Activation of the NLRP1 Inflammasome and Its Role in Transmissible Gastroenteritis Coronavirus Infection.

The inflammasome pathway is a critical early response mechanism of the host that detects pathogens, initiates the production of inflammatory cytokines, and recruits effector cells to the infection site. Nonetheless, the mechanism of inflammasome activation in coronavirus infection and its biological functions in host defense remain unclear. Transmissible gastroenteritis virus (TGEV), a member of the genus Alphacoronavirus, is a significant pathogen that mainly infects piglets and causes intestinal inflammation and inflammatory cell infiltration. Here, we investigated the mechanism of inflammasome activation in intestinal epithelial cells (IECs) infected with TGEV. We observed a substantial increase in interleukin 1ß (IL-1ß) and IL-18 levels in both IECs and TGEV-infected porcine intestinal tissues. Furthermore, TGEV infection resulted in increased activation of caspase-1 and the NLRP1 (NOD-like receptor [NLR]-containing pyrin domain [PYD]) inflammasome. Our findings revealed that TGEV infection impeded the interaction between porcine NLRP1 (pNLRP1) and porcine dipeptidyl peptidases 9 (pDPP9), yet it did not reduce the expression of pDPP9. Importantly, the ZU5 domain, not the function-to-find domain (FIIND) reported in human NLRP1, was identified as the minimal domain of pNLRP1 for pDPP9 binding. In addition, the robust type I IFN expression induced by TGEV infection also upregulated pNLRP1 expression and pNLRP1 itself acts as an interferon-stimulated gene to counteract TGEV infection. Our data demonstrate that pNLRP1 has antiviral capabilities against coronavirus infection, which highlights its potential as a novel therapeutic target for coronavirus antiviral therapy. IMPORTANCE Coronavirus primarily targets the epithelial cells of the respiratory and gastrointestinal tracts, leading to damage in both humans and animals. NLRP1 is a direct sensor for RNA virus infection which is highly expressed in epithelial barrier tissues. However, until recently, the precise molecular mechanisms underlying its activation in coronavirus infection and subsequent downstream events remained unclear. In this study, we demonstrate that the alphacoronavirus TGEV induces the production of IL-1ß and IL-18 and upregulates the expression of pNLRP1. Furthermore, we found that pNLRP1 can serve as an interferon-stimulated gene (ISG) to inhibit the infection of enterovirus TGEV. Our research highlights the crucial role of NLRP1 as a regulator of innate immunity in TGEV infection and shows that it may serve as a potential therapeutic target for the treatment of coronavirus infection.

Glycyrrhizin inhibits swine acute diarrhea syndrome coronavirus infection via the HMGB1/TLR4/JNK signal pathway

