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.

Porcine enteric coronaviruses: an updated overview of the pathogenesis, prevalence, and diagnosis.

The recent prevalence of coronavirus (CoV) poses a serious threat to animal and human health. Currently, porcine enteric coronaviruses (PECs), including the transmissible gastroenteritis virus (TGEV), the novel emerging swine acute diarrhoea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and re-emerging porcine epidemic diarrhoea virus (PEDV), which infect pigs of different ages, have caused more frequent occurrences of diarrhoea, vomiting, and dehydration with high morbidity and mortality in piglets. PECs have the potential for cross-species transmission and are causing huge economic losses in the pig industry in China and the world, which therefore needs to be urgently addressed. Accordingly, this article summarises the pathogenicity, prevalence, and diagnostic methods of PECs and provides an important reference for their improved diagnosis, prevention, and control.

Bioinformatics Analysis of Spike Proteins of Porcine Enteric Coronaviruses.

This article is aimed at analyzing the structure and function of the spike (S) proteins of porcine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) by applying bioinformatics methods. The physical and chemical properties, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, phosphorylation and glycosylation sites, epitope, functional domains, and motifs of S proteins of porcine enteric coronaviruses were predicted and analyzed through online software. The results showed that S proteins of TGEV, PEDV, SADS-CoV, and PDCoV all contained transmembrane regions and signal peptide. TGEV S protein contained 139 phosphorylation sites, 24 glycosylation sites, and 53 epitopes. PEDV S protein had 143 phosphorylation sites, 22 glycosylation sites, and 51 epitopes. SADS-CoV S protein had 109 phosphorylation sites, 20 glycosylation sites, and 43 epitopes. PDCoV S protein had 124 phosphorylation sites, 18 glycosylation sites, and 52 epitopes. Moreover, TGEV, PEDV, and PDCoV S proteins all contained two functional domains and two motifs, spike_rec_binding and corona_S2. The corona_S2 consisted of S2 subunit heptad repeat 1 (HR1) and S2 subunit heptad repeat 2 (HR2) region profiles. Additionally, SADS-CoV S protein was predicted to contain only one functional domain, the corona_S2. This analysis of the biological functions of porcine enteric coronavirus spike proteins can provide a theoretical basis for the design of antiviral drugs.

Activation of Interleukin-1ß 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-1ß (pro-IL-1ß) 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-1ß 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-1ß release and pyroptosis is dependent on NLRP3 inflammasome, thus NLRP3 inflammasome may play a central role in the innate immune response to TGEV infection.

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.

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.

Circular RNA CircEZH2 Suppresses Transmissible Gastroenteritis Coronavirus-induced Opening of Mitochondrial Permeability Transition Pore via Targeting MiR-22 in IPEC-J2.

Transmissible gastroenteritis (TGE) is a contagious and infectious disease that is characterized by severe vomiting and diarrhea of swine , especially piglet, and caused by transmissible gastroenteritis coronavirus (TGEV) . TGEV infection provokes mitochondrial damage of porcine intestinal epthelial cell (IPEC), which is responsible for inflammation and cell death. In our previous study, we have demonstrated that circular RNA circEZH2 was down-regulated during TGEV infection and promoted the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) via targeting miR-22 in porcine intestinal epithelial cell line (IPEC-J2). Activation of NF-κB is an important factor for mitochondrial damage. Mitochondrial permeability transition pore (mPTP) opening is a key reason for mitochondrial damage. So, we speculate that circEZH2 may regulate TGEV-induced mPTP opening via NF-kB pathway. In the present study, we found that mPTP opening of IPEC-J2 was occured during TGEV infection and suppressed by circEZH2 via attaching miR-22. Hexokinase 2 (HK2) and interleukin 6 (IL-6) were identified as the targets of miR-22. Silencing HK2 enhanced TGEV-induced mPTP opening, while no effect on NF-κB pathway. Silencing IL-6 promoted TGEV-induced mPTP opening and inhibited NF-κB pathway. Inhibitor of NF-κB increased TGEV-induced mPTP opening. The data revealed that TGEV-induced mPTP opening was regulated via two pathways: circEZH2/miR-22/HK2 axis and circEZH2/miR-22/IL-6/NF-κB axis.

