transcription factor, X-box-binding protein 1 (Xbp1s). Xbp1s inhibited the transcription of miR-27 and ultimately reduced the production of miR-27b-3p. Therefore, our findings indicate that TGEV inhibits the expression of an anti-coronavirus microRNA through the IRE1 pathway and suggest a novel way in which coronavirus regulates the host cell response to infection.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-1967-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

L-Leucine Promotes STAT1 and ISGs Expression in TGEV-Infected IPEC-J2 Cells via mTOR Activation.

L-leucine (Leu), as one of the effective amino acids to activate the mTOR signaling pathway, can alleviate transmissible gastroenteritis virus (TGEV) infection. However, the underlying mechanism by which Leu alleviates the virus infection has not been fully characterized. In particular, how Leu impacts TGEV replication through mTOR signaling has yet to be elucidated. In the present study, we found that TGEV proliferated efficiently in intestinal porcine epithelial cells (IPEC-J2 cells) as evidenced by the increase in viral contents by flow cytometry, the inhibition of cell proliferation by CCK-8 assay as well as the reduction of PCNA level by western blot. Besides, western blot analysis showed that STAT1 expression was markedly reduced in TGEV-infected cells. The results of ELISA revealed the inhibition of ISGs (ISG56, MxA, and PKR) expressions by TGEV infection. TGEV-induced mTOR and its downstream p70 S6K and 4E-BP1, STAT1 and ISGs downregulation were blocked by an mTOR activator-MHY1485 but not by an mTOR inhibitor-RAPA. Concurrently, mTOR activation by MHY1485 reduced the contents of TGEV and vice versa. Furthermore, Leu reversed the inhibition of STAT1 and ISGs by activating mTOR and its downstream p70 S6K and 4E-BP1 in TEGV-infected cells. Our findings demonstrated that Leu promoted the expressions of STAT1 and ISGs via activating mTOR signaling in IPEC-J2 cells, aiming to prevent TGEV infection.

Activation of Interleukin-1β Release and Pyroptosis by Transmissible Gastroenteritis Virus Is Dependent on the NOD-Like Receptor Protein 3 Inflammasome in Porcine Intestinal Epithelial Cell Line.

Transmissible gastroenteritis virus (TGEV) is a coronavirus, which causes fatal severe diarrhea and leads to high mortality in newborn piglets. Inflammasomes are hub molecules that induce proinflammatory cytokine production and maturation to initiate innate immune defenses upon cellular infection. To date, the potential role of inflammasome in TGEV infection in porcine intestinal epithelial cells has not been elucidated. The present study aims to investigate the function of the inflammasome in response to TGEV infection in porcine intestinal epithelial cells. Our results revealed that TGEV infection induced the production of pro-interleukin-1β (pro-IL-1β) and enhanced its processing and maturation in porcine intestinal epithelial cells through caspase-1 activation. In addition, TGEV infection in porcine intestinal epithelial cells induced pyroptosis, indicated by cell death and the production and cleavage of gasdermin D (GSDMD). Meanwhile, TGEV infection sufficiently activated the expression and assembly of the NOD-like receptor protein 3 (NLRP3) inflammasome in porcine intestinal epithelial cells, and inhibition of NLRP3 blocked TGEV-induced IL-1β release. We also found that inhibition of NLRP3 enhanced the replication of TGEV without inducing cell death. In conclusion, these data demonstrated that activation of IL-1β release and pyroptosis is dependent on NLRP3 inflammasome, thus NLRP3 inflammasome may play a central role in the innate immune response to TGEV infection.

Rhodanine derivative LJ001 inhibits TGEV and PDCoV replication in vitro

Transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are members of the family coronaviridae and mainly cause acute diarrhea/vomiting, dehydration and mortality in piglets, which lead to huge economic losses to the swine industry. Rhodanine derivative LJ001 has been verified to be effective against some enveloped virus infections in vitro. In this study, we evaluated the antiviral activity of LJ001 towards TGEV and PDCoV replication on swine testicular(ST) cells. Our results showed the 50 % cellular cytotoxicity (CC50) value of LJ001 was 146.4 μM on ST cell. The virus titers of TGEV and PDCoV were obviously decreased in the presence of LJ001 with the concentrations of 3.125 and 12.5 μM, and LJ001 potently inhibited TGEV and PDCoV infection at the replication stages of viral life cycle. Further study indicated that LJ001 inhibited TGEV and PDCoV replication by inhibition of viral RNA and protein synthesis, and reducing virus yields at 12 and 24 h post-inoculation. These data indicated that LJ001 had antiviral activities on TGEV and PDCoV replications in vitro, which may serve as a new candidate for treatment of coronaviruses infections.

Antiviral and virucidal effects of curcumin on transmissible gastroenteritis virus in vitro.

