Transmissible gastroenteritis virus induces inflammatory responses via RIG-I/NF-κB/HIF-1α/glycolysis axis in intestinal organoids and in vivo.

Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.

Differential Transcriptomics Analysis of IPEC-J2 Cells Single or Coinfected With Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus.

Porcine epidemic diarrhea (PED) and transmissible gastroenteritis (TGE) caused by porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are two highly contagious intestinal diseases in the swine industry worldwide. Notably, coinfection of TGEV and PEDV is common in piglets with diarrhea-related diseases. In this study, intestinal porcine epithelial cells (IPEC-J2) were single or coinfected with PEDV and/or TGEV, followed by the comparison of differentially expressed genes (DEGs), especially interferon-stimulated genes (ISGs), between different groups via transcriptomics analysis and real-time qPCR. The antiviral activity of swine interferon-induced transmembrane protein 3 (sIFITM3) on PEDV and TGEV infection was also evaluated. The results showed that DEGs can be detected in the cells infected with PEDV, TGEV, and PEDV+TGEV at 12, 24, and 48 hpi, and the number of DEGs was the highest at 24 hpi. The DEGs are mainly annotated to the GO terms of protein binding, immune system process, organelle part, and intracellular organelle part. Furthermore, 90 ISGs were upregulated during PEDV or TGEV infection, 27 of which were associated with antiviral activity, including ISG15, OASL, IFITM1, and IFITM3. Furthermore, sIFITM3 can significantly inhibit PEDV and TGEV infection in porcine IPEC-J2 cells and/or monkey Vero cells. Besides, sIFITM3 can also inhibit vesicular stomatitis virus (VSV) replication in Vero cells. These results indicate that sIFITM3 has broad-spectrum antiviral activity.

All-Trans Retinoic Acid Attenuates Transmissible Gastroenteritis Virus-Induced Inflammation in IPEC-J2 Cells via Suppressing the RLRs/NF-κB Signaling Pathway.

Transmissible gastroenteritis virus (TGEV) infection can cause transmissible gastroenteritis (TGE), especially in suckling piglets, resulting in a significant economic loss for the global pig industry. The pathogenesis of TGEV infection is closely related to intestinal inflammation. All-trans retinoic acid (ATRA) has anti-inflammatory activity and immunomodulatory properties, but it is unclear whether ATRA can attenuate the inflammatory response induced by TGEV. This study aimed to investigate the protective effect of ATRA on TGEV-induced inflammatory injury in intestinal porcine epithelial cells (IPEC-J2) and to explore the underlying molecular mechanism. The results showed that TGEV infection triggered inflammatory response and damaged epithelial barrier integrity in IPEC-J2 cells. However, ATRA attenuated TGEV-induced inflammatory response by inhibiting the release of pro-inflammatory cytokines, including IL-1ß, IL-6, IL-8 and TNF-α. ATRA also significantly reversed the reduction of ZO-1 and Occludin protein levels induced by TGEV infection and maintained epithelial barrier integrity. Moreover, ATRA treatment significantly prevented the upregulation of IкBα and NF-κB p65 phosphorylation levels and the nuclear translocation of NF-кB p65 induced by TGEV. On the other hand, treatment of TGEV-infected IPEC-J2 cells with the NF-κB inhibitors (BAY11-7082) significantly decreased the levels of inflammatory cytokines. Furthermore, ATRA treatment significantly downregulated the mRNA abundance and protein levels of TLR3, TLR7, RIG-I and MDA5, and downregulated their downstream signaling molecules TRIF, TRAF6 and MAVS mRNA expressions in TGEV-infected IPEC-J2 cells. However, the knockdown of RIG-I and MDA5 but not TLR3 and TLR7 significantly reduced the NF-κB p65 phosphorylation level and inflammatory cytokines levels in TGEV-infected IPEC-J2 cells. Our results indicated that ATRA attenuated TGEV-induced IPEC-J2 cells damage via suppressing inflammatory response, the mechanism of which is associated with the inhibition of TGEV-mediated activation of the RLRs/NF-κB signaling pathway.

Swine Enteric Coronaviruses (PEDV, TGEV, and PDCoV) Induce Divergent Interferon-Stimulated Gene Responses and Antigen Presentation in Porcine Intestinal Enteroids.

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.

Dynamics of transmissible gastroenteritis virus internalization unraveled by single-virus tracking in live cells.