Production of porcine aminopeptidase N (pAPN) site-specific edited pigs.

Porcine viral diarrhea is an acute and highly contagious enteric disease in pigs which causes huge economic losses in pig industry worldwide. Transmissible gastroenteritis virus (TGEV) is main pathogens responsible for piglets viral diarrhea. Knockout the host cellular surface receptor for TGEV may be an effective way to accelerate the breeding of resistant pigs. In this study, we applied site-specific editing pAPN which is effective in swine testis (ST) cells. Site-specific editing of pAPN reduced TGEV proliferation in ST cells by 96%-99% at different time periods post-infection. Next, the site-specific editing of pAPN porcine fetal fibroblasts were produced, and then the cell colonies were used as donor cells to generate the site-specific editing of pAPN pigs. Our research findings will not only offer a more thorough understanding of the pathogenesis of piglet diarrhea and lay the foundation for breeding TGEV-resistant piglets, but also understanding the molecular mechanisms involved in coronaviral infections.

Resistance to coronavirus infection in amino peptidase N-deficient pigs.

The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.

Prospects for the use of spherical gold nanoparticles in immunization.

Recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. We used spherical gold nanoparticles (average diameter, 15 nm) as a platform for the antigen for swine transmissible gastroenteritis virus (TGEV). The literature data demonstrate that immunization of animals with the TGEV antigen coupled to gold nanoparticles (GNPs) not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. The contents of γ-IFN, IL-1ß, and IL-6 in animals immunized with GNP-antigen conjugates were found to be higher than those in intact animals or in animals given the antigen alone. The increased concentration of IL-1ß in the immunized animals directly correlated with the activity of macrophages and stimulated B cells, which produce this cytokine when activated. The increased concentration of IL-6 indicates that the injected preparations are stimulatory to cellular immunity. Immunization with the TGEV antigen conjugated to GNPs as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to the activation of antibody generation. Furthermore, the use of this conjugate allows marked improvement of the structure of the animals' immune organs and restores the morphological-functional state of these organs. The microanatomical changes (increased number of follicles) indicate the activation of the B-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction. The degradative processes observed in the animals immunized with TGEV antigen alone are evidence of weak resistance to pathogen attack. These results can be used to develop vaccines against this infection by employing TGEV antigen coupled to gold nanoparticles as a carrier.

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.

Transmissible gastroenteritis virus N protein causes endoplasmic reticulum stress, up-regulates interleukin-8 expression and its subcellular localization in the porcine intestinal epithelial cell.

This essay focuses on transmissible gastroenteritis virus (TGEV), which is an enteropathogenic virus related to contagious and acute diseases in suckling piglets. Previous literature suggests that the TGEV nucleocapsid protein (N) plays a significant role in viral transcriptional process, however, there is a need to examine other functions of TGEV N protein in the porcine intestinal epithelial cell (IEC) which is the target cell of TGEV. In the present study, we investigated the degradation, subcellular localisation, and function of TGEV N protein by examining its effects on cycle progression, endoplasmic reticulum (ER) stress, interleukin-8 (IL-8) expression, and cell survival. The results showed that TGEV N protein localised in the cytoplasm, inhibited IEC growth, prolonged the S-phase cell cycle by down-regulating cell cycle protein cyclin A, and was mainly degraded through the proteasome pathway. Moreover, TGEV N protein induced ER stress and activated NF-κB, which was responsible for the up-regulation of IL-8 and Bcl-2 expression. This report mainly considers the functions of TGEV N protein in IEC. To be specific, in IEC, TGEV N protein induces cell cycle prolongation at the S-phase, ER stress and up-regulates IL-8 expression. These results provide a better understanding of the functions and structural mechanisms of TGEV N protein.

Transmissible Gastroenteritis Virus Papain-Like Protease 1 Antagonizes Production of Interferon-ß through Its Deubiquitinase Activity.