Emerging coronaviruses represent serious threats to human and animal health worldwide, and no approved therapeutics are currently available. Here, we used Transmissible gastroenteritis virus (TGEV) as the alpha-coronavirus model, and investigated the antiviral properties of curcumin against TGEV. Our results demonstrated that curcumin strongly inhibited TGEV proliferation and viral protein expression in a dose-dependent manner. We also observed that curcumin exhibited direct virucidal abilities in a dose-, temperature- and time-dependent manner. Furthermore, time-of-addition assays showed that curcumin mainly acted in the early phase of TGEV replication. Notably, in an adsorption assay, curcumin at 40 µM resulted in a reduction in viral titres of 3.55 log TCID50 ml-1, indicating that curcumin possesses excellent inhibitory effects on the adsorption of TGEV. Collectively, we demonstrate for the first time that curcumin has virucidal activity and virtual inhibition against TGEV, suggesting that curcumin might be a candidate drug for effective control of TGEV infection.

Transmissible gastroenteritis virus targets Paneth cells to inhibit the self-renewal and differentiation of Lgr5 intestinal stem cells via Notch signaling

Infection with transmissible gastroenteritis virus (TGEV) has been associated with villous atrophy within 48 h, which seriously disrupts intestinal homeostasis. However, the underlying mechanisms remain elusive. In this study, we found that TGEV infection severely disrupted intestinal homeostasis via inhibition of self-renewal and differentiation in Lgr5 intestinal stem cells (ISCs). Profoundly, TGEV-encoded NSP10/NSP16 protein complex-mediated the inactivation of Notch signaling provided a mechanistic explanation for this phenomenon. Initial invasions by TGEV-targeted Paneth cells through aminopeptidase N (APN) receptor, then inducing mitochondrial damage and ROS generation in them, ultimately causing Paneth cell decrease and loss of Notch factors (DII4 and Hes5), which are essential for Lgr5 ISCs self-renewal and differentiation. Interestingly, loss of Notch signaling induced goblet cells differentiation at the cost of absorptive enterocytes and promoted mucins secretion, which accelerated TGEV replication. Therefore, the more differentiation of goblet cells, the greater TGEV infection in jejunum. These results provide a detailed mechanistic pathway by which villous atrophy sharply occurs in TGEV-infected jejunum within 48 h. Thus, the pathogenesis of TGEV can be described as a “bottom up scenario”, which is contrary to the traditional “top down” hypothesis. Together, our findings provide a potential link between diarrheal virus infection and crypt cells response that regulates Paneth cells function and Lgr5 ISCs fate and could be exploited for therapeutic application.

Receptor tyrosine kinase inhibitors block proliferation of TGEV mainly through p38 mitogen-activated protein kinase pathways

Emerging coronaviruses (CoVs) primarily cause severe gastroenteric or respiratory diseases in humans and animals, and no approved therapeutics are currently available. Here, A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, is identified as a robust inhibitor of transmissible gastroenteritis virus (TGEV) infection in cell-based assays. Moreover, A9 exhibited potent antiviral activity against the replication of various CoVs, including murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV). We further performed a comparative phosphoproteomic analysis to investigate the mechanism of action of A9 against TGEV infection in vitro. We specifically identified p38 and JNK1, which are the downstream molecules of receptor tyrosine kinases (RTKs) required for efficient TGEV replication, as A9 targets through plaque assays, qRT-PCR and Western blotting assays. p38 and JNK1 inhibitors and RNA interference further showed that the inhibitory activity of A9 against TGEV infection was mainly mediated by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. All these findings indicated that the RTKI A9 directly inhibits TGEV replication and that its inhibitory activity against TGEV replication mainly occurs by targeting p38, which provides vital clues to the design of novel drugs against CoVs.

The Coronavirus Transmissible Gastroenteritis Virus Evades the Type I Interferon Response through IRE1α-Mediated Manipulation of the MicroRNA miR-30a-5p/SOCS1/3 Axis.

