Porcine IKKε is involved in the STING-induced type I IFN antiviral response of the cytosolic DNA signaling pathway.

The cyclic GMP-AMP synthase and stimulator of interferon (IFN) genes (cGAS-STING) pathway serves as a crucial component of innate immune defense and exerts immense antiviral activity by inducing the expression of type I IFNs. Currently, STING-activated production of type I IFNs has been thought to be mediated only by TANK-binding kinase 1 (TBK1). Here, we identified that porcine IKKε (pIKKε) is also directly involved in STING-induced type I IFN expression and antiviral response by using IKKε-/- porcine macrophages. Similar to pTBK1, pIKKε interacts directly with pSTING on the C-terminal tail. Furthermore, the TBK1-binding motif of pSTING C-terminal tail is essential for its interaction with pIKKε, and within the TBK1-binding motif, the leucine (L) 373 is also critical for the interaction. On the other hand, both kinase domain and scaffold dimerization domain of pIKKε participate in the interactions with pSTING. Consistently, the reconstitution of pIKKε and its mutants in IKKε-/- porcine macrophages corroborated that IKKε and its kinase domain and scaffold dimerization domain are all involved in the STING signaling and antiviral function. Thus, our findings deepen the understanding of porcine cGAS-STING pathway, which lays a foundation for effective antiviral therapeutics against porcine viral diseases.

The Porcine and Chicken Innate DNA Sensing cGAS-STING-IRF Signaling Axes Exhibit Differential Species Specificity.

The innate immune DNA sensing cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) signaling pathway plays a key role in host antiviral function. Although the cGAS-STING pathway has been extensively studied, the cGAS-STING signaling in livestock and poultry is not well understood, and whether the species specificity exists is still unknown. In this study, we found that porcine and chicken STING, but not cGAS, exhibit species differences in regulation of IFN; that is, porcine (p)STING mediates good induction of IFN in mammalian cells and low IFN induction in chicken DF-1 cells; on the contrary, chicken (ch)STING mediates IFN induction only in chicken cells but not in mammalian cells. Furthermore, it was found that the motifs pLxIS of pSTING and pLxVS of chSTING are responsible for the species disparity, with the IFN activity of pSTING and chSTING exchanged by swapping the two pLxI/VS motifs. The pLxI/VS motifs mediated the interactions of various STING with downstream IFN regulatory factors (IRFs), reflecting the species-specific pIRF3 and chIRF7. Next, the STING, IRFs, and STING-IRFs were reconstituted in porcine and chicken macrophages that were genetically knocked out for STING and/or IRFs by the CRISPR-Cas9 approach. The results showed that pSTING plus pIRF3 or chIRF7 are able to induce IFN; however, chSTING plus chIRF7 but not pIRF3 are able to induce IFN, suggesting that pIRF3 is specific and stringent, which underlies the inability of chSTING to induce IFN in mammalian cells. In summary, our findings reveal the differential species specificity in the cGAS-STING pathway and the underlying mechanisms, thus providing valuable insights on the cGAS-STING-IRF signaling axis for comparative immunology.

[Viral co-infections targeting the porcine respiratory system: Consequences and limits of the experimental systems]. / Les co-infections virales affectant le système respiratoire porcin : conséquences et limites des systèmes d'étude.

Coinfections affecting the porcine respiratory system have often been overlooked, in favor of mono-infections, even though they are significantly more common in the field. In pigs, the term 'porcine respiratory complex' is used to describe coinfections involving both viruses, such as, for example, the swine influenza type A virus (swIAV), the porcine respiratory and reproductive syndrome virus (PRRSV), and the porcine circovirus type 2 (PCV-2), as well as bacteria. Until recently, most studies were primarily focused on clinical aspects and paid little attention to the molecular consequences of coinfections. This narrative review addresses the consequences of coinfections in the porcine respiratory system involving viruses. When possible, interactions that can occur between viruses are briefly presented. Conversely, research involving bacteria, protozoa, and fungi has not been considered at all. Finally, the main limitations complicating the interpretation of results from coinfection/superinfection studies are considered, and prospects in this exciting field of health research are presented.

