Porcine Epidemic Diarrhea Virus Antagonizes Host IFN-λ-Mediated Responses by Tilting Transcription Factor STAT1 toward Acetylation over Phosphorylation To Block Its Activation.

Porcine epidemic diarrhea virus (PEDV) is the main etiologic agent causing acute swine epidemic diarrhea, leading to severe economic losses to the pig industry. PEDV has evolved to deploy complicated antagonistic strategies to escape from host antiviral innate immunity. Our previous study demonstrated that PEDV downregulates histone deacetylase 1 (HDAC1) expression by binding viral nucleocapsid (N) protein to the transcription factor Sp1, inducing enhanced protein acetylation. We hypothesized that PEDV inhibition of HDAC1 expression would enhance acetylation of the molecules critical in innate immune signaling. Signal transducer and activator of transcription 1 (STAT1) is a crucial transcription factor regulating expression of interferon (IFN)-stimulated genes (ISGs) and anti-PEDV immune responses, as shown by overexpression, chemical inhibition, and gene knockdown in IPEC-J2 cells. We further show that PEDV infection and its N protein overexpression, although they upregulated STAT1 transcription level, could significantly block poly(I·C) and IFN-λ3-induced STAT1 phosphorylation and nuclear localization. Western blotting revealed that PEDV and its N protein promote STAT1 acetylation via downregulation of HDAC1. Enhanced STAT1 acetylation due to HDAC1 inhibition by PEDV or MS-275 (an HDAC1 inhibitor) impaired STAT1 phosphorylation, indicating that STAT1 acetylation negatively regulated its activation. These results, together with our recent report on PEDV N-mediated inhibition of Sp1, clearly indicate that PEDV manipulates the Sp1-HDAC1-STAT1 signaling axis to inhibit transcription of OAS1 and ISG15 in favor of its replication. This novel immune evasion mechanism is realized by suppression of STAT1 activation through preferential modulation of STAT1 acetylation over phosphorylation as a result of HDAC1 expression inhibition. IMPORTANCE PEDV has developed sophisticated evasion mechanisms to escape host IFN signaling via its structural and nonstructural proteins. STAT1 is one of the key transcription factors in regulating expression of ISGs. We found that PEDV and its N protein inhibit STAT1 phosphorylation and nuclear localization via inducing STAT1 acetylation as a result of HDAC1 downregulation, which, in turn, dampens the host IFN signaling activation. Our study demonstrates a novel mechanism that PEDV evades host antiviral innate immunity through manipulating the reciprocal relationship of STAT1 acetylation and phosphorylation. This provides new insights into the pathogenetic mechanisms of PEDV and even other coronaviruses.

Coronaviruses Nsp5 Antagonizes Porcine Gasdermin D-Mediated Pyroptosis by Cleaving Pore-Forming p30 Fragment.

Coronaviruses (CoVs) are a family of RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans. Here, we use porcine epidemic diarrhea virus (PEDV) as a model of CoVs to illustrate the reciprocal regulation between CoV infection and pyroptosis. For the first time, we elucidate the molecular mechanism of porcine gasdermin D (pGSDMD)-mediated pyroptosis and demonstrate that amino acids R238, T239, and F240 within pGSDMD-p30 are critical for pyroptosis. Furthermore, 3C-like protease Nsp5 from SARS-CoV-2, MERS-CoV, PDCoV, and PEDV can cleave pGSDMD at the Q193-G194 junction to produce two fragments unable to trigger pyroptosis. The two cleaved fragments could not inhibit PEDV replication. In addition, Nsp5 from SARS-CoV-2 and MERS-CoV also cleave human GSDMD (hGSDMD). Therefore, we provide clear evidence that PEDV may utilize the Nsp5-GSDMD pathway to inhibit pyroptosis and, thus, facilitate viral replication during the initial period, suggesting an important strategy for the coronaviruses to sustain their infection. IMPORTANCE Recently, GSDMD has been reported as a key executioner for pyroptosis. This study first demonstrates the molecular mechanism of pGSDMD-mediated pyroptosis and that the pGSDMD-mediated pyroptosis protects host cells against PEDV infection. Notably, PEDV employs its Nsp5 to directly cleave pGSDMD in favor of its replication. We found that Nsp5 proteins from other coronaviruses, such as porcine deltacoronavirus, severe acute respiratory syndrome coronavirus 2, and Middle East respiratory syndrome coronavirus, also had the protease activity to cleave human and porcine GSDMD. Thus, we provide clear evidence that the coronaviruses might utilize Nsp5 to inhibit the host pyroptotic cell death and facilitate their replication during the initial period, an important strategy for their sustaining infection. We suppose that GSDMD is an appealing target for the design of anticoronavirus therapies.