Coronaviruses (CoVs), such as human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome CoV (SARS-CoV), murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), encode papain-like (PL) proteases that inhibit Sendai virus- (SeV-) induced interferon (IFN-ß) production. Recently, the crystal structure of transmissible gastroenteritis virus (TGEV) PL1 has been solved, which was similar to that of SARS-CoV PL2pro, which may antagonize host innate immunity. However, very little is known about whether TGEV PL1 can antagonize host innate immune response. Here, we presented evidence that TGEV PL1 encoded by the replicase gene could suppress the IFN-ß expression and inhibit the nuclear translocation of interferon regulatory factor 3 (IRF3). The ability to antagonize IFN-ß production was dependent on the intact catalytic activity of PL1. Furthermore, TGEV PL1 exerted deubiquitinase (DUB) activity which strongly inhibited the retinoic acid-induced gene I- (RIG-1-) and stimulator of interferon gene- (STING-) dependent IFN expression. Our data collectively suggest that TGEV PL1 can inhibit the IFN-ß expression and interfere with RIG-1- and STING-mediated signaling through a viral DUB activity. Our study has yielded strong evidence for the TGEV PL1 mechanisms that counteract the host innate immunity.

Proteomic Analysis of IPEC-J2 Cells in Response to Coinfection by Porcine Transmissible Gastroenteritis Virus and Enterotoxigenic Escherichia coli K88.

SCOPE: Piglet diarrhea causes large economic losses to the swine industry. Epidemiological investigations show that piglet diarrhea is often caused by mixed infections, but the mechanisms by which multiple microorganisms cause disease are unclear. EXPERIMENTAL DESIGN: Because transmissible gastroenteritis virus (TGEV) and enterotoxigenic Escherichia coli K88 (ETEC K88) are important contributors to piglet diarrhea, coinfection experiments are conducted using porcine intestinal columnar epithelial cells (IPEC-J2) as a model system. In order to evaluate piglet diarrhea caused TGEV and ETEC K88, the authors examin the effects of coinfection in IPEC-J2 cells. In TGEV pre-infected IPEC-J2 cells, ETEC K88 adhesion is enhanced over uninfected cells. ETEC K88 is also found to inhibit the proliferation of TGEV. Additionally, cytokine levels (IL-1ß, IL-6, IL-8, and TNF-α) in coinfected cells are lower than cells infected by TGEV alone, and higher than cells infected by ETEC K88 alone. LCMS/MS coupled to isobaric tags for relative and absolute quantification (iTRAQ) is used to profile expressed proteins in IPEC-J2 cells infected by TGEV alone, ETEC K88 alone, and by both agents together. RESULTS: 77, 89, and 136 differentially expressed proteins are identified in TGEV infected, ETEC K88 infected, and coinfected cells, respectively. CONCLUSION AND CLINICAL RELEVANCE: Based on these data, the authors suspect that integrin α5 might enable TGEV to promote ETEC K88 adhesion. This study is the first to analyze piglet diarrhea caused by TGEV-ETEC K88 coinfection using high-throughput quantitative proteomics. The results advance the understanding of coinfection and its role in causing piglet diarrhea.

Persistent Transmissible Gastroenteritis Virus Infection Enhances Enterotoxigenic Escherichia coli K88 Adhesion by Promoting Epithelial-Mesenchymal Transition in Intestinal Epithelial Cells.