Transmissible gastroenteritis virus (TGEV) is a swine enteropathogenic coronavirus that causes significant economic losses in swine industry. Current studies on TGEV internalization mainly focus on viral receptors, but the internalization mechanism is still unclear. In this study, we used single-virus tracking to obtain the detailed insights into the dynamic events of the TGEV internalization and depict the whole sequential process. We observed that TGEVs could be internalized through clathrin- and caveolae-mediated endocytosis, and the internalization of TGEVs was almost completed within ~2 minutes after TGEVs attached to the cell membrane. Furthermore, the interactions of TGEVs with actin and dynamin 2 in real time during the TGEV internalization were visualized. To our knowledge, this is the first report that single-virus tracking technique is used to visualize the entire dynamic process of the TGEV internalization: before the TGEV internalization, with the assistance of actin, clathrin, and caveolin 1 would gather around the virus to form the vesicle containing the TGEV, and after ~60 seconds, dynamin 2 would be recruited to promote membrane fission. These results demonstrate that TGEVs enter ST cells via clathrin- and caveolae-mediated endocytic, actin-dependent, and dynamin 2-dependent pathways.

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.

microRNA-222 Attenuates Mitochondrial Dysfunction During Transmissible Gastroenteritis Virus Infection.

Transmissible gastroenteritis virus (TGEV) is a member of Coronaviridae family. Our previous research showed that TGEV infection could induce mitochondrial dysfunction and upregulate miR-222 level. Therefore, we presumed that miR-222 might be implicated in regulating mitochondrial dysfunction induced by TGEV infection. To verify the hypothesis, the effect of miR-222 on mitochondrial dysfunction was tested and we showed that miR-222 attenuated TGEV-induced mitochondrial dysfunction. To investigate the underlying molecular mechanism of miR-222 in TGEV-induced mitochondrial dysfunction, a quantitative proteomic analysis of PK-15 cells that were transfected with miR-222 mimics and infected with TGEV was performed. In total, 4151 proteins were quantified and 100 differentially expressed proteins were obtained (57 upregulated, 43 downregulated), among which thrombospondin-1 (THBS1) and cluster of differentiation 47 (CD47) were downregulated. THBS1 was identified as the target of miR-222. Knockdown of THBS1 and CD47 decreased mitochondrial Ca2+ level and increased mitochondrial membrane potential (MMP) level. Reversely, overexpression of THBS1 and CD47 elevated mitochondrial Ca2+ level and reduced mitochondrial membrane potential (MMP) level. Together, our data establish a significant role of miR-222 in regulating mitochondrial dysfunction in response to TGEV infection.

EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication.

Transmissible gastroenteritis coronavirus (TGEV) is enteropathogenic coronavirus that causes diarrhea in pigs, and is associated with high morbidity and mortality in sucking piglets. The TGEV membrane (M) protein is a decisive protein for the proliferation of viral proteins, and is associated with virus assembly and budding. To identify the cellular proteins that interact with the TGEV M protein, yeast two-hybrid screening was employed, and seven cellular proteins were identified M-binding partners. Using the GST pull-down approach and a CO-IP assay, the M protein was found to interact with porcine intestinal cells via eukaryotic translation initiation factor 4-alpha (EIF4A2), an essential component of the cellular translational machinery. Additionally, confocal microscopy revealed that EIF4A2 and M were colocalized in the cytoplasm. Furthermore, the function of EIF4A2 in intestinal cells during TGEV infection was examined. A knockdown of EIF4A2 by siRNA markedly decreased M protein proliferation and TGEV replication in target cells. Thus demonstrating that EIF4A2 plays a significant role in TGEV replication. The present study provides mechanistic insight into the interaction between the TGEV M protein and intestinal cells which contributes to the understanding of coronavirus replication and may be useful for the development of novel therapeutic strategies for TGEV infection.

Transmissible Gastroenteritis Virus Infection Enhances SGLT1 and GLUT2 Expression to Increase Glucose Uptake.

Transmissible gastroenteritis virus (TGEV) is a coronavirus that causes villus atrophy, followed by crypt hyperplasia, reduces the activities of intestinal digestive enzymes, and disrupts the absorption of intestinal nutrients. In vivo, TGEV primarily targets and infects intestinal epithelial cells, which play an important role in glucose absorption via the apical and basolateral transporters Na+-dependent glucose transporter 1 (SGLT1) and facilitative glucose transporter 2 (GLUT2), respectively. In this study, we therefore sought to evaluate the effects of TGEV infection on glucose uptake and SGLT1 and GLUT2 expression. Our data demonstrate that infection with TGEV resulted in increased glucose uptake and augmented expression of EGFR, SGLT1 and GLUT2. Moreover, inhibition studies showed that EGFR modulated glucose uptake in control and TGEV infected cells. Finally, high glucose absorption was subsequently found to promote TGEV replication.

Tubulins interact with porcine and human S proteins of the genus Alphacoronavirus and support successful assembly and release of infectious viral particles.