In host innate immunity, type I interferons (IFN-I) are major antiviral molecules, and coronaviruses have evolved diverse strategies to counter the IFN-I response during infection. Transmissible gastroenteritis virus (TGEV), a member of the Alphacoronavirus family, induces endoplasmic reticulum (ER) stress and significant IFN-I production after infection. However, how TGEV evades the IFN-I antiviral response despite the marked induction of endogenous IFN-I has remained unclear. Inositol-requiring enzyme 1 α (IRE1α), a highly conserved ER stress sensor with both kinase and RNase activities, is involved in the IFN response. In this study, IRE1α facilitated TGEV replication via downmodulating the host microRNA (miR) miR-30a-5p abundance. miR-30a-5p normally enhances IFN-I antiviral activity by directly targeting the negative regulators of Janus family kinase (JAK)-signal transducer and activator of transcription (STAT), the suppressor of cytokine signaling protein 1 (SOCS1), and SOCS3. Furthermore, TGEV infection increased SOCS1 and SOCS3 expression, which dampened the IFN-I antiviral response and facilitated TGEV replication. Importantly, compared with mock infection, TGEV infection in vivo resulted in decreased miR-30a-5p levels and significantly elevated SOCS1 and SOCS3 expression in the piglet ileum. Taken together, our data reveal a new strategy used by TGEV to escape the IFN-I response by engaging the IRE1α-miR-30a-5p/SOCS1/3 axis, thus improving our understanding of how TGEV escapes host innate immune defenses.IMPORTANCE Type I interferons (IFN-I) play essential roles in restricting viral infections. Coronavirus infection induces ER stress and the interferon response, which reflects different adaptive cellular processes. An understanding of how coronavirus-elicited ER stress is actively involved in viral replication and manipulates the host IFN-I response has remained elusive. Here, TGEV inhibited host miR-30a-5p via the ER stress sensor IRE1α, which led to the increased expression of negative regulators of JAK-STAT signaling cascades, namely, SOCS1 and SOCS3. Increased SOCS1 or SOCS3 expression impaired the IFN-I antiviral response, promoting TGEV replication. These findings enhance our understanding of the strategies used by coronaviruses to antagonize IFN-I innate immunity via IRE1α-mediated manipulation of the miR-30a-5p/SOCS axis, highlighting the crucial role of IRE1α in innate antiviral resistance and the potential of IRE1α as a novel target against coronavirus infection.

Porcine transmissible gastroenteritis virus nonstructural protein 2 contributes to inflammation via NF-κB activation

ABSTRACT Transmissible gastroenteritis virus (TGEV) infection causes acute enteritis in swine of all ages, and especially in suckling piglets. Small intestinal inflammation is considered a central event in the pathogenesis of TGEV infections, and nuclear factor-kappa B (NF-κB) is a key transcription factor in the inflammatory response. However, it is unclear whether NF-κB is crucial for inducing inflammation during a TGEV infection. Our results show that NF-κB was activated in swine testicular (ST) cells and intestinal epithelial cell lines J2 (IPEC-J2) cells infected with TGEV, which is consistent with the up-regulation of pro-inflammatory cytokines. Treatment of TGEV-infected ST cells and IPEC-J2 cells with the NF-κB-specific inhibitor caused the down-regulation of pro-inflammatory cytokine expression, but did not significantly affect TGEV replication. Individual TGEV protein screening results demonstrated that Nsp2 exhibited a high potential for activating NF-κB and enhancing the expression of pro-inflammatory cytokines. Functional domain analyzes indicated that the first 120 amino acid residues of Nsp2 were essential for NF-κB activation. Taken together, these data suggested that NF-κB activation was a major contributor to TGEV infection-induced inflammation, and that Nsp2 was the key viral protein involved in the regulation of inflammation, with amino acids 1–120 playing a critical role in activating NF-κB. Abbreviations: TCID50: 50% tissue culture infectious dose; DMEM: Dulbecco’s Modified Eagle Medium; eNOS: Endothelial nitric oxide synthase; FBS: fetal bovine serum; IFA: Indirect immunofluorescence; IκB: inhibitor of nuclear factor kappa-B; IL: interleukin; IPEC-J2: intestinal epithelial cell lines J2; IKK: IκB kinase; Luc: luciferase reporter gene; mAbs: monoclonal antibodies; MOI: multiple of infection; Nsp: nonstructural protein; NF-κB: nuclear factor-kappa ; ORFs: open reading frames; PBS: phosphate-buffered saline; p65 p-p65: phosphorylated; RT-PCR: reverse transcription PC; SeV: Sendai virus; ST: swine testicular; TGEV: Transmissible gastroenteritis virus; TNF-α: tumor necrosis factor α; UV-TGEV: Ultraviolet light-inactivated TGEV; ZnF: zinc finger

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein.

The coronavirus membrane (M) protein acts as a dominant immunogen and is a major player in virus assembly. In this study, we prepared two monoclonal antibodies (mAbs; 1C3 and 4C7) directed against the transmissible gastroenteritis virus (TGEV) M protein. The 1C3 and 4C7 mAbs both reacted with the native TGEV M protein in western blotting and immunofluorescence (IFA) assays. Two linear epitopes, 243YSTEART249 (1C3) and 243YSTEARTDNLSEQEKLLHMV262 (4C7), were identified in the endodomain of the TGEV M protein. The 1C3 mAb can be used for the detection of the TGEV M protein in different assays. An IFA method for the detection of TGEV M protein was optimized using mAb 1C3. Furthermore, the ability of the epitope identified in this study to stimulate antibody production was also evaluated. An immunodominant epitope in the TGEV membrane protein endodomain was identified. The results of this study have implications for further research on TGEV replication.