DNAJA3 Interacts with PEDV S1 Protein and Inhibits Virus Replication by Affecting Virus Adsorption to Host Cells.

Porcine epidemic diarrhea virus (PEDV) infection causes huge economic losses to the pig industry worldwide. DNAJA3, a member of the Hsp40 family proteins, is known to play an important role in the replication of several viruses. However, it remains unknown if it interacts with PEDV. We found that DNAJA3 interacted with PEDV S1, initially with yeast two-hybrid screening and later with Co-IP, GST pull-down, and confocal imaging. Further experiments showed the functional relationship between DNAJA3 and PEDV in the infected IPEC-J2 cells. DNAJA3 overexpression significantly inhibited PEDV replication while its knockdown had the opposite effect, suggesting that it is a negative regulator of PEDV replication. In addition, DNAJA3 expression could be downregulated by PEDV infection possibly as the viral strategy to evade the suppressive role of DNAJA3. By gene silencing and overexpression, we were able to show that DNAJA3 inhibited PEDV adsorption to IPEC-J2 cells but did not affect virus invasion. In conclusion, our study provides clear evidence that DNAJA3 mediates PEDV adsorption to host cells and plays an antiviral role in IPEC-J2 cells.

Porcine Epidemic Diarrhea Virus Inhibits HDAC1 Expression To Facilitate Its Replication via Binding of Its Nucleocapsid Protein to Host Transcription Factor Sp1.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the HDAC1 promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to HDAC1 transcription. The putative nuclear localization sequence 261PKKNKSR267 contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for HDAC1 expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. IMPORTANCE The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.

Development and application of an indirect enzyme-linked immunosorbent assay using recombinant S1 for serological testing of porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) causes severe infectious diseases in all ages of swine and leads to serious economic losses. Serologic tests are widely accepted and used to detect anti-PEDV antibodies that could indicate PEDV infection or vaccination. In this study, PEDV recombinant S1 protein (rS1) was expressed with the Bac-to-Bac system and purified by nickel-affinity chromatography. An indirect enzyme-linked immunosorbent assay based on rS1 (rS1-ELISA) was then developed and optimized by checkerboard assays with serial dilutions of antigen and serum. Serum samples from 453 domestic pigs and 42 vaccinated pigs were analyzed by the indirect fluorescent antibody (IFA) test and rS1-ELISA. Taking IFA as a gold standard, rS1-ELISA produced a high sensitivity (90.7%) and specificity (94.6%) by a receiver operating characteristic (ROC) curve. In addition, ROC analysis also revealed that rS1-ELISA was consistent with IFA (area under the curve 0.9583 +or- 0.0082). This rS1-ELISA was then applied to antibody detection in inactivated PEDV vaccinated pigs. The antibody could be detected 2-4 weeks after the first inoculation. These results indicated that the rS1-ELISA established in this study provides a promising and reliable tool for serologic detection of anti-PEDV IgG antibodies in infected or vaccinated pigs.

