An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

RBM14 inhibits the replication of porcine epidemic diarrhea virus by recruiting p62 to degrade nucleocapsid protein through the activation of autophagy and interferon pathway.

Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host's antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication.

Advances in Helicobacter pylori vaccine research: From candidate antigens to adjuvants-A review.

BACKGROUND: Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that infects approximately 50% of the world's population and has been strongly associated with chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoma, and gastric cancer. The elimination of H. pylori is currently considered one of the most effective strategies for the treatment of gastric-related diseases, so antibiotic therapy is the most commonly used regimen for the treatment of H. pylori infection. Although this therapy has some positive effects, antibiotic resistance has become another clinically prominent problem. Therefore, the development of a safe and efficient vaccine has become an important measure to prevent H. pylori infection. METHODS: PubMed and ClinicalTrials.gov were systematically searched from January 1980 to March 2023 with search terms-H. pylori vaccine, adjuvants, immunization, pathogenesis, and H. pylori eradication in the title and/or abstract of literature. A total of 5182 documents were obtained. Based on the principles of academic reliability, authority, nearly publicated, and excluded the similar documents, finally, 75 documents were selected, organized, and analyzed. RESULTS: Most of the candidate antigens used as H. pylori vaccines in these literatures are whole-cell antigens and virulence antigens such as UreB, VacA, CagA, and HspA, and the main types of vaccines for H. pylori are whole bacteria vaccines, vector vaccines, subunit vaccines, nucleic acid vaccines, epitope vaccines, etc. Some vaccines have shown good immune protection in animal trials; however, few vaccines show good in clinical trials. The only H. pylori vaccine passed phase 3 clinical trial is a recombinant subunit vaccine using Urease subunit B (UreB) as the vaccine antigen, and it shows good prophylactic effects. Meanwhile, the adjuvant system for vaccines against this bacterium has been developed considerably. In addition to the traditional mucosal adjuvants such as cholera toxin (CT) and E. coli heat labile enterotoxin (LT), there are also promising safer and more effective mucosal adjuvants. All these advances made safe and effective H. pylori vaccines come into service as early as possible. CONCLUSIONS: This review briefly summarized the advances of H. pylori vaccines from two aspects, candidates of antigens and adjuvants, to provide references for the development of vaccine against this bacterium. We also present our prospects of exosomal vaccines in H. pylori vaccine research, in the hope of inspiring future researchers.

Identification of Linear Epitopes in the C-Terminal Region of ASFV p72 Protein.

African swine fever, which is induced by the African swine fever virus (ASFV), poses a significant threat to the global pig industry due to its high lethality in domestic pigs and wild boars. Despite the severity of the disease, there is a lack of effective vaccines and drugs against the ASFV. The p72 protein, constituting 31 to 33% of the total virus particle mass, serves as the primary capsid protein of ASFV. It is a crucial antigen for the development of ASF subunit vaccines and serological diagnostic methods. In this investigation, 27 monoclonal antibodies (mAbs) were generated through mouse immunization with the truncated C-terminal p72 protein expressed by Escherichia coli. Among these, six mAbs exhibited binding to the p72 trimer, with their respective recognized epitopes identified as 542VTAHGINLIDKF553, 568GNAIKTP574, and 584FALKPREEY592. All three epitopes were situated within the interval sequences of functional units of the C-terminal jelly-roll barrel of p72. Notably, two epitopes, 568GNAIKTP574 and 584FALKPREEY592, were internal to the p72 trimer, while the epitope 542VTAHGINLIDKF553 was exposed on the surface of the trimer and consistently conserved across all ASFV genotypes. These findings enhance our comprehension of the antigenic function and structure of the p72 protein, facilitating the utilization of p72 in the development of diagnostic techniques for ASFV.

The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity.