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly discovered enteric coronavirus, is the etiological agent that causes severe clinical diarrhea and intestinal pathological damage in piglets. In this study, Vero E6 and IPI-2I cells were pretreated with different concentrations of glycyrrhizin (GLY) for 2 hours, and then infected with different concentrations of SADSCoV, aiming to investigate the inhibitory effect of GLY on SADS-CoV. Western blot and TCID50 results revealed a significantly decreased N protein expression and viral titer, indicating that GLY can inhibit the infection of SADS-CoV. Vero E6 and IPI-2I cells were pretreated with different concentrations of GLY for 2 hours and infected with SADS-CoV. Western blot results showed that when the concentration of GLY was 0.8 mmol/L, the expression of N protein decreased significantly, indicating that GLY inhibited the invasion of the virus. At first, cells were treated with 0.4 mmol/L GLY, and cell samples were collected at 2 hours, 6 hours and 12 hours after being infected with SADS-CoV for analysis, and the expression of N protein were found to be significantly reduced at all points, indicating that GLY had a significant inhibitory effect on the replication of the virus. GLY is a competitive inhibitor of high mobility group box 1 (HMGB1), and the receptors of HMGB1 mainly include TLR4 and RAGE. Based on this fact, the mutant plasmid at the key sites of HMGB1 (C45S, C106S, C45/106S) and the siRNA of the RAGE receptor were transfected to Vero E6 cells and infected with SADS-CoV, and the cell supernatant and samples were harvested. The western blot and TCID50 results showed that the expression of N protein and the virus titer were decreased, suggesting that GLY exerts its function by affecting the binding of HMGB1/TLR4/RAGE during SADS-CoV infection. To further explore the signaling pathway through which GLY functions, Vero E6 and IPI-2I cells were inoculated with SADS-CoV, and cell samples were harvested, western blot was used to detect the changes of MAPK proteins. The results showed that the protein expression levels of p-p38, p-JNK and p-ERK were up-regulated in the early and late stages, indicating that the MAPK pathway was activated by SADS-CoV infection. Vero E6 and IPI-2I were pretreated with different concentrations of GLY and TLR4 inhibitor TAK for 2 hours and infected with SADS-CoV. Protein samples were harvested and analysed by western blot which showed a decreased p-JNK and N proteins, while other proteins showed no significant changes. These results indicated that GLY and TAK regulated the phosphorylation of JNK but did not regulate the phosphorylation of p38 and ERK. Also, Vero E6 cells were treated with HMGB1 antibody, the siRNA of HMGB1 and HMGB1 mutants plasmid, and infected with SADS-CoV. Protein samples were harvested, western blot results showed that phosphorylation of JNK decreased, indicating that HMGB1 affected JNK phosphorylation. Finally, Vero E6 and IPI-2I cells were pretreated with different concentrations of JNK inhibitor SP600125 to infect SADS-CoV, western blot, TCID50 and IFA results showed that the expression of N protein and virus titer, as well as virus replication were reduced, indicating that SP600125 inhibited virus replication. In conclusion, our results revealed that GLY can inhibit in vitro replication of SADS- CoV, mainly through the HMGB1/TLR4/JNK signaling pathway. The discovery of this pathway provides theoretical support for the research of novel anti-SADS-CoV drugs.

Octyl gallate targeting the 3C-like protease exhibits highly efficient antiviral activity against swine enteric coronavirus PEDV.

Infection with porcine epidemic diarrhea virus (PEDV) causes severe watery diarrhea in newborn piglets, leading to substantial financial losses for the swine industry. In this study, we screened small molecule drugs targeting 3 C-like protease (3CLpro) by molecular docking, and further evaluated the antiviral activity of the screened drugs against PEDV. Results showed that octyl gallate (OG), a widely used food additive, exhibited strong binding affinity with the 3CLpro active sites of PEDV. Bio-layer interferometry and fluorescence resonance energy transfer revealed that OG directly interacts with PEDV 3CLpro (KD = 549 nM) and inhibits 3CLpro activity (IC50 = 22.15 µM). OG showed a strong inhibition of PEDV replication in vitro. Virus titers were decreased by 0.58 and 0.71 log10 TCID50/mL for the CV777 and HM2017 strains, respectively. In vivo, all piglets in the PEDV-infected group died at 48 h post-infection (hpi), while 75% of piglets in the OG treatment group showed significant relief from the clinical symptoms, pathological damage, and viral loads in the jejunum and ileum. Moreover, the western blotting results showed that OG also has strong antiviral activity against other swine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). Our findings revealed that OG could be developed as a novel antiviral drug against PEDV. The OG exhibited a potential broad-spectrum antiviral drug for control of other swine enteric coronaviruses.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

The TGEV Membrane Protein Interacts with HSC70 To Direct Virus Internalization through Clathrin-Mediated Endocytosis.