Coronavirus spike proteins mediate host-cell-attachment and virus entry. Virus replication takes place within the host cell cytosol, whereas assembly and budding occur at the endoplasmic reticulum-Golgi intermediate compartment. In this study we demonstrated that the last 39 amino acid stretches of Alphacoronavirus spike cytoplasmic domains of the human coronavirus 229E, NL63, and the porcine transmissible gastroenteritis virus TGEV interact with tubulin alpha and beta chains. In addition, a partial co-localization of TGEV spike proteins with authentic host cell ß-tubulin was observed. Furthermore, drug-induced microtubule depolymerization led to changes in spike protein distribution, a reduction in the release of infectious virus particles and less amount of spike protein incorporated into virions. These data demonstrate that interaction of Alphacoronavirus spike proteins with tubulin supports S protein transport and incorporation into virus particles.

TGEV infection up-regulates FcRn expression via activation of NF-κB signaling.

It has been well characterized that the neonatal Fc receptor (FcRn) transports maternal IgG to a fetus or newborn and protects IgG from degradation. We previously reported that FcRn is expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2). Transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (TGEV). How porcine FcRn (pFcRn) expression is regulated by pathogenic infection remains unknown. Our research shows that IPEC-J2 cells infected with TGEV had up-regulated pFcRn expression. In addition, the NF-κB signaling pathway was activated in IPEC-J2 cells by TGEV infection. Furthermore, treatment of TGEV-infected IPEC-J2 cells with the NF-κB-specific inhibitor BAY 11-7082 resulted in down-regulation of pFcRn expression. Transient transfection of pFcRn promoter luciferase report plasmids with overexpression of NF-κB p65 transcription factor enhanced the activation of the luciferase report plasmids. We identified four NF-κB transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. Together, the data provide the first evidence that TGEV infection up-regulates pFcRn expression via activation of NF-κB signaling.

Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are emerged and reemerging viruses in pigs, and together with transmissible gastroenteritis virus (TGEV), pose significant economic concerns to the swine industry. These viruses infect epithelial cells of the small intestine and cause watery diarrhea, dehydration, and a high mortality in neonatal piglets. Type I interferons (IFN-α/ß) are major antiviral cytokines forming host innate immunity, and in turn, these enteric coronaviruses have evolved to modulate the host innate immune signaling during infection. Accumulating evidence however suggests that IFN induction and signaling in the intestinal epithelial cells differ from other epithelial cells, largely due to distinct features of the gut epithelial mucosal surface and commensal microflora, and it appears that type III interferon (IFN-λ) plays a key role to maintain the antiviral state in the gut. This review describes the recent understanding on the immune evasion strategies of porcine enteric coronaviruses and the role of different types of IFNs for intestinal antiviral innate immunity.

The porcine microRNA transcriptome response to transmissible gastroenteritis virus infection.

Transmissible gastroenteritis virus (TGEV; Coronaviridae family) causes huge economic losses to the swine industry. MicroRNAs (miRNAs) play a regulatory role in viral infection and may be involved in the mammalian immune response. Here, we report a comprehensive analysis of host miRNA expression in TGEV-infected swine testis (ST) cells. Deep sequencing generated 3,704,353 and 2,763,665 reads from uninfected ST cells and infected ST cells, respectively. The reads were aligned to known Sus scrofa pre-miRNAs in miRBase 19, identifying 284 annotated miRNAs. Certain miRNAs were differentially regulated during TGEV infection. 59 unique miRNAs displayed significant differentially expression between the normal and TGEV-infected ST cell samples: 15 miRNAs were significantly up-regulated and 44 were significantly down-regulated. Stem-loop RT-PCR was carried out to determine the expression levels of specific miRNAs in the two samples, and the results were consistent with those of sequencing. Gene ontology enrichment analysis of host target genes demonstrated that the differentially expressed miRNAs are involved in regulatory networks, including cellular process, metabolic process, immune system process. This is the first report of the identification of ST cell miRNAs and the comprehensive analysis of the miRNA regulatory mechanism during TGEV infection, which revealed the miRNA molecular regulatory mechanisms for the viral infection, expression of viral genes and the expression of immune-related genes. The results presented here will aid research on the prevention and treatment of viral diseases.

Identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus.

Nucleocapsid (N) protein of transmissible gastroenteritis virus (TGEV) packages viral RNA genome to form a ribonucleoprotein complex. In addition to its function as a structural protein, N protein is involved in cell apoptosis or cell-cycle regulation. N protein possibly interacts with host factors to modulate cellular functions. To identify cellular proteins that interacted with N protein of TGEV, methods of GST pull-down and Co-IP were utilized to precipitate cellular proteins of swine testicular (ST). Bound cellular proteins were resolved by SDS-PAGE. Analysis of interacting proteins by mass spectrometry allowed identification of 15 cellular protein bands representative of 12 cellular proteins including vimentin that bound to N protein. Furthermore, the function of vimentin cytoskeleton in ST cells during TGEV infection was examined. Vimentin cytoskeleton was required for virus replication. The present study thus provides protein-related information about interaction of TGEV N protein with host cell that should be useful for understanding host cell response to coronavirus pathogenesis infection and the underlying mechanism of coronavirus replication.

