Evaluation of the Effect of Inactivated Transmissible Gastroenteritis Virus Vaccine with Nano Silicon on the Phenotype and Function of Porcine Dendritic Cells.

A transmissible gastroenteritis virus (TGEV) is a porcine enteropathogenic coronavirus, causing acute swine enteric disease especially in suckling piglets. Mesoporous silica nanoparticles (MSNs) are safe vaccine adjuvant, which could enhance immune responses. Our previous research confirmed that nano silicon had immune-enhancing effects with inactivated TGEV vaccine. In this study, we further clarified the immune-enhancing mechanism of the inactivated TGEV vaccine with MSNs on porcine dendritic cells (DCs). Our results indicated that the inactivated TGEV vaccine with MSNs strongly enhanced the activation of the DCs. Expressions of TLR3, TLR5, TLR7, TLR9, and TLR10, cytokines IFN-α, IL-1ß, IL-6, IL-12, and TNF-α, cytokine receptor CCR-7 of immature DCs were characterized and showed themselves to be significantly higher in the inactivated TGEV vaccine with the MSN group. In summary, the inactivated TGEV vaccine with MSNs has effects on the phenotype and function of porcine DCs, which helps to better understand the immune-enhancing mechanism.

Inhibitory effects of recombinant porcine interferon-α on porcine transmissible gastroenteritis virus infections in TGEV-seronegative piglets.

Our previous research obtained purified recombinant porcine interferon-α (rPoIFN-α) containing thioredoxin (Trx) fusion tag in E. coli Rosetta (DE3). Here, we evaluate the efficacy of this rPoIFN-α to prevent piglets from the infection of the transmissible gastroenteritis virus (TGEV) attack. In this experiment, twenty-five TGEV-seronegative piglets were randomly divided into five groups. Group 1 was positive control and only challenged with TGEV; Pigs in groups 2-4 were pretreated with 2 × 10(7)IU/pig, 2 × 10(6)IU/pig, and 2 × 10(5)IU/pig rPoIFN-α before TGEV challenge. The fifth group is a negative control group. The animals of this group are pretreated only with Trx protein-containing PBS solution without TGEV challenge. After 48 h of rPoIFN-α pretreatment, the pigs in groups 1-4 were challenged by TGEV, and the pigs in group 5 were administered with PBS. The surveillance results show that Pigs pre-treated with 2 × 10 (7) IU/pig rPoIFN-α are fully aligned with the violent TGEV attack. Pigs pretreated with 2 × 10 (6) IU/pig rPoIFN-α are partially aligned with the violent TGEV attack. Though piglets pretreated with 2 × 10(6) IU/pig or 2 × 10(5)IU/pig rPoIFN-α cannot be adapted to the challenge of TGEV. However, the use of this dose of rPoIFN-α could put off the clinical signs of pigs than the positive control group of the above. These results indicate that rPoIFN-α can protect pigs from the infection of potential TGEV or delay the appearance of clinical symptoms, and its effect is dose-dependent.

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.

Next-Generation Porcine Intestinal Organoids: an Apical-Out Organoid Model for Swine Enteric Virus Infection and Immune Response Investigations.

The intestinal organoid culture system is a pathbreaking working model for investigating pathogen-host interactions in the intestines. However, due to the limitations of the first generation of intestinal organoids, basal-out structure and growth in Matrigel, most pathogens can rarely attach to the apical membrane directly and hardly initiate infection. In this study, we first developed a next-generation porcine intestinal organoid culture system, characterized by an apical membrane on the surface, called apical-out. To investigate the infectivity and antiviral immune responses of this apical-out porcine intestinal organoid, a swine enteric virus, transmissible gastroenteritis virus (TGEV), was employed to inoculate the culture system. Both reverse transcription-quantitative PCR (RT-qPCR) and immunofluorescence assay (IFA) analysis demonstrated that TGEV replicated in the apical-out porcine intestinal organoid culture system. Additionally, our results illustrated that TGEV infection significantly upregulated the expression levels of alpha interferon (IFN-α), IFN-λ1, interferon-stimulated gene 15 (ISG15), ISG58, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) in this culture system. Hence, we successfully developed a porcine intestinal apical-out organoid culture system, which will facilitate the investigation of pathogen-host interactions in pig intestines.IMPORTANCE Intestinal organoids are a newly developed culture system for investigating pathogen-host interactions. Intestinal organoid models have been widely used since their development, because the results obtained from this type of culture model better represent physiological conditions than those from well-established cell lines. The three-dimensional (3D) porcine intestinal organoid model was reported in 2018 and 2019 for the investigation of intestinal pathogens. However, those organoid culture models were basal-out intestinal organoids, which are not suitable for porcine enteric virus research because they invade the intestines via the apical side of epithelial cells on villi. In this study, we developed a porcine apical-out intestinal organoid culture system and verified its infectivity, type I and type III interferon (IFN) antiviral responses, and inflammatory responses following infection by a swine enteric virus. Our results imply that this apical-out porcine intestinal organoid culture system is an ideal model for the investigation of interactions between swine enteric viruses and the intestines.