Coronavirus membrane protein is a major component of the viral envelope and plays a central role in the viral life cycle. Studies of the coronavirus membrane protein (M) have mainly focused on its role in viral assembly and budding, but whether M protein is involved in the initial stage of viral replication remains unclear. In this study, eight proteins in transmissible gastroenteritis virus (TGEV)-infected cells coimmunoprecipitated with monoclonal antibodies (MAb) against M protein in PK-15 cells, heat shock cognate protein 70 (HSC70), and clathrin were identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF MS). Further studies demonstrated that HSC70 and TGEV M colocalized on the cell surface in early stages of TGEV infection; specifically, HSC70 bound M protein through its substrate-binding domain (SBD) and preincubation of TGEV with anti-M serum to block the interaction of M and HSC70 reduced the internalization of TGEV, thus demonstrating that the M-HSC70 interaction mediates the internalization of TGEV. Remarkably, the process of internalization was dependent on clathrin-mediated endocytosis (CME) in PK-15 cells. Furthermore, inhibition of the ATPase activity of HSC70 reduced the efficiency of CME. Collectively, our results indicated that HSC70 is a newly identified host factor involved in TGEV infection. Taken together, our findings clearly illustrate a novel role for TGEV M protein in the viral life cycle and present a unique strategy used by HSC70 to promote TGEV infection in which the interaction with M protein directs viral internalization. These studies provide new insights into the life cycle of coronaviruses. IMPORTANCE TGEV is the causative agent of porcine diarrhea, a viral disease that economically affects the pig industry in many countries. However, the molecular mechanisms underlying viral replication remain incompletely understood. Here, we provide evidence of a previously undescribed role of M protein in viral replication during early stages. We also identified HSC70 as a new host factor affecting TGEV infection. We demonstrate that the interaction between M and HSC70 directs TGEV internalization in a manner dependent on CME, thus revealing a novel mechanism for TGEV replication. We believe that this study may change our understanding of the first steps of infection of cells with coronavirus. This study should facilitate the development of anti-TGEV therapeutic agents by targeting the host factors and may provide a new strategy for the control of porcine diarrhea.

Innate Immune Evasion of Porcine Epidemic Diarrhea Virus through Degradation of the FBXW7 Protein via the Ubiquitin-Proteasome Pathway.

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. Different antagonistic strategies have been identified, and the mechanism by which PEDV infection impairs the production of interferon (IFN) and delays the activation of the IFN response to escape host innate immunity has been determined, but the pathogenic mechanisms of PEDV infection remain enigmatic. Our preliminary results revealed that endogenous F-box and WD repeat domain-containing 7 (FBXW7) protein, the substrate recognition component of the SCF-type E3 ubiquitin ligase, is downregulated in PEDV-infected Vero E6 cells, according to the results from an isobaric tags for relative and absolute quantification (iTRAQ) analysis. Overexpression of FBXW7 in target cells makes them more resistant to PEDV infection, whereas ablation of FBXW7 expression by small interfering RNA (siRNA) significantly promotes PEDV infection. In addition, FBXW7 was verified as an innate antiviral factor capable of enhancing the expression of RIG-I and TBK1, and it was found to induce interferon-stimulated genes (ISGs), which led to an elevated antiviral state of the host cells. Moreover, we revealed that PEDV nonstructural protein 2 (nsp2) interacts with FBXW7 and targets FBXW7 for degradation through the K48-linked ubiquitin-proteasome pathway. Consistent with the results proven in vitro, FBXW7 reduction was also confirmed in different intestinal tissues from PEDV-infected specific-pathogen-free (SPF) pigs. Taken together, the data indicated that PEDV has evolved with a distinct antagonistic strategy to circumvent the host antiviral response by targeting the ubiquitin-proteasome-mediated degradation of FBXW7. Our findings provide novel insights into PEDV infection and pathogenesis. IMPORTANCE To counteract the host antiviral defenses, most viruses, including coronaviruses, have evolved with diverse strategies to dampen host IFN-mediated antiviral response, by interfering with or evading specific host regulators at multiple steps of this response. In this study, a novel antagonistic strategy was revealed showing that PEDV infection could circumvent the host innate response by targeted degradation of endogenous FBXW7 in target cells, a process that was verified to be a positive modulator for the host innate immune system. Degradation of FBXW7 hampers host innate antiviral activation and facilitates PEDV replication. Our findings reveal a new mechanism exploited by PEDV to suppress the host antiviral response.

Genome Analysis of the G6P6 Genotype of Porcine Group C Rotavirus in China.