Prokaryotic expression of porcine type III interferon and studying on its antiviral activity

Normally, type III interferon (IFN-lambda) was highly expressed in intestinal mucosa and other mucosal systems, with relatively stronger broad-spectrum antiviral ability and immune regulating ability. Porcine epidemic diarrhea virus (PEDV) and porcine deltacorona virus (PDCoV) were two major intestinal pathogens causing diarrhea in piglets, which hindered the swine industry severely. In this study, the recombinant plasmid containing PoIFN-lambda3 fragment was constructed. The recombinant plasmid was then transformed into E.coli Rosaetta cells for recombinant expression. Analyzed by SDS-PAGE and western blotting, the results showed that the recombinant PoIFN-lambda3 (27 ku) was successfully expressed in the inclusion bodies of E. coli cells. After denaturation, purification and renaturation, the active PoIFN-lambda3 recombinant protein was obtained and was used to treat IPEC-J2 cells. Challenged by recombinant PoIFN-lambda3, the immune-stimulating genes (ISGs), such as ISG15-, OAS1, Mx-1, IFIT1, IFITM1 and IFITM3 were all up-regulated significantly and reached to the peak (P 0.001) at 12 h post challenge. For PEDV and PDCoV infection in IPEC-J2, the virus replication was detected after pretreatment, simultaneous treatment and post-infection treatment of recombinant PoIFN-lambda3. Compared with the control, the viral copy numbers of both PEDV and PDCoV were decreased significantly (P 0.05) after treatment of recombinant PoIFN-lambda3. The above results indicated that the purified and renatured PoIFN-lambda3 recombinant protein could have good biological activity. The recombinant PoIFN-lambda3 can induce high expression of different ISGs in IPEC-J2, and can also inhibit PEDV and PDCoV replication in the host cells. In summary, our study provided a basis for preventing and treating viral diarrhea in piglets.

Higher immune response induced by vaccination in Houhai acupoint relates to the lymphatic drainage of the injection site.

Houhai acupoint (HA) is a site for acupuncture stimulation, located in the fossa between the anus and tail base in animals. To evaluate HA as a potential immunization site, the immune responses were compared when HA and the conventional site nape were vaccinated in rats. The results showed that injection of a porcine epidemic diarrhea virus (PEDV) vaccine in HA induced significantly higher IgG, IgG1, IgG2, splenocyte proliferation and mRNA expression of IL-2, IL-4 and IFN-γ than in the nape. To search for the underlying mechanisms, the draining lymph nodes for HA and the nape were investigated. When rats were injected in HA with Indian ink, 11 lymph nodes including caudal mesenteric lymph node and bilateral gluteal lymph nodes, posterior inguinal lymph nodes, lumbar lymph nodes, internal iliac lymph nodes and popliteal lymph nodes were visibly stained with the ink and injection of a model antigen ovalbumin (OVA) in HA resulted in detection of OVA by western blotting while in the same lymph nodes only a pair of lymph nodes (central brachial lymph nodes) were observed when Indian ink or OVA was injected in the nape. IL-2 mRNA expression was detected in all the lymph nodes when PEDV vaccine was injected. Therefore, the enhanced immune response elicited by vaccination in HA may be attributed to more lymphocytes activated.

Utility Evaluation of Porcine Enteroids as PDCoV Infection Model in vitro.

Porcine deltacoronavirus (PDCoV) is a novel emerging enteric coronavirus found in pigs. Intestinal enteroids, which partially recreate the structure and function of intestinal villi-crypts, have many physiological similarities to the intestinal tissues in vivo. Enteroids exhibit advantages in studying the interactions between intestines and enteric pathogens. To create a novel infection model for PDCoV, we developed an in vitro system to generate porcine intestinal enteroids from crypts of duodenum, jejunum, and ileum of pigs. Enterocytes, enteroendocrine cells, Paneth cells, stem cells, proliferating cells, and goblet cells were found in the differentiated enteroids. Replication of PDCoV was detected in the cultured enteroids by immunofluorescence and quantitative RT-PCR. Double immunofluorescence labeling demonstrated that PDCoV was present in Sox9-positive intestinal cells and Villin1-positive enterocytes. There were multiple cellular responses shown as changes of transcription of genes related to mucosal immunity, antiviral genes, and marker genes of stem cells and other cells in the enteroids infected with PDCoV. We conclude that the 2-D enteroids derived from porcine jejunum can be used as an in vitro multicellular model for the investigation of pathogenesis and host immune responses to porcine enteric pathogens, such as PDCoV.