African swine fever virus (ASFV) is a highly contagious and deadly virus that leads to high mortality rates in domestic swine populations. Although the envelope protein CD2v of ASFV has been implicated in immunomodulation, the molecular mechanisms underlying CD2v-mediated immunoregulation remain unclear. In this study, we generated a stable CD2v-expressing porcine macrophage (PAM-CD2v) line and investigated the CD2v-dependent transcriptomic landscape using RNA-seq. GO terms enrichment analysis and gene set enrichment analysis revealed that CD2v predominantly affected the organization and assembly process of the extracellular matrix. Wound healing and Transwell assays showed that CD2v inhibited swine macrophage migration. Further investigation revealed a significant decrease in the expression of transcription factor early growth response 1 (EGR1) through inhibiting the activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Notably, EGR1 knockout in swine macrophages restricted cell migration, whereas EGR1 overexpression in PAM-CD2v restored the ability of macrophage migration, suggesting that CD2v inhibits swine macrophage motility by downregulating EGR1 expression. Furthermore, we performed chromatin immunoprecipitation and sequencing for EGR1 and the histone mark H3K27 acetylation (H3K27ac), and we found that EGR1 co-localized with the activated histone modification H3K27ac neighboring the transcriptional start sites. Further analysis indicated that EGR1 and H3K27ac co-occupy the promoter regions of cell locomotion-related genes. Finally, by treating various derivatives of swine macrophages with lipopolysaccharides, we showed that depletion of EGR1 decreased the expression of inflammatory cytokines including TNFα, IL1α, IL1ß, IL6, and IL8, which play essential roles in inflammation and host immune response. Collectively, our results provide new insights into the immunomodulatory mechanism of ASFV CD2v.

PGAM5 degrades PDCoV N protein and activates type I interferon to antagonize viral replication.

IMPORTANCE: As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.

PEDV N protein capture protein translation element PABPC1 and eIF4F to promote viral replication.

Porcine epidemic diarrhea (PED) is an acute, highly infectious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which seriously endangers the healthy development of the pig industry. PEDV N protein is the most abundant viral structural protein, which can be combined with viral genomic RNA to form ribonucleoprotein complexes, thereby participating in the transcription and replication of the virus. However, how PEDV hijacks the host transcription translation system to promote viral proliferation remains unclear. In this study, we found that there is an interaction between PEDV N, polyadenylate-binding protein cytoplasmic 1 (PABPC1) and eukaryotic initiation factor 4F (eIF4F) proteins through coimmunoprecipitation, GST pulldown and fluorescence microscopy experiments. PABPC1 could bind to the poly(A) tail of the mRNA, and eIF4F could bind to the 5' end cap structure of the mRNA, so the interaction of PABPC1 and eIF4F could facilitate mRNA forming a circular shape to promote translation to the proteins. To further explore the effect of N protein capture protein translation element PABPC1 and eIF4F on PEDV replication, we overexpressed PABPC1, eIF4F (containing eIF4A, eIF4E and eIF4G) separately on Vero cells and LLC-PK1 cells, and we found that the PABPC1 and eIF4F protein could promote PEDV replication. Taken together, our data suggested that PEDV N protein promoted cyclization of viral mRNA carried by N protein through binding with PABPC1 and eIF4F proteins, thus promoting viral transcription and facilitating viral replication.

Expression of ASFV p17 in CHO cells and identification of one novel epitope using a monoclonal antibody.

As a highly pathogenic large DNA virus, African swine fever virus (ASFV) has huge particles and numerous encoded proteins. At present, few of the existing studies on ASFV proteins have investigated the function of p17. Specific antibodies against p17 to promote the development of prevention techniques against African swine fever (ASF) are urgently needed. Herein, we successfully expressed ASFV p17 in CHO cells using a suspension culture system and generated a monoclonal antibody (mAb) against p17. The mAb recognized a novel linear epitope (8LLSHNLSTREGIK20) and exhibited specific reactivity, which was conducive to the identification of ectopically expressed p17, the recombinant porcine reproductive and respiratory syndrome virus expressing p17, and the ASFV-SY18. The epitope was conservative among genotype I and genotype II ASFV strains. Overall, the mAb against p17 revealed efficient detection and promising application prospects, making it a useful tool for future vaccine research on ASF. Determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of ASFV antigen protein.

PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production.

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication.

Screening and identification of the dominant antigens of the African swine fever virus.

African swine fever is a highly lethal contagious disease of pigs for which there is no vaccine. Its causative agent African swine fever virus (ASFV) is a highly complex enveloped DNA virus encoding more than 150 open reading frames. The antigenicity of ASFV is still unclear at present. In this study, 35 proteins of ASFV were expressed by Escherichia coli, and ELISA was developed for the detection of antibodies against these proteins. p30, p54, and p22 were presented as the major antigens of ASFV, positively reacting with all five clinical ASFV-positive pig sera, and 10 pig sera experimentally infected by ASFV. Five proteins (pB475L, pC129R, pE199L, pE184L, and pK145R) reacted well with ASFV-positive sera. The p30 induced a rapid and strong antibody immune response during ASFV infection. These results will promote the development of subunit vaccines and serum diagnostic methods against ASFV.

N protein of PEDV plays chess game with host proteins by selective autophagy.