Swine enteric disease is the predominant cause of morbidity and mortality, and viral species involved in swine enteric disease include rotaviruses and coronaviruses, among others. Awareness of the circulating porcine rotavirus group C (PoRVC) in pig herds is critical to evaluate the potential impact of infection. At present, due to the lack of disease awareness and molecular diagnostic means, the research on RVC infection in China is not well-studied. In this study, diarrhea samples collected from pig farms were detected positive for RVC by PCR, and the full-length RVC was not previously reported for Chinese pig farms. This rotavirus strain was designated as RVC/Pig/CHN/JS02/2018/G6P6. A natural recombination event was observed with breakpoints at nucleotides (nt) 2509 to 2748 of the VP2 gene. Phylogenetic analysis based on nsp1 revealed that a new branch A10 formed. Collectively, our data suggest a potentially novel gene recombination event of RVC in the VP2 gene. These findings provide a new insight into the evolution of the rotavirus.

Porcine deltacoronavirus E protein induces interleukin-8 production via NF-κB and AP-1 activation.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and interleukin-6 (IL-6). Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present study, we found that interleukin-8 (IL-8) was upregulated by PDCoV infection. We then demonstrated that PDCoV E protein induced IL-8 production and that the TM domain and the C-terminal domain of the E protein were important for IL-8 production. Subsequently, we showed here that deleting the AP-1 and NF-κB binding motif in porcine IL-8 promoter abrogated its activation, suggesting that IL-8 expression was dependent on AP-1 and NF-κB. Furthermore, PDCoV E induced IL-8 production, which was also dependent on the NF-κB pathway through activating nuclear factor p65 phosphorylation and NF-κB inhibitor alpha (IκBα) protein phosphorylation, as well as inducing the nuclear translocation of p65, eventually resulting in the promotion of IL-8 production. PDCoV E also activated c-fos and c-jun, both of which are members of the AP-1 family. These findings provide new insights into the molecular mechanisms of PDCoV-induced IL-8 production and help us further understand the pathogenesis of PDCoV infection.

Screening and identification of host proteins interacting with PDCoV S1-CTD

The C-terminal domain (CTD) of porcine deltacoronavirus S1 subunit is the main region which induces the neutralizing antibody. S1-CTD was expressed by HEK-293T eukaryotic expression system and purified, and porcine ileal epithelium cells membrane proteins were extracted to investigate porcine host proteins that interact with it. Thirty-two suspected interacting host proteins were obtained by co-inmunprecipitation (Co-IP) and mass spectrometry. Eukaryotic expression plasmid of KIF1 binding protein (KIFBP) was constructed, and the interaction between KIFBP and S1-CTD was identified by Co-IP and laser confocal microscopy. All results proved that KIFBP interacted with S1-CTD and co-located in cytoplasm. Further research indicated that overexpression of KIFBP could effectively reduce the viral mRNA level and the viral titer in which the mRNA level decreased by about 70%, and the viral titer decreased by 101.6TCID50. In conclusion, a host protein KIFBP interacting with PDCoV S1-CTD was screened and identified in this study which provides a theoretical basis for understanding the pathogenesis of PDCoV.

A New Neutralization Epitope in the Spike Protein of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) infects piglets and causes serious diarrhea as well as vomiting, dehydration, and death. The trimeric S protein plays a crucial role in the induction of neutralizing antibodies, and many neutralizing monoclonal antibodies (mAbs) against PEDV S protein have been developed. However, these mAbs exclusively target the S1 protein. In this study, we obtained a neutralizing mAb, 5F7, against the S2 protein of PEDV, and this mAb could neutralize new variant genotype 2 PEDV strains (LNCT2), as well as a genotype 1 PEDV strain (CV777), in vitro. The core sequence of the epitope was found in amino acid sequence 1261 aa~1337 aa. These findings confirm that the S2 protein possessed neutralizing epitopes and provided knowledge to aid further research on this virus.

Stromal Antigen 2 Deficiency Induces Interferon Responses and Restricts Porcine Deltacoronavirus Infection.