Agrodiag PorCoV: A multiplex immunoassay for the differential diagnosis of porcine enteric coronaviruses.

Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV.

Detecting and Monitoring Porcine Hemagglutinating Encephalomyelitis Virus, an Underresearched Betacoronavirus.

Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

Porcine IL-12 plasmid as an adjuvant improves the cellular and humoral immune responses of DNA vaccine targeting transmissible gastroenteritis virus spike gene in a mouse model.

Transmissible gastroenteritis (TGE), caused by transmissible gastroenteritis virus (TGEV), is a highly infectious disease in pigs. Vaccination is an effective approach to prevent TGEV infection. Here, we evaluated the potential of TGEV S1 as a DNA vaccine and porcine interleukin (pIL)-12 as an adjuvant in a mouse model. A DNA vaccine was constructed with the TGEV S1 gene to induce immune response in an experimental mouse model; pIL-12 was chosen as the immunological adjuvant within this DNA vaccine. The pVAX1-(TGEV-S1) and pVAX1-(pIL-12) vectors were transfected into BHK-21 cells and expressed in vitro. Experimental mice were separately immunized with each of the recombinant plasmids and controls through the intramuscular route. The lymphocytes isolated from the blood and spleen were analyzed for proliferation, cytotoxic activities, and populations of CD4+ and CD8+ cells. The titers of TGEV S1 in an enzyme-linked immunosorbent assay (ELISA) and TGEV neutralizing antibodies and the concentrations of interferon (IFN)-γ and IL-4 were also analyzed in the serum. The plasmids pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could be expressed in BHK-21 cells, and the combination of pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could induce a significant increase in all markers. pIL-12 could act as an immunological adjuvant in the DNA vaccine for TGEV-S1. Furthermore, the DNA vaccine prepared using TGEV-S1 and porcine IL-12 could induce excellent humoral and cellular immune responses.

Interferon gamma inhibits transmissible gastroenteritis virus infection mediated by an IRF1 signaling pathway.

Interferon gamma (IFN-γ) is best known for its ability to regulate host immune responses; however, its direct antiviral activity is less well studied. Transmissible gastroenteritis virus (TGEV) is an economically important swine enteric coronavirus and causes acute diarrhea in piglets. At present, little is known about the function of IFN-γ in the control of TGEV infection. In this study, we demonstrated that IFN-γ inhibited TGEV infection directly in ST cells and intestine epithelial IPEC-J2 cells and that the anti-TGEV activity of IFN-γ was independent of IFN-α/ß. Moreover, IFN-γ suppressed TGEV infection in ST cells more efficiently than did IFN-α, and the combination of IFN-γ and IFN-α displayed a synergistic effect against TGEV. Mechanistically, using overexpression and functional knockdown experiments, we demonstrated that porcine interferon regulatory factor 1 (poIRF1) elicited by IFN-γ primarily mediated IFN-γ signaling cascades and the inhibition of TGEV infection by IFN-γ. Importantly, we found that TGEV elevated the expression of poIRF1 and IFN-γ in infected small intestines and peripheral blood mononuclear cells. Thus, IFN-γ plays a crucial role in curtailing enteric coronavirus infection and may serve as an effective prophylactic and/or therapeutic agent against TGEV infection.

Porcine transmissible gastroenteritis virus inhibits NF-κB activity via nonstructural protein 3 to evade host immune system.