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. The protein degradation role of autophagy has been widely used to control viral infection at multiple levels. In the ongoing evolutionary arms race, viruses have developed various ways to hijack and subvert autophagy in favor of its replication. It is still unclear exactly how autophagy affects or inhibits viruses. In this study, we have found a novel host restriction factor, HNRNPA1, that could inhibit PEDV replication by degrading viral nucleocapsid (N) protein. The restriction factor activates the HNRNPA1-MARCHF8/MARCH8-CALCOCO2/NDP52-autophagosome pathway with the help of transcription factor EGR1 targeting the HNRNPA1 promoter. HNRNPA1 could also promote the expression of IFN to facilitate the host antiviral defense response for antagonizing PEDV infection through RIGI protein interaction. During viral replication, we found that PEDV can, in contrast, degrade the host antiviral proteins HNRNPA1 and others (FUBP3, HNRNPK, PTBP1, and TARDBP) through its N protein through the autophagy pathway. These results reveal the dual function of selective autophagy in PEDV N and host proteins, which could promote the ubiquitination of viral particles and host antiviral proteins and degradation both of the proteins to regulate the relationship between virus infection and host innate immunity.Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; Baf A1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PEDV: porcine epidemic diarrhea virus; siRNA: small interfering RNA; TCID50: 50% tissue culture infectious doses.

The rPRRSV-E2 strain exhibited a low level of potential risk for virulence reversion.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Classical Swine Fever Virus (CSFV) are two important pathogens, which cause serious impact on swine industry worldwide. In our previous research, rPRRSV-E2, the recombinant PRRSV expressing CSFV E2 protein, could provide sufficient protection against the lethal challenge of highly pathogenic PRRSV and CSFV, and could maintained genetically stable in vitro. Here, to evaluate the virulence reversion potential risk, rPRRSV-E2 had been continuously passaged in vivo, the stability of E2 expression and virulence of the passage viruses were analyzed. The results showed that no clinical symptoms or pathological changes could be found in the inoculated groups, and there were no significant differences of viraemia among the test groups. Sequencing and IFA analysis showed that the coding gene of exogenous CSFV E2 protein existed in the passaged viruses without any sequence mutations, deletions or insertions, and could expressed steadily. It could be concluded that the foreign CSFV E2 gene in the genome of rPRRSV-E2 could be maintained genetically stable in vivo, and rPRRSV-E2 strain had relatively low level of potential risk for virulence reversion.

PSMB1 Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Recruiting NBR1 To Degrade Nonstructural Protein 12 by Autophagy.

The nonstructural proteins (Nsps) of porcine reproductive and respiratory syndrome virus (PRRSV) play essential roles in virus replication-a multistep process that requires the participation of host factors. It is of great significance for the development of antiviral drugs to characterize the host proteins that interact with PRRSV Nsps and their functions in PRRSV replication. Here, we determined that proteasome subunit ß type 1 (PSMB1) interacted with viral Nsp12 to inhibit PRRSV replication in target and permissive cells. PSMB1 could be downregulated by PRRSV infection through interaction with the transcription factor EBF1. Proteasome and autophagy inhibitor assays showed that PSMB1 was regulated by the autophagic pathway to degrade Nsp12. Cotransfection of PSMB1 and Nsp12 increased the level of intracellular autophagy; both molecules were colocated in lysosomes. We also found that the selective autophagy cargo receptor protein NBR1 and E3 ubiquitin ligase STUB1 interacted with PSMB1 and Nsp12, respectively, in the autophagic degradation of Nsp12. Furthermore, the degradation of Nsp12 by PSMB1 was mainly dependent on the ubiquitination of Nsp12 at lysine site 130. Our results indicate for the first time that PSMB1 is an anti-PRRSV host protein that inhibits the replication of PRRSV by degradation of Nsp12 through the selective autophagy pathway. IMPORTANCE PRRS is a major threat to the global pig industry and urgently requires an effective and sustainable control strategy. PRRSV Nsps have important roles in viral RNA synthesis, proteinase activity, induction of replication-associated membrane rearrangements, replicative endoribonuclease activity, determination of virulence, and regulation of host immune response. Research associated with PRRSV Nsps can provide vital guidance to modify the PRRSV genome through reverse genetics in the development of vaccines and diagnostics. The function of Nsp12, which generally plays essential roles in virus replication, remains unclear. We demonstrated that PSMB1 interacted with and degraded Nsp12 through an autophagic pathway to inhibit PRRSV replication. Our data confirmed a novel antiviral function of PSMB1 and allowed us to elaborate on the roles of Nsp12 in PRRSV pathogenesis. These findings suggest a valid and highly conserved candidate target for the development of novel therapies and more effective vaccines and demonstrate the complex cross talk between selective autophagy and PRRSV infection.