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV)-infected swine testicular (ST) cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-ß1), caspase-8, and heat shock protein 90 alpha (HSP90α) were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling.

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.

Regulation of ROS in transmissible gastroenteritis virus-activated apoptotic signaling.

Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, causes severe lethal watery diarrhea and dehydration in piglets. Previous studies indicate that TGEV infection induces cell apoptosis in host cells. In this study, we investigated the roles and regulation of reactive oxygen species (ROS) in TGEV-activated apoptotic signaling. The results showed that TGEV infection induced ROS accumulation, whereas UV-irradiated TGEV did not promote ROS accumulation. In addition, TGEV infection lowered mitochondrial transmembrane potential in PK-15 cell line, which could be inhibited by ROS scavengers, pyrrolidinedithiocarbamic (PDTC) and N-acetyl-l-cysteine (NAC). Furthermore, the two scavengers significantly inhibited the activation of p38 MAPK and p53 and further blocked apoptosis occurrence through suppressing the TGEV-induced Bcl-2 reduction, Bax redistribution, cytochrome c release and caspase-3 activation. These results suggest that oxidative stress pathway might be a key element in TGEV-induced apoptosis and TGEV pathogenesis.

Transmissible gastroenteritis virus infection induces apoptosis through FasL- and mitochondria-mediated pathways.

Transmissible gastroenteritis virus (TGEV) has been reported to induce apoptosis in swine testis (ST) cells. However, the mechanisms underlying TGEV-induced apoptosis are still unclear. In this study we observed that TGEV infection induced apoptosis in porcine kidney (PK-15) cells in a time- and dose-dependent manner. TGEV infection up-regulated FasL, activated FasL-mediated apoptotic pathway, leading to activation of caspase-8 and cleavage of Bid. In addition, TGEV infection down-regulated Bcl-2, up-regulated Bax expression, promoted translocation of Bax to mitochondria, activated mitochondria-mediated apoptotic pathway, which in turn caused the release of cytochrome c and the activation of caspase-9. Both extrinsic and intrinsic pathways activated downstream effector caspase-3, followed by the cleavage of PARP, resulting in cell apoptosis. Moreover, TGEV infection did not induce significant DNA fragmentation in ammonium chloride (NH(4)Cl) pretreated PK-15 cells or cells infected with UV-inactivated TGEV. In turn, block of caspases activation also did not affect TGEV replication. Taken together, this study demonstrates that TGEV-induced apoptosis is dependent on viral replication in PK-15 cells and occurs through activation of FasL- and mitochondria-mediated apoptotic pathways.

The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus.

BACKGROUND: Transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that is believed to be important for enteropathogenicity, but that has so far appeared to be dispensable for infection of cultured cells. The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions, and comparison of TGEV strains and mutants, as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity. METHODS: The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5, 20, and 60 min. Prior to infection, cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface, or mock-treated. In a second approach, pre-treatment of the virus with porcine intestinal mucin was performed, followed by the plaque assay after a 5 min adsorption time. A student's t-test was used to verify the significance of the results. RESULTS: Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection. However, when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60%. A TGEV PUR46 mutant (HAD3) deficient in sialic acid binding showed a 77% lower titer than the parental virus after a 5 min adsorption time. After an adsorption time of 60 min the titer of HAD3 was 58% lower than that of TGEV PUR46. Another TGEV strain, TGEV Miller, and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time. CONCLUSIONS: Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions. The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains.

Enhanced protection against infection with transmissible gastroenteritis virus in piglets by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interferon-α and interleukin-18.

The enhanced effect of cytokine combinations has been assessed empirically, based on their immunobiological mechanisms. However, far less is known of the enhanced protection of practical cytokine combinations against viral infection in the livestock industry, due to cost and production issues associated with mass administration. This study demonstrates the enhanced protection of oral co-administration of swine interferon-α (swIFN-α) and interleukin-18 (swIL-18) against infection with transmissible gastroenteritis virus (TGEV) in piglets using attenuated Salmonella enterica serovar Typhimurium as carrier of cytokine proteins. A single oral co-administration of S. enterica serovar Typhimurium expressing swIFN-α and swIL-18 induced enhanced alleviation of the severity of diarrhea caused by TGEV infection, compared to piglets administered S. enterica serovar Typhimurium expressing swIFN-α or swIL-18 alone. This enhancement was further observed by the reduction of TGEV shedding and replication, and the expression of IFN-stimulated gene products in the intestinal tract. The results suggest that the combined administration of the swIFN-α and swIL-18 cytokines using attenuated S. enterica serovar Typhimurium as an oral carrier provides enhanced protection against intestinal tract infection with TGEV.