Porcine deltacoronavirus (PDCoV) is a recently discovered enteropathogenic coronavirus and has caused significant economic impacts on the pork industry. Although studies have partly uncovered the molecular mechanism of PDCoV-host interaction, it requires further research. In this study, we explored the roles of Stromal Antigen 2 (STAG2) in PDCoV infection. We found that STAG2-deficient cells inhibited infection with vesicular stomatitis virus (VSV) and PDCoV, whereas restoration of STAG2 expression in STAG2-depleted (STAG2-/-) IPEC-J2 cells line restored PDCoV infection, suggesting that STAG2 is involved in the PDCoV replication. Furthermore, we found that STAG2 deficiency results in robust interferon (IFN) expression. Subsequently, we found that STAG2 deficiency results in the activation of JAK-STAT signaling and the expression of IFN stimulated gene (ISG), which establish an antiviral state. Taken together, the depletion of STAG2 activates the JAK-STAT signaling and induces the expression of ISG, thereby inhibiting PDCoV replication. Our study provides new insights and potential therapeutic targets for unraveling the mechanism of PDCoV replication.

Identification and integrated analysis of lncRNAs and miRNAs in IPEC-J2 cells provide novel insight into the regulation of the innate immune response by PDCoV infection.

BACKGROUND: Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), are pivotal regulators involved in the pathogenic mechanism of multiple coronaviruses. Porcine deltacoronavirus (PDCoV) has evolved multiple strategies to escape the innate immune response of host cells, but whether ncRNAs are involved in this process during PDCoV infection is still unknown. RESULTS: In this study, the expression profiles of miRNAs, lncRNAs and mRNAs in IPEC-J2 cells infected with PDCoV at 0, 12 and 24 hours postinfection (hpi) were identified through small RNA and RNA sequencing. The differentially expressed miRNAs (DEmiRNAs), lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were screened from the comparison group of IPEC-J2 cells at 0 and 12 hpi as well as the comparison group of IPEC-J2 cells at 12 and 24 hpi. The target genes of these DEncRNAs were predicted. The bioinformatics analysis of the target genes revealed multiple significantly enriched functions and pathways. Among them, the genes that were associated with innate immunity were specifically screened. The expression of innate immunity-related ncRNAs and mRNAs was validated by RT-qPCR. Competing endogenous RNA (ceRNA) regulatory networks among innate immunity-related ncRNAs and their target mRNAs were established. Moreover, we found that the replication of PDCoV was significantly inhibited by two innate immunity-related miRNAs, ssc-miR-30c-3p and ssc-miR-374b-3p, in IPEC-J2 cells. CONCLUSIONS: This study provides a data platform to conduct studies of the pathogenic mechanism of PDCoV from a new perspective and will be helpful for further elucidation of the functional role of ncRNAs involved in PDCoV escaping the innate immune response.

In-patient Expenditure Between 2012 and 2020 Concerning Patients With Liver Cirrhosis in Chongqing: A Hospital-Based Multicenter Retrospective Study.

Background: Liver cirrhosis is a major global health and economic challenge, placing a heavy economic burden on patients, families, and society. This study aimed to investigate medical expenditure trends in patients with liver cirrhosis and assess the drivers for such medical expenditure among patients with liver cirrhosis. Methods: Medical expenditure data concerning patients with liver cirrhosis was collected in six tertiary hospitals in Chongqing, China, from 2012 to 2020. Trends in medical expenses over time and trends according to subgroups were described, and medical expenditure compositions were analyzed. A multiple linear regression model was constructed to evaluate the factors influencing medical expenditure. All expenditure data were reported in Chinese Yuan (CNY), based on the 2020 value, and adjusted using the year-specific health care consumer price index for Chongqing. Results: Medical expenditure for 7,095 patients was assessed. The average medical expenditure per patient was 16,177 CNY. An upward trend in medical expenditure was observed in almost all patient subgroups. Drug expenses were the largest contributor to medical expenditure in 2020. A multiple linear regression model showed that insurance type, sex, age at diagnosis, marital status, length of stay, smoking status, drinking status, number of complications, autoimmune liver disease, and the age-adjusted Charlson comorbidity index score were significantly related to medical expenditure. Conclusion: Conservative estimates suggest that the medical expenditure of patients with liver cirrhosis increased significantly from 2012 to 2020. Therefore, it is necessary to formulate targeted measures to reduce the personal burden on patients with liver cirrhosis.