PRPF19 Limits Porcine Epidemic Diarrhea Virus Replication through Targeting and Degrading Viral Capsid Protein.

Porcine epidemic diarrhea (PED) indicates the disease of the acute and highly contagious intestinal infection due to porcine epidemic diarrhea virus (PEDV), with the characteristics of watery diarrhea, vomiting, and dehydration. One of the reasons for diarrhea and death of piglets is PEDV, which leads to 100% mortality in neonatal piglets. Therefore, it is necessary to explore the interaction between virus and host to prevent and control PEDV. This study indicated that the host protein, pre-mRNA processing factor 19 (PRPF19), could be controlled by the signal transducer as well as activator of transcription 1 (STAT1). Thus, PEDV replication could be hindered through selective autophagy. Moreover, PRPF19 was found to recruit the E3 ubiquitin ligase MARCH8 to the N protein for ubiquitination. For the purpose of degradation, the ubiquitin N protein is acknowledged by the cargo receptor NDP52 and transported to autolysosomes, thus inhibiting virus proliferation. To conclude, a unique antiviral mechanism of PRPF19-mediated virus restriction was shown. Moreover, a view of the innate immune response and protein degradation against PEDV replication was provided in this study. IMPORTANCE The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV. This virus has caused devastating economic losses in the pork industry worldwide. Studying the relationship between virus and host antiviral factors is important to develop the new antiviral strategies. This study identified the pre-mRNA processing factor 19 (PRPF19) as a novel antiviral protein in PEDV replication and revealed its viral restriction mechanisms for the first time. PRPF19 recruited the E3 ubiquitin ligase MARCH8 to the PEDV N protein for ubiquitination, and the ubiquitin N protein was acknowledged by the cargo receptor NDP52 and transported to autolysosomes for degradation. Our findings provide new insights in host antiviral factors PRPF19 that regulate the selective autophagy protein degradation pathway to inhibit PEDV replication.

Identification and Characterization of Cell Lines HepG2, Hep3B217 and SNU387 as Models for Porcine Epidemic Diarrhea Coronavirus Infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

hnRNP K Degrades Viral Nucleocapsid Protein and Induces Type I IFN Production to Inhibit Porcine Epidemic Diarrhea Virus Replication.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteric coronavirus currently spreading in several nations and inflicting substantial financial damages on the swine industry. The currently available coronavirus vaccines do not provide adequate protection against the newly emerging viral strains. It is essential to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. This study shows that heterogeneous nuclear ribonucleoprotein K (hnRNP K), the host protein determined by the transcription factor KLF15, inhibits the replication of PEDV by degrading the nucleocapsid (N) protein of PEDV in accordance with selective autophagy. hnRNP K was found to be capable of recruiting the E3 ubiquitin ligase, MARCH8, aiming to ubiquitinate N protein. Then, it was found that the ubiquitinated N protein could be delivered into autolysosomes for degradation by the cargo receptor NDP52, thereby inhibiting PEDV proliferation. Moreover, based on the enhanced MyD88 expression, we found that hnRNP K activated the interferon 1 (IFN-1) signaling pathway. Overall, the data obtained revealed a new mechanism of hnRNP K-mediated virus restriction wherein hnRNP K suppressed PEDV replication by degradation of viral N protein using the autophagic degradation pathway and by induction of IFN-1 production based on upregulation of MyD88 expression. IMPORTANCE The spread of the highly virulent PEDV in many countries is still leading to several epidemic and endemic outbreaks. To elucidate effective antiviral mechanisms, it is important to study the relationship between host antiviral factors and the virus and to investigate the mechanisms underlying host immune response against PEDV infection. In the work, we detected hnRNP K as a new host restriction factor which can hinder PEDV replication through degrading the nucleocapsid protein based on E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. In addition, via the upregulation of MyD88 expression, hnRNP K could also activate the interferon (IFN) signaling pathway. This study describes a previously unknown antiviral function of hnRNP K and offers a new vision toward host antiviral factors that regulate innate immune response as well as a protein degradation pathway against PEDV infection.

KLF16 inhibits PEDV replication by activating the type I IFN signaling pathway.