The Porcine Cyclic GMP-AMP Synthase-STING Pathway Exerts an Unusual Antiviral Function Independent of Interferon and Autophagy.

The innate immune DNA-sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon (IFN) gene (STING) pathway exerts strong antiviral activity through downstream IFN production; however, it has been recently recognized that an IFN-independent activity of STING also plays an important role in antiviral functions. Nevertheless, the IFN-independent antiviral activity of STING is not fully understood. Here, we showed that porcine STING (pSTING) played a critical role against herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) infections, and IFN-defective mutants, including pSTING pLxIS sub, S365A, and △CTT, all exhibited similar antiviral functions, compared to wild-type (WT) pSTING. Furthermore, all of these IFN-defective pSTING mutants possessed comparable autophagy activity, relative to WT pSTING, as expected. From pSTING WT, S365A, and △CTT, the residues responsible for autophagy, including L333A/R334A, Y167A/L170A, and Y245A/L248A, were mutated. Surprisingly, all of these autophagy-defective pSTING mutants still resisted the two viral infections, demonstrating that the pSTING antiviral function is independent of IFN as well as autophagy. On the other hand, all of the autophagy-defective pSTING mutants triggered cell apoptosis, which was associated with and participated in the antiviral functions. Additionally, pSTING lost its antiviral activity in TANK-binding kinase 1 (TBK1)-/- and IFN regulatory factor 3 (IRF3)-/- porcine macrophages, indicating the involvement of TBK1 and IRF3 in other STING activities such as apoptosis. Collectively, our results revealed that STING exerts both IFN- and autophagy-independent antiviral activity, and they also suggested that STING-triggered cell apoptosis resists viral infections. IMPORTANCE The IFN-independent antiviral function of the cGAS-STING pathway has attracted great attention in recent years; however, the nature of this IFN-independent antiviral function is unknown, although STING-induced autophagy has been shown to mediate the STING antiviral activity. First, we analyzed the antiviral activity through the porcine cGAS-pSTING pathway and established that pSTING signaling exerts an IFN-independent antiviral function. Second, we found that pSTING-induced IFN-independent autophagy and the antiviral activity of pSTING are independent of both IFN and autophagy. Finally, pSTING signaling activates cell apoptosis independently of IFN and autophagy, and the apoptosis is associated with antiviral activity. Our results suggest that pSTING-activated apoptosis at least partially mediates the antiviral activity or multiple pSTING-activated signals, including IFN production, nuclear factor κ light chain enhancer of activated B cells (NF-κB) expression, autophagy, and apoptosis, exert a redundant antiviral role. Thus, the work reveals a new layer of complexity in STING antiviral activity.

How the Innate Immune DNA Sensing cGAS-STING Pathway Is Involved in Apoptosis.

The cGAS-STING signaling axis can be activated by cytosolic DNA, including both non-self DNA and self DNA. This axis is used by the innate immune system to monitor invading pathogens and/or damage. Increasing evidence has suggested that the cGAS-STING pathway not only facilitates inflammatory responses and the production of type I interferons (IFN), but also activates other cellular processes, such as apoptosis. Recently, many studies have focused on analyzing the mechanisms of apoptosis induced by the cGAS-STING pathway and their consequences. This review gives a detailed account of the interplay between the cGAS-STING pathway and apoptosis. The cGAS-STING pathway can induce apoptosis through ER stress, NLRP3, NF-κB, IRF3, and IFN signals. Conversely, apoptosis can feed back to regulate the cGAS-STING pathway, suppressing it via the activation of caspases or promoting it via mitochondrial DNA (mtDNA) release. Apoptosis mediated by the cGAS-STING pathway plays crucial roles in balancing innate immune responses, resisting infections, and limiting tumor growth.