Transmissible gastroenteritis virus (TGEV) is a coronavirus characterized by diarrhea and high morbidity rates, and the mortality rate is 100% in piglets less than 2 weeks old. Pigs infected with TGEV often suffer secondary infection by other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelial-mesenchymal transition (EMT), and thus enterotoxigenic Escherichia coli (ETEC) can more easily adhere to generating cells. Intestinal epithelial cells are the primary targets of TGEV and ETEC infections. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2) and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes; exhibit increases in levels of mesenchymal markers with a corresponding loss of epithelial markers; have enhanced expression levels of interleukin-1ß (IL-1ß), IL-6, IL-8, transforming growth factor ß (TGF-ß), and tumor necrosis factor alpha (TNF-α) mRNAs; and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways via TGF-ß is critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin α5 and fibronectin and exhibit enhanced ETEC K88 adhesion. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin α5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection.IMPORTANCE Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection by other pathogens. In this study, we showed that persistent TGEV infection induces an EMT in porcine intestinal columnar epithelial cells (IPEC-J2) and enhances the adhesion of the secondary pathogen ETEC K88. Additional experiments suggest that integrin α5 and fibronectin play an important role in TGEV-enhanced ETEC K88 adhesion. Reversal of EMT reduces the expression of integrin α5 and fibronectin and also reduces ETEC K88 adhesion. We conclude that TGEV infection triggers EMT and facilitates dual infection. Our results provide new insights into secondary infection and suggest that targeted anti-EMT therapy may have implications for the prevention and treatment of secondary infection.

Identification of a natural recombinant transmissible gastroenteritis virus between Purdue and Miller clusters in China.

Transmissible gastroenteritis virus (TGEV) is an infective coronavirus (CoV) that causes diarrhea-related morbidity and mortality in piglets. For the first time, a natural recombination strain of a TGEV Anhui Hefei (AHHF) virus between the Purdue and the Miller clusters was isolated from the small intestine content of piglets in China. A phylogenetic tree based on a complete genome sequence placed the TGEV AHHF strain between the Purdue and the Miller clusters. The results of a computational analysis of recombination showed that the TGEV AHHF strain is a natural recombinant strain between these clusters. Two breakpoints located in the open reading frame 1a (ORF1a) and spike (S) genes were identified. The pathogenicity of the TGEV AHHF strain was evaluated in piglets, and the results show that TGEV AHHF is an enteric pathogenic strain. These results provide valuable information about the recombination and evolution of CoVs and will facilitate future investigations of the molecular pathogenesis of TGEV.

Inhibition of porcine transmissible gastroenteritis virus infection in porcine kidney cells using short hairpin RNAs targeting the membrane gene.

The membrane (M) protein is the most abundant component of the porcine transmissible gastroenteritis virus (TGEV) particle. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, three plasmids (pRNAT-1, pRNAT-2, and pRNAT-3) expressing short hairpin RNAs were designed to target three different coding regions of the M gene of TGEV. The plasmids were constructed and transiently transfected into a porcine kidney cells, PK-15, to determine whether these constructs inhibited TGEV production. The analysis of cytopathic effects demonstrated that pRNAT-2 and pRNAT-3 could protect PK-15 cells against pathological changes specifically and efficiently. Additionally, indirect immunofluorescence and 50% tissue culture infectious dose (TCID50) assays showed that pRNAT-2 and pRNAT-3 inhibited the multiplication of the virus at the protein level effectively. Quantitative real-time PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the three plasmids were reduced by 13, 68, and 70%, respectively. This is the first report showing that RNAi targeting of the M gene. Our results could promote studies of the specific function of viral genes associated with TGEV infection and might provide a theoretical basis for potential therapeutic applications.

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.

Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

[ANTIMICROBIAL ACTIVITY OF POLYAZOLIDINAMMONIUM, MODIFIED WITH HYDRATE-IONS OF IODINE].

AIM: Study of antimicrobial activity of a polymer compound--polyazolidinammonium, modified with hydrate-ions of iodine. MATERIALS AND METHODS: Antimicrobial activity of polyazolidinammonium, modified with hydrate-ions of iodine, against reference strains and clinical isolates of Gram positive and negative bacteria, microscopical fungi, as well as RNA viruses was studied. RESULTS: High antibacterial activity of the studied compound was established, especially against Gram positive bacteria. A higher concentration of the preparation (125-250 µg/ml) was characterized by anti-fungal effect. A high sensitivity to polymer of swine transmissible gastroenteritis virus was noted. CONCLUSION: The polymer compound, based on the results of the studies, is a perspective antiseptic and etiotropic means for control of infectious disease causative agents.