Porcine deltacoronavirus infection is inhibited by Griffithsin in cell culture.

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteric coronavirus that causes severe diarrhea in piglets and results in serious economic losses. There are no effective vaccines and antiviral drugs to prevent and treat PDCoV infection currently. Griffithsin (GRFT) is a lectin with potent antiviral activity against enveloped viruses because of its ability to specifically bind N-linked high-mannose oligosaccharides. GRFT has been reported to possess antiviral activity against severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and porcine epidemic diarrhea virus (PEDV). Here, we first confirmed the antiviral activity of GRFT against PDCoV in vitro. The infected cells (%) and virus titers were significantly decreased at concentration 1 µg/mL or above of GRFT. Time-course experiments revealed that GRFT inhibits PDCoV infection at the adsorption and penetration step. GRFT binding to PDCoV spike (S) protein on the surface wraps the virus and blocks its entry. The outstanding antiviral potency indicates that GRFT has the potential value as a candidate drug for the prevention and treatment of PDCoV infection.

Impact of COVID-19 Pandemic on Mechanical Reperfusion in ST-Segment-Elevation Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention: A Multicenter Retrospective Study From a Non-epicenter Region.

Objective: The COVID-19 pandemic placed heavy burdens on emergency care and posed severe challenges to ST-segment-elevation myocardial infarction (STEMI) treatment. This study aimed to investigate the impact of COVID-19 pandemic on mechanical reperfusion characteristics in STEMI undergoing primary percutaneous coronary intervention (PPCI) in a non-epicenter region. Methods: STEMI cases undergoing PPCI from January 23 to March 29 between 2019 and 2020 were retrospectively compared. PPCI parameters mainly included total ischemic time (TIT), the period from symptom onset to first medical contact (S-to-FMC), the period from FMC to wire (FMC-to-W) and the period from door to wire (D-to-W). Furthermore, the association of COVID-19 pandemic with delayed PPCI risk was further analyzed. Results: A total of 14 PPCI centers were included, with 100 and 220 STEMI cases undergoing PPCI in 2020 and 2019, respectively. As compared to 2019, significant prolongations occurred in reperfusion procedures (P < 0.001) including TIT (420 vs. 264 min), S-to-FMC (5 vs. 3 h), FMC-to-W (113 vs. 95 min) and D-to-W (83 vs. 65 min). Consistently, delayed reperfusion surged including TIT ≥ 12 h (22.0 vs.3.6%), FMC-to-W ≥ 120 min (34.0 vs. 6.8%) and D-to-W ≥ 90 min (19.0 vs. 4.1%). During the pandemic, the patients with FMC-to-W ≥ 120 min had longer durations in FMC to ECG completed (6 vs. 5 min, P = 0.007), FMC to DAPT (24 vs. 21 min, P = 0.001), catheter arrival to wire (54 vs. 43 min, P < 0.001) and D-to-W (91 vs. 78 min, P < 0.001). The pandemic was significantly associated with high risk of delayed PPCI (OR = 7.040, 95% CI 3.610-13.729, P < 0.001). Conclusions: Even in a non-epicenter region, the risk of delayed STEMI reperfusion significantly increased due to cumulative impact of multiple procedures prolongation.

Aminopeptidase N Expression, Not Interferon Responses, Determines the Intestinal Segmental Tropism of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.

Porcine epidemic diarrhea virus antagonizes host antiviralresponse by downregulating the expression of FBXW7

To investigate the interaction between FBXW7 and PEDV infection, the endogenous expression level of FBXW7was assessed after PEDV infection. The results showed that PEDV infection inhibited the expression of FBXW7 in Vero E6 cells. Then the effects of FBXW7 on PEDV replication was evaluated by western blot after transfection of pCAGGS-HA-FBXW7 to VeroE6 cells. The results showed that PEDV N proteins were significantly suppressed by FBXW7 overexpression, indicating theinhibitory effect of FBXW7 on PEDV replication. Further using fluorescent quantitative kit to detect the influence of overexpressingFBXW7 on interferon and interferon-stimulated genes under PEDV infection conditions, the results showed that compared with thepCAGGS-HA control group, The transcription level of IFN-beta, ISG15, ISG54 and ISG56 were significantly enhanced in host cellstransfected with pGAGGS-HA-FBXW7 (p0.05). Collectively, PEDV replication was facilitated by down regulating the expressionof FBXW7, which provides a novel strategy utilizing by PEDV to antagonize the host innate immune response.