BACKGROUND: Transmissible gastroenteritis virus (TGEV), a member of the family Coronaviridae, causes lethal watery diarrhea in piglets. Previous studies have revealed that the coronaviruses develop various strategies to evade the host innate immunity through the inhibition of nuclear factor kappa B (NF-κB) signaling pathway. However, the ability of TGEV to inhibit the host innate immune response by modulating the NF-κB signaling pathway is not clear. METHODS: In this study, a dual luciferase reporter assay was used to confirm the inhibition of NF-κB by TGEV infection and to identify the major viral proteins involved in the inhibition of NF-κB signaling. Real-time quantitative PCR was used to quantify the mRNA expression of inflammatory factors. The deubiquitination of Nsp3 domains and its effect on IκBα and p65 were analyzed by western blotting. The ubiquitination level of IκBα was analyzed by immunoprecipitation. RESULTS: In ST and IPEC-J2 cells, TGEV exhibited a dose-dependent inhibition of NF-κB activity. Individual TGEV protein screening revealed the high potential of non-structural protein 3 (Nsp3) to inhibit NF-κB signaling, and leading to the downregulation of the NF-κB-induced cytokine production. We demonstrated that the inhibitory effect of Nsp3 was mainly mediated through the suppression of IκBα degradation as well as the inhibition of p65 phosphorylation and nuclear translocation. Furthermore, the amino acid residues at positions 590-1,215 in Nsp3 were demonstrated to inhibit the degradation of IκBα by inhibiting the IκBα ubiquitination. CONCLUSION: TGEV infection can inhibit the activation of the NF-κB signaling pathway, which is mainly mediated by Nsp3 through the canonical pathway. The amino acid residues at positions 590-1,215 in Nsp3 compose the critical domain that mediates NF-κB inhibition. We speculate that this inhibitory effect is likely to be related to the structure of PLP2 with deubiquitinating enzyme activity of the amino acid residues at positions 590-1,215 in Nsp3. Our study provides a better understanding of the TGEV-mediated innate immune modulation and lays the basis for studies on the pathogenesis of coronavirus.

Evaluation of the Serologic Cross-Reactivity between Transmissible Gastroenteritis Coronavirus and Porcine Respiratory Coronavirus Using Commercial Blocking Enzyme-Linked Immunosorbent Assay Kits.

This study compared the performances of three commercial transmissible gastroenteritis virus/porcine respiratory coronavirus (TGEV/PRCV) blocking enzyme-linked immunosorbent assays (ELISAs) using serum samples (n = 528) collected over a 49-day observation period from pigs inoculated with TGEV strain Purdue (n = 12), TGEV strain Miller (n = 12), PRCV (n = 12), or with virus-free culture medium (n = 12). ELISA results were evaluated both with "suspect" results interpreted as positive and then as negative. All commercial kits showed excellent diagnostic specificity (99 to 100%) when testing samples from pigs inoculated with virus-free culture medium. However, analyses revealed differences between the kits in diagnostic sensitivity (percent TGEV- or PRCV-seropositive pigs), and all kits showed significant (P < 0.05) cross-reactivity between TGEV and PRCV serum antibodies, particularly during early stages of the infections. Serologic cross-reactivity between TGEV and PRCV seemed to be TGEV strain dependent, with a higher percentage of PRCV-false-positive results for pigs inoculated with TGEV Purdue than for TGEV Miller. Moreover, the overall proportion of false positives was higher when suspect results were interpreted as positive, regardless of the ELISA kit evaluated.IMPORTANCE Current measures to prevent TGEV from entering a naive herd include quarantine and testing for TGEV-seronegative animals. However, TGEV serology is complicated due to the cross-reactivity with PRCV, which circulates subclinically in most swine herds worldwide. Conventional serological tests cannot distinguish between TGEV and PRCV antibodies; however, blocking ELISAs using antigen containing a large deletion in the amino terminus of the PRCV S protein permit differentiation of PRCV and TGEV antibodies. Several commercial TGEV/PRCV blocking ELISAs are available, but performance comparisons have not been reported in recent research. This study demonstrates that the serologic cross-reactivity between TGEV and PRCV affects the accuracy of commercial blocking ELISAs. Individual test results must be interpreted with caution, particularly in the event of suspect results. Therefore, commercial TGEV/PRCV blocking ELISAs should only be applied on a herd basis.

Construction and characterization of porcine single-chain fragment variable antibodies that neutralize transmissible gastroenteritis virus in vitro.