How Does cGAS Avoid Sensing Self-DNA under Normal Physiological Conditions?

cGAS is a cytosolic DNA sensor that activates innate immune responses by producing the second messenger 2'3'-cGAMP, which activates the adaptor STING. cGAS senses dsDNA in a length-dependent but sequence-independent manner, meaning it cannot discriminate self-DNA from foreign DNA. In normal physiological conditions, cellular DNA is sequestered in the nucleus by a nuclear envelope and in mitochondria by a mitochondrial membrane. When self-DNA leaks into the cytosol during cellular stress or mitosis, the cGAS can be exposed to self-DNA and activated. Recently, many studies have investigated how cGAS keeps inactive and avoids being aberrantly activated by self-DNA. Thus, this narrative review aims to summarize the mechanisms by which cGAS avoids sensing self-DNA under normal physiological conditions.

How the Innate Immune DNA Sensing cGAS-STING Pathway Is Involved in Autophagy.

The cGAS-STING pathway is a key component of the innate immune system and exerts crucial roles in the detection of cytosolic DNA and invading pathogens. Accumulating evidence suggests that the intrinsic cGAS-STING pathway not only facilitates the production of type I interferons (IFN-I) and inflammatory responses but also triggers autophagy. Autophagy is a homeostatic process that exerts multiple effects on innate immunity. However, systematic evidence linking the cGAS-STING pathway and autophagy is still lacking. Therefore, one goal of this review is to summarize the known mechanisms of autophagy induced by the cGAS-STING pathway and their consequences. The cGAS-STING pathway can trigger canonical autophagy through liquid-phase separation of the cGAS-DNA complex, interaction of cGAS and Beclin-1, and STING-triggered ER stress-mTOR signaling. Furthermore, both cGAS and STING can induce non-canonical autophagy via LC3-interacting regions and binding with LC3. Subsequently, autophagy induced by the cGAS-STING pathway plays crucial roles in balancing innate immune responses, maintaining intracellular environmental homeostasis, alleviating liver injury, and limiting tumor growth and transformation.

Coinfections and their molecular consequences in the porcine respiratory tract.

Understudied, coinfections are more frequent in pig farms than single infections. In pigs, the term "Porcine Respiratory Disease Complex" (PRDC) is often used to describe coinfections involving viruses such as swine Influenza A Virus (swIAV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine CircoVirus type 2 (PCV2) as well as bacteria like Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Bordetella bronchiseptica. The clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. In this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. Coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. We discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health.

First demonstration of the circulation of a pneumovirus in French pigs by detection of anti-swine orthopneumovirus nucleoprotein antibodies.

The presence of pneumoviruses in pigs is poorly documented. In this study, we used the published sequence of the nucleoprotein (N) of the recently identified Swine Orthopneumovirus (SOV) to express and purify SOV N as a recombinant protein in Escherichia coli. This protein was purified as nanorings and used to set up an enzyme-linked immunosorbent assay, which was used to analyse the presence of anti-pneumovirus N antibodies in swine sera. Sera collected from different pig farms in the West of France and from specific pathogen free piglets before colostrum uptake showed indirectly that a pneumovirus is circulating in pig populations with some variations between animals. Piglets before colostrum uptake were sero-negative for anti-pneumovirus antibodies while most of the other pigs showed positivity. Interestingly, in two farms presenting respiratory clinical signs and negative or under control for some common respiratory pathogens, pigs were detected positive for anti-pneumovirus antibodies. Globally, anti-pneumovirus N antibody concentrations were variable between and within farms. Further studies will aim to isolate the circulating virus and determine its potential pathogenicity. SOV could potentially become a new member of the porcine respiratory complex, important on its own or in association with other viral and bacterial micro-organisms.

High dosage of zinc modulates T-cells in a time-dependent manner within porcine gut-associated lymphatic tissue.