Development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific SIgA in colostrum.

Porcine epidemic diarrhea virus (PEDV) causes very high mortality in newborn piglets. The mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. Antibodies derived from the secretory immunoglobulin A (SIgA) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. Therefore, the measurement of SIgA levels is an important index in evaluating PEDV infections and immune status. A simple and rapid method for the detection of PEDV-specific SIgA using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-SIgA secretory component (SC) mAb probe for the detection of anti-PEDV-specific SIgA in swine. On the strip, a gold-labeled anti-SIgA SC mAb was applied to a conjugate pad; purified PEDV particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. Results showed that the immunochromatographic test strip had high sensitivity and specificity. When compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect PEDV specific SIgA in colostrum samples. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. We found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of PEDV specific SIgA, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying PEDV.

Porcine Epidemic Diarrhea Virus nsp15 Antagonizes Interferon Signaling by RNA Degradation of TBK1 and IRF3.

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. The type I interferon (IFN-I or IFN α/ß) is a key mediator of innate antiviral response during virus infection. Different antagonistic strategies have been identified and determined as to how PEDV infection inhibits the host's IFN responses to escape the host innate immune pathway, but the pathogenic mechanisms of PEDV infection are not fully elucidated. Our preliminary results revealed that endogenous TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), the key components in the IFN signaling pathway were downregulated in PEDV infected IPEC-J2 cells by iTRAQ analysis. In this study, we screened nsp15 as the most important viral encoded protein involved in TBK1 and IRF3 reduction. Endoribonuclease (EndoU) activity has been well determined for coronavirus nsp15. Three residues (H226, H241, and K282) of PEDV nsp15 were identified as critical amino acids for PEDV EndoU but not D265, which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (SARS-CoV). Moreover, PEDV nsp15 can directly degrade the RNA levels of TBK1 and IRF3 dependent on its EndoU activity to suppress IFN production and constrain the induction of IFN stimulated genes (ISGs), by which PEDV antagonizes the host innate response to facilitate its replication. Collectively, these results have confirmed that PEDV nsp15 was capable of subverting the IFN response by the RNA degradation of TBK1 and IRF3.

Swine acute diarrhea syndrome coronavirus-induced apoptosis is caspase- and cyclophilin D- dependent.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly discovered enteric coronavirus, is the aetiological agent that causes severe clinical diarrhea and intestinal pathological damage in piglets. To understand the effect of SADS-CoV on host cells, we characterized the apoptotic pathways and elucidated mechanisms underlying the process of apoptotic cell death after SADS-CoV infection. SADS-CoV-infected cells showed evidence of apoptosis in vitro and in vivo. The use of a pan-caspase inhibitor resulted in the inhibition of SADS-CoV-induced apoptosis and reduction in SADS-CoV replication, suggestive of the association of a caspase-dependent pathway. Furthermore, SADS-CoV infection activated the initiators caspase-8 and -9 and upregulated FasL and Bid cleavage, demonstrating a crosstalk between the extrinsic and intrinsic pathways. However, the proapoptotic proteins Bax and Cytochrome c (Cyt c) relocalized to the mitochondria and cytoplasm, respectively, after infection by SADS-CoV. Moreover, Vero E6 and IPI-2I cells treated with cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPTP) opening, were completely protected from SADS-CoV-induced apoptosis and viral replication, suggesting the involvement of cyclophilin D (CypD) in these processes. Altogether, our results indicate that caspase-dependent FasL (extrinsic)- and mitochondria (intrinsic)- mediated apoptotic pathways play a central role in SADS-CoV-induced apoptosis that facilitates viral replication. In summary, these findings demonstrate mechanisms by which SADS-CoV induces apoptosis and improve our understanding of SADS-CoV pathogenesis.