KLF16, a member of KLFs (Krüppel-like factors), contributes to the progression of a variety of cancer types. There is, however, still uncertain regarding the role of KLF16 in viral replication and the signaling mechanism of type I IFN. It was discovered that KLF16 inhibited the replication of porcine epidemic diarrhea virus (PEDV) through the type I IFN signaling pathway. Besides, it can also be found that the expression of KLF16 was down-regulated after PEDV infection of LLC-PK1 cells. Furthermore, overexpression of KLF16 inhibited the replication of PEDV in Vero cells as well as LLC-PK1 cells, whereas the replication of PEDV was promoted by the knockdown of KLF16. KLF16 up-regulated the expression of interferons (IFNs) via the TRAF6-pTBK1-pIRF3 pathway with the aim of promoting the host antiviral innate immune response. In addition, the obtained findings proved that KLF16 plays a novel role in antiviral action, thereby offering novel possibilities for preventing and controlling PEDV.

ATG4B hinders porcine epidemic diarrhea virus replication through interacting with TRAF3 and activating type-I IFN signaling.

Autophagy-related 4B (ATG4B) is found to exert a vital function in viral replication, although the mechanism through which ATG4B activates type-I IFN signaling to hinder viral replication remains to be explained, so far. The current work revealed that ATG4B was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. In addition, ATG4B overexpression inhibited PEDV replication in both Vero cells and LLC-PK1 cells. On the contrary, ATG4B knockdown facilitated PEDV replication. Moreover, ATG4B was observed to hinder PEDV replication by activating type-I IFN signaling. Further detailed analysis revealed that the ATG4B protein targeted and upregulated the TRAF3 protein to induce IFN expression via the TRAF3-pTBK1-pIRF3 pathway. The above data revealed a novel mechanism underlying the ATG4B-mediated viral restriction, thereby providing novel possibilities for preventing and controlling PEDV.

Exploration of shared TF-miRNA‒mRNA and mRNA-RBP-pseudogene networks in type 2 diabetes mellitus and breast cancer.

Type 2 diabetes mellitus (T2DM) has been confirmed to be closely associated with breast cancer (BC). However, the shared mechanisms between these diseases remain unclear. By comparing different datasets, we identified shared differentially expressed (DE) RNAs in T2DM and BC, including 427 mRNAs and 6 miRNAs from the GEO(Gene Expression Omnibus) database. We used databases to predict interactions to construct two critical networks. The transcription factor (TF)-miRNA‒mRNA network contained 236 TFs, while the RNA binding protein (RBP)-pseudogene-mRNA network showed that the pseudogene S-phase kinase associated protein 1 pseudogene 1 (SKP1P1) might play a key role in regulating gene expression. The shared mRNAs between T2DM and BC were enriched in cytochrome (CYP) pathways, and further analysis of CPEB1 and COLEC12 expression in cell lines, single cells and other cancers showed that they were strongly correlated with the survival and prognosis of patients with BC. This result suggested that patients with T2DM presenting the downregulation of CPEB1 and COLEC12 might have a higher risk of developing BC. Overall, our work revealed that high expression of CYPs in patients with T2DM might be a susceptibility factor for BC and identified novel gene candidates and immune features that are promising targets for immunotherapy in patients with BC.

Porcine epidemic diarrhea virus strain FJzz1 infection induces type I/III IFNs production through RLRs and TLRs-mediated signaling.

Interferons (IFNs) including type I/III IFNs are the major components of the host innate immune response against porcine epidemic diarrhea virus (PEDV) infection, and several viral proteins have been identified to antagonize type I/III IFNs productions through diverse strategies. However, the modulation of PEDV infection upon the activation of the host's innate immune response has not been fully characterized. In this study, we observed that various IFN-stimulated genes (ISGs) were upregulated significantly in a time- and dose-dependent manner in LLC-PK1 cells infected with the PEDV G2 strain FJzz1. The transcriptions of IRF9 and STAT1 were increased markedly in the late stage of FJzz1 infection and the promotion of the phosphorylation and nuclear translocation of STAT1, implicating the activation of the JAK-STAT signaling pathway during FJzz1 infection. In addition, abundant type I/III IFNs were produced after FJzz1 infection. However, type I/III IFNs and ISGs decreased greatly in FJzz1-infected LLC-PK1 cells following the silencing of the RIG-I-like receptors (RLRs), including RIG-I and MDA5, and the Toll-like receptors (TLRs) adaptors, MyD88 and TRIF. Altogether, FJzz1 infection induces the production of type-I/III IFNs in LLC-PK1 cells, in which RLRs and TLRs signaling pathways are involved, followed by the activation of the JAK-STAT signaling cascade, triggering the production of numerous ISGs to exert antiviral effects of innate immunity.