Zn serves as a powerful feed additive to reduce post-weaning diarrhoea in pigs. However, the mechanisms responsible for Zn-associated effects on the adaptive immune responses following feeding of a very high dosage of Zn remain elusive. In this study, we examined the T-cell response in gut-associated lymphatic tissues of seventy-two weaned piglets. Piglets received diets with 57 mg Zn/kg (low Zn concentration, LZn), 164 mg Zn/kg (medium Zn concentration, MZn) or 2425 mg Zn/kg (high Zn concentration, HZn) mg Zn/kg feed for 1, 2 or 4 weeks. We observed that feeding the HZn diet for 1 week increased the level of activated T-helper cells (CD4+ and CD8α dim) compared with feeding MZn and LZn (P<0·05). In addition, we observed higher transcript amounts of interferon γ and T-box 21 (TBET) in the HZn group compared with the MZn and LZn groups (P<0·05). A gene set enrichment analysis revealed an over-representation of genes associated with 'cytokine signalling in immune system'. Remarkably, feeding of a very high Zn dosage led to a switch in the immune response after 2 weeks. We detected higher relative cell counts of CD4+CD25high regulatory T-helper cells (P<0·05) and a higher expression of forkhead box P3 (FOXP3) transcripts (P<0·05). After 4 weeks of feeding a high-dosage Zn diet, the relative CD4+ T-cell count (P<0·05) and the relative CD8ß + T-cell count (P<0·1) were reduced compared with the MZn group. We hypothesise that after 1 week the cellular T-helper 1 response is switched on and after 2 weeks it is switched off, leading to decreased numbers of T-cells.

Mycoplasma hyopneumoniae does not affect the interferon-related anti-viral response but predisposes the pig to a higher level of inflammation following swine influenza virus infection.

In pigs, influenza A viruses and Mycoplasma hyopneumoniae (Mhp) are major contributors to the porcine respiratory disease complex. Pre-infection with Mhp was previously shown experimentally to exacerbate the clinical outcomes of H1N1 infection during the first week after virus inoculation. In order to better understand the interactions between these pathogens, we aimed to assess very early responses (at 5, 24 and 48 h) after H1N1 infection in pigs pre-infected or not with Mhp. Clinical signs and macroscopic lung lesions were similar in both infected groups at early times post-H1N1 infection; and Mhp pre-infection affected neither the influenza virus replication nor the IFN-induced antiviral responses in the lung. However, it predisposed the animals to a higher inflammatory response to H1N1 infection, as revealed by the massive infiltration of neutrophils and macrophages into the lungs and the increased production of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α). Thus, it seems it is this marked inflammatory state that would play a role in exacerbating the clinical signs subsequent to H1N1 infection.

Newborn pig trachea cell line cultured in air-liquid interface conditions allows a partial in vitro representation of the porcine upper airway tissue.

BACKGROUND: The domestic pig is an excellent animal model to study human microbial diseases due to its similarity to humans in terms of anatomy, physiology, and genetics. We assessed the suitability of an in vitro air-liquid interface (ALI) culture system for newborn pig trachea (NPTr) cells as a practical tool for analyzing the immune response of respiratory epithelial cells to aggressors. This cell line offers a wide microbial susceptibility spectrum to both viruses and bacteria. The purpose of our study was to evaluate and characterize diverse aspects of cell differentiation using different culture media. After the NPTr cells reached confluence, the apical medium was removed and the cells were fed by medium from the basal side. RESULTS: We assessed the cellular layer's capacity to polarize and differentiate in ALI conditions. Using immunofluorescence and electronic microscopy we evaluated the presence of goblet and ciliated cells, the epithelial junction organization, and the transepithelial electrical resistance. We found that the cellular layer develops a variable density of mucus producing cells and acquires a transepithelial resistance. We also identified increased development of cellular junctions over the culture period. Finally, we observed variable expression of transcripts associated to proteins such as keratin 8, mucins (MUC1, MUC2, and MUC4), occludin, and villin 1. CONCLUSIONS: The culture of NPTr cells in ALI conditions allows a partial in vitro representation of porcine upper airway tissue that could be used to investigate some aspects of host/respiratory pathogen interactions.