Transmissible gastroenteritis virus (TGEV) infection causes severe diarrhea in piglets and imposes a significant economic burden on pig farms. Single-chain fragment variable (scFv) antibodies effectively inhibit virus infection and could be a potential therapeutic reagent for preventing disease. In this study, a recombinant scFv antibody phage display library was constructed from peripheral blood lymphocytes of piglets infected with TGEV. The library was screened with four rounds of biopanning using purified TGEV antigen, and scFv antibodies that bound to TGEV were obtained. The scFv gene was subcloned into the pET-28a(+), and the constituted plasmid was introduced into Escherichia coli BL21 (DE3) for protein expression. All three scFv clones identified had neutralizing activity against TGEV. An immunofluorescence assay and western blot analysis demonstrated that two scFv antibodies reacted with the spike protein of TGEV. These results indicate that scFv antibodies provide protection against viral infection in vitro and may be a therapeutic candidate for both prevention and treatment of TGEV infection in swine.

Two critical N-terminal epitopes of the nucleocapsid protein contribute to the cross-reactivity between porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus.

Both porcine epidemic diarrhoea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV), which cause high mortality in piglets and produce similar clinical symptoms and histopathological morphology, belong to the genus Alphacoronavirus. Serological diagnosis plays an important role in distinguishing pathogen species. Together with the spike (S) protein, the nucleocapsid (N) protein is one of the immunodominant regions among coronaviruses. In this study, two-way antigenic cross-reactivity between the N proteins of PEDV and TGEV was observed by indirect immunofluorescence assay (IFA) and Western blot analysis. Furthermore, the PEDV N protein harbouring truncations of amino acids (aa) 1 to 170 or aa 125 to 301 was demonstrated to cross-react with the anti-TGEV N polyclonal antibody (PAb), whereas the truncation-expressing aa 302 to 401 resulted in a specific reaction with the anti-PEDV N PAb but not with the anti-TGEV N PAb. Mutants of the PEDV N protein were generated based on sequence alignment and structural analysis; we then confirmed that the N-terminal residues 58-RWRMRRGERIE-68 and 78-LGTGPHAD-85 contributed to the cross-reactivity. All the results provide vital clues for the development of precise diagnostic assays for porcine coronaviruses.

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.

p53 mediated IFN-ß signaling to affect viral replication upon TGEV infection.

TGEV can induce IFN-ß production, which in turn plays a vital role in host antiviral immune responses. Our previous studies showed that TGEV infection activated p53 signaling to induce host cell apoptosis, which might influence virus replication. However, whether there be an interaction between p53 and IFN-ß signaling in the process of TGEV infection is unknown. In the present study, we used low dose of TGEV to infect p53 wild-type PK-15 cells (WT PK-15 cells) and p53 deficient cells (p53-/- PK-15 cells), to investigate the modulation of IFN signaling and virus replication by p53. The results showed that the IFN-ß expression and production were notably inhibited in p53-/- PK-15 cells compared with that in WT PK-15 cells at early stage of TGEV infection. In addition, TGEV-induced the changes in mRNA levels of TRIF, TRAM, MDA5, RIG-I, IPS-1, IRF9, IRF3, ISG15 and ISG20 were notably hindered in p53-/- PK-15 cells before 36 h post infection (p.i.). Moreover, TGEV genomic RNA and sub genomic mRNA (N gene and ORF7) levels showed significant increase in p53-/- PK-15 cells compared with WT PK-15 cells after TGEV infection. And viral titers were observably enhanced in p53-/- PK-15 cells. Furthermore, exogenous IFN-ß and polyinosinic-polycytidylic acid (poly (I:C)) treatment markedly inhibited the mRNA levels of TGEV gRNA, N and ORF7 in WT PK-15 cells and p53-/- PK-15 cells compared to control. Taken together, these results demonstrated that p53 may mediate IFN-ß signaling to inhibit viral replication early after TGEV infection.

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

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.

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.

Immune responses induced by recombinant Lactobacillus plantarum expressing the spike protein derived from transmissible gastroenteritis virus in piglets.