Innate immune response to a H3N2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices.

Viral respiratory diseases remain of major importance in swine breeding units. Swine influenza virus (SIV) is one of the main known contributors to infectious respiratory diseases. The innate immune response to swine influenza viruses has been assessed in many previous studies. However most of these studies were carried out in a single-cell population or directly in the live animal, in all its complexity. In the current study we report the use of a trachea epithelial cell line (newborn pig trachea cells - NPTr) in comparison with alveolar macrophages and lung slices for the characterization of innate immune response to an infection by a European SIV of the H3N2 subtype. The expression pattern of transcripts involved in the recognition of the virus, interferon type I and III responses, and the host-response regulation were assessed by quantitative PCR in response to infection. Some significant differences were observed between the three systems, notably in the expression of type III interferon mRNA. Then, results show a clear induction of JAK/STAT and MAPK signaling pathways in infected NPTr cells. Conversely, PI3K/Akt signaling pathways was not activated. The inhibition of the JAK/STAT pathway clearly reduced interferon type I and III responses and the induction of SOCS1 at the transcript level in infected NPTr cells. Similarly, the inhibition of MAPK pathway reduced viral replication and interferon response. All together, these results contribute to an increased understanding of the innate immune response to H3N2 SIV and may help identify strategies to effectively control SIV infection.

Systematic analysis of the codon usage patterns of African swine fever virus genome coding sequences reveals its host adaptation phenotype.

African swine fever (ASF) is a severe haemorrhagic disease caused by the African swine fever virus (ASFV), transmitted by ticks, resulting in high mortality among domestic pigs and wild boars. The global spread of ASFV poses significant economic threats to the swine industry. This study employs diverse analytical methods to explore ASFV's evolution and host adaptation, focusing on codon usage patterns and associated factors. Utilizing phylogenetic analysis methods including neighbour-joining and maximum-likelihood, 64 ASFV strains were categorized into four clades. Codon usage bias (CUB) is modest in ASFV coding sequences. This research identifies multiple factors - such as nucleotide composition, mutational pressures, natural selection and geographical diversity - contributing to the formation of CUB in ASFV. Analysis of relative synonymous codon usage reveals CUB variations within clades and among ASFVs and their hosts. Both Codon Adaptation Index and Similarity Index analyses confirm that ASFV strains are highly adapted to soft ticks (Ornithodoros moubata) but less so to domestic pigs, which could be a result of the long-term co-evolution of ASFV with ticks. This study sheds light on the factors influencing ASFV's codon usage and fitness dynamics, enriching our understanding of its evolution, adaptation and host interactions.

The pig as a model for investigating the role of neutrophil serine proteases in human inflammatory lung diseases.

The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084-34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837-1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.

Physical Treatments to Control Clostridium botulinum Hazards in Food.

Clostridium botulinum produces Botulinum neurotoxins (BoNTs), causing a rare but potentially deadly type of food poisoning called foodborne botulism. This review aims to provide information on the bacterium, spores, toxins, and botulisms, and describe the use of physical treatments (e.g., heating, pressure, irradiation, and other emerging technologies) to control this biological hazard in food. As the spores of this bacterium can resist various harsh environmental conditions, such as high temperatures, the thermal inactivation of 12-log of C. botulinum type A spores remains the standard for the commercial sterilization of food products. However, recent advancements in non-thermal physical treatments present an alternative to thermal sterilization with some limitations. Low- (<2 kGy) and medium (3-5 kGy)-dose ionizing irradiations are effective for a log reduction of vegetative cells and spores, respectively; however, very high doses (>10 kGy) are required to inactivate BoNTs. High-pressure processing (HPP), even at 1.5 GPa, does not inactivate the spores and requires heat combination to achieve its goal. Other emerging technologies have also shown some promise against vegetative cells and spores; however, their application to C. botulinum is very limited. Various factors related to bacteria (e.g., vegetative stage, growth conditions, injury status, type of bacteria, etc.) food matrix (e.g., compositions, state, pH, temperature, aw, etc.), and the method (e.g., power, energy, frequency, distance from the source to target, etc.) influence the efficacy of these treatments against C. botulinum. Moreover, the mode of action of different physical technologies is different, which provides an opportunity to combine different physical treatment methods in order to achieve additive and/or synergistic effects. This review is intended to guide the decision-makers, researchers, and educators in using physical treatments to control C. botulinum hazards.