Transmissible gastroenteritis coronavirus (TGEV) is one of the most severe threats to the swine industry. In this study, we constructed a suite of recombinant Lactobacillus plantarum with surface displaying the spike (S) protein coming from TGEV and fused with DC cells targeting peptides (DCpep) to develop an effective, safe, and convenient vaccine against transmissible gastroenteritis. Our research results found that the recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA-S-DCpep) group expressing S fused with DCpep could not only significantly increase the percentages of MHC-II+CD80+ B cells and CD3+CD4+ T cells but also the number of IgA+ B cells and CD3+CD4+ T cells of ileum lamina propria, which elevated the specific secretory immunoglobulin A (SIgA) titers in feces and IgG titers in serum. Taken together, these results suggest that NC8-pSIP409-pgsA-S-DCpep expressing the S of TGEV fused with DCpep could effectively induce immune responses and provide a feasible original strategy and approach for the design of TGEV vaccines.

Immune response characterization of mice immunized with Lactobacillus plantarum expressing spike antigen of transmissible gastroenteritis virus.

The highly infectious porcine transmissible gastroenteritis virus (TGEV), which belongs to the coronaviruses (CoVs), causes diarrhea and high mortality rates in piglets, resulting in severe economic losses in the pork industry worldwide. In this study, we used Lactobacillus plantarum (L. plantarum) to anchor the expression of TGEV antigen (S) to dendritic cells (DCs) via dendritic cell-targeting peptides (DCpep). The results show that S antigen could be detected on the surface of L. plantarum by different detection methods. Furthermore, flow cytometry and ELISA techniques were used to measure the cellular, mucosal, and humoral immune responses of the different orally gavaged mouse groups. The obtained results demonstrated the significant effect of the constructed L. plantarum expressing S-DCpep fusion proteins in inducing high expression levels of B7 molecules on DCs, as well as high levels of IgG, secretory IgA, and IFN-γ and IL-4 cytokines compared with the other groups. Accordingly, surface expression of DC-targeted antigens successfully induced cellular, mucosal, and humoral immunity in mice and could be used as a vaccine.

A nano silicon adjuvant enhances inactivated transmissible gastroenteritis vaccine through activation the Toll-like receptors and promotes humoral and cellular immune responses.

Inactivated transmissible gastroenteritis virus (TGEV) vaccines are widely used in swine herds in China. These are limited, however, by the need to elicit both humoral and cellular immunity, as well as the efficiency of adjuvants. In this study, a 70-nm nano silicon particle was applied with inactivated TGEV vaccine in mice, and its immune-enhancing effects and mechanism of action investigated. We found that nano silicon applied with inactivated TGEV vaccine induced high antibody titers, increase IL-6, TNF-α and IFN-γ expression, and stimulate CD3+ T cell proliferation with a high CD4+/CD8+ T lymphocyte ratio. Nano silicon could quickly activate innate and adaptive immunity by stimulating Toll-like receptor signaling pathways, indicating that the nano silicon adjuvant enhanced long-term humoral and early cellular immune responses when combined with inactivated TGEV vaccine. Nano silicon could be considered for use as an antigen- carrier and adjuvant for veterinary vaccines.

Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus.

Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the causative agents of highly fatal acute diarrhea in pigs, resulting in enormous losses in the pig industry worldwide. To develop an effective bivalent oral vaccine against TGEV and PEDV infection, the D antigenic site of the TGEV spike (S) protein and the major antigen site (core neutralizing epitope-COE) of the PEDV S protein were used as immunogens, and the enhanced green fluorescent protein (eGFP) gene was used as a reporter to construct genetically engineered Lactobacillus casei rLpPGF-T7g10-eGFP-6D-COE. The expression of proteins of interest by the recombinant L. casei was confirmed by confocal laser scanning microscopy and a Western blot assay, and the immunogenicity of rLpPGF-T7g10-eGFP-6D-COE in orally immunized mice was evaluated. The results showed that levels of anti-PEDV and anti-TGEV serum immunoglobulin G (IgG) and mucosal secreted immunoglobulin A (sIgA) antibodies obtained from the mice immunized with rLpPGF-T7g10-eGFP-6D-COE, as well as the proliferation levels of lymphocytes, were significantly higher than those in mice orally administered phosphate-buffered saline (PBS) or rLpPG-T7g10. Moreover, the serum IgG antibodies showed neutralizing effects against PEDV and TGEV. Our data suggest that the antibiotic resistance-free genetically engineered L. casei bivalent oral vaccine provides a safe and promising strategy for vaccine development against PEDV and TGEV.