The Chicken cGAS-STING Pathway Exerts Interferon-Independent Antiviral Function via Cell Apoptosis.

It has been recently recognized that the DNA sensing innate immune cGAS-STING pathway exerts an IFN-independent antiviral function; however, whether and how chicken STING (chSTING) exerts such an IFN-independent antiviral activity is still unknown. Here, we showed that chSTING exerts an antiviral activity in HEK293 cells and chicken cells, independent of IFN production. chSTING was able to trigger cell apoptosis and autophagy independently of IFN, and the apoptosis inhibitors, rather than autophagy inhibitors, could antagonize the antiviral function of chSTING, suggesting the involvement of apoptosis in IFN-independent antiviral function. In addition, chSTING lost its antiviral function in IRF7-knockout chicken macrophages, indicating that IRF7 is not only essential for the production of IFN, but also participates in the other activities of chSTING, such as the apoptosis. Collectively, our results showed that chSTING exerts an antiviral function independent of IFN, likely via apoptosis.

Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L.

African swine fever virus (ASFV) is a large double-stranded DNA virus that is highly infectious and seriously affects domestic pigs and wild boars. African swine fever (ASF) has caused huge economic losses to endemic countries and regions. At present, there is still a lack of effective vaccines and therapeutics. Therefore, rapid and accurate detection is essential for the prevention and control of ASF. The portable DNA endonuclease (Cas12a)-mediated lateral flow strip detection method (Cas12a-LFS) combined with recombinant polymerase amplification (RPA) has been gradually recognized as effective for virus detection including ASFV. In this study, based on the ASFV structural protein p17 gene (D117L), an RPA-Cas12a-LFS detection method was established. The detection method exhibits a sensitivity of up to two gene copies and has no cross-reaction with nine other swine viruses. Thus, the method is highly sensitive and specific. In 68 clinical samples, the coincidence rate of the p17 strip was 100%, compared to the traditional quantitative PCR (qPCR). In conclusion, we have developed a simple, rapid, sensitive, and specific ASFV visual detection method and demonstrated the potential of on-site detection of ASFV.

Multiple Porcine Innate Immune Signaling Pathways Are Involved in the Anti-PEDV Response.

Porcine epidemic diarrhea virus (PEDV) has caused great damage to the global pig industry. Innate immunity plays a significant role in resisting viral infection; however, the exact role of innate immunity in the anti-PEDV response has not been fully elucidated. In this study, we observed that various porcine innate immune signaling adaptors are involved in anti-PEDV (AJ1102-like strain) activity in transfected Vero cells. Among these, TRIF and MAVS showed the strongest anti-PEDV activity. The endogenous TRIF, MAVS, and STING were selected for further examination of anti-PEDV activity. Agonist stimulation experiments showed that TRIF, MAVS, and STING signaling all have obvious anti-PEDV activity. The siRNA knockdown assay showed that TRIF, MAVS, and STING are also all involved in anti-PEDV response, and their remarkable effects on PEDV replication were confirmed in TRIF-/-, MAVS-/- and STING-/- Vero cells via the CRISPR approach. For further verification, the anti-PEDV activity of TRIF, MAVS, and STING could be reproduced in porcine IPEC-DQ cells treated with siRNAs. In summary, this study reveals that multiple pattern-recognition receptor (PRR) signaling pathways of porcine innate immunity play an important role in the anti-PEDV infection, providing new and useful antiviral knowledge for prevention and control of PEDV spreading.