Porcine epidemic diarrhea virus (PEDV) is a re-emerging pathogen that causes severe economic losses in the pig industry. Commercial PEDV vaccines provide limited protection against PEDV virulent strains. Therefore, the development of novel vaccines and antiviral drugs is urgently required. In this study, we investigated the inhibitory effects of Salinomycin (SLM) against PEDV infection in vitro. First, the half-maximal cytotoxic concentration (CC50) and half-maximal inhibitory concentration (IC50) of SLM were measured by a cell counting kit 8 (CCK-8) and cytopathic effect (CPE). The results showed that the CC50 of SLM on Vero cells was 7.698 μmol·L-1, and the IC50 for PEDV was 0.998 μmol·L-1. SLM dose-dependently suppressed the PEDV-QY strain infection in vitro. In addition, SLM mainly acted on the internalization and replication stages of the PEDV-QY strain, and had no significant effect on viral inactivation, attachment, and release. Finally, SLM inhibited PEDV infection by suppressing PEDV-induced Wnt/β-catenin activation. Collectively, these results suggest that SLM exerts anti-PEDV effects in vitro and presents a potential as an anti-PEDV drug.

Porcine epidemic diarrhea virus (PEDV) infection in pigs is characterized by vomiting, dehydration, and diarrhoea. The structural proteins of PEDV play crucial roles in viral entry, release, assembly, outgrowth, and host immune regulation. Similar to other viruses, PEDV primarily relies on host cellular mechanisms for productive infection. However, the host factors associated with PEDV infection remain undefined. Therefore, an in-depth understanding of the pathogenic mechanisms of PEDV is essential for comprehending this disease. Zinc-containing finger CCHC-type protein 3 (ZCCHC3) is an antiviral factor known to interact with RIG-I and cGAS, inhibiting the replication of pseudorabies virus (PRV). In this study, we investigated the role of porcine ZCCHC3 in PEDV proliferation. We first demonstrated that the expression of ZCCHC3 in LLC-PK1 cells is downregulated upon PEDV infection. Overexpression of ZCCHC3 inhibited PEDV replication, whereas knockdown of ZCCHC3 increased viral titer and N protein levels. Further studies revealed that ZCCHC3 interacts and co-localizes with N proteins, and that ZCCHC3-mediated antiviral effects depend on its zinc finger protease activity. Taken together, these findings provide valuable insights into the role of ZCCHC3 in PEDV proliferation and enhance our understanding of host-virus interactions.

Coronaviruses have undergone evolutionary changes and mutations in response to the immune pressures exerted by vaccines and environmental factors, resulting in more severe consequences during breakthrough infections. Nevertheless, the specific correlation between the evolutionary mutations of coronaviruses and immune pressures remains ambiguous. Swine coronavirus-porcine epidemic diarrhea virus (PEDV)-has existed for decades. This study utilized in vivo preparation of polyclonal antibodies against the PEDV and identified critical neutralizing epitopes through serial in vitro passaging. Then, the recombinant mutated strains were successfully constructed. In vitro experiments confirmed the ability of the rA1273P strain to escape neutralization by polyclonal antibodies. Both in vitro cell studies and in vivo animal experiments revealed that the strain maintains virulence and pathogenicity while evading antibody pressure post-vaccination. The pathogenicity of the strain while evading immune pressure is comparable to wild-type strains. A comparison of the S protein gene between vaccine strains and clinical strains identified mutations in 1273 amino acid positions in clinical strains. In conclusion, this study identified a novel PEDV S protein neutralizing site under immune pressure through serial passaging, indicating that the 1,273th amino acid position is prone to mutation under prolonged antibody pressure, enhancing the virus's ability to escape hosts. This study offers new insights into the interpretation of coronavirus escape immune pressure and provides technical support for monitoring and predicting the variation and evolution of coronavirus.IMPORTANCECoronaviruses represent an ongoing public health threat because of high variability. Since 2010, the emergence of highly pathogenic porcine epidemic diarrhea virus (PEDV) strains has resulted in significant economic losses to the global pig industry. PEDV undergoes evolution and mutation under external immune pressure, rendering it an increasingly challenging target for prevention and control measures. Here, we prepared the polyclonal antibodies against PEDV and identified a novel neutralization epitope on the S protein (1,273th amino acids) through serial in vitro passaging. Furthermore, our findings indicate that the mutation of A1273P in the S protein did not alter the virulence of the PEDV but significantly enhanced its ability to escape and infect the host in vitro and in vivo. Finally, we found that the 1,273 amino acid position of the S gene has been mutated to varying degrees in clinical PEDV strains. This work provides a specific correlation between the evolutionary mutations of coronaviruses and immune pressures.

The nucleocapsid (N) protein is the most abundant viral protein during porcine epidemic diarrhea virus (PEDV) infection and exhibits strong immunogenicity, prompting the host immune system to produce large quantities of antibodies against it. However, N-specific antibodies are non-neutralizing, as the N protein is typically located inside viral particles. Understanding the roles of N-specific antibodies during PEDV infection is crucial for developing more effective vaccines. In this study, we demonstrated that a significant amount of PEDV-N protein is expressed on the surface of living cells during PEDV infection or transient N expression. Mechanistically, the PEDV-N protein may reach the cell surface via the exosome secretion pathway. Furthermore, anti-N antibodies can bind to the surface N protein and induce antibody-dependent natural killer (NK) cell-mediated cytotoxicity (ADCC), directly killing PEDV-infected cells. Although anti-N antibodies lack neutralizing effects against PEDV infection, they can target the N protein present on the surface of PEDV-infected cells to induce NK cell-mediated ADCC, thus protecting the host from viral infection. Our results indicate that the N protein holds significant potential as a candidate for designing vaccines against porcine enteric viruses.

Porcine epidemic diarrhea virus (PEDV) has caused significant losses in the pork industry, but the mechanism of PEDV infection is still unclear. On the basis of our RNA-Seq data and due to the potential role of sialic acid as a coreceptor, we investigated the function of sialic acid-binding Ig-like lectin 15 (Siglec-15) to determine its role as a receptor in PEDV infection. We found that Siglec-15 enhances PEDV infection by promoting viral adsorption to host cells. Coimmunoprecipitation and immunofluorescence assays revealed that Siglec-15 binds to the S1 subunit and M protein of PEDV. PEDV infectivity was significantly reduced in Siglec-15 knockout mice. In addition, we developed a monoclonal antibody targeting Siglec-15 that can effectively inhibit PEDV infection both in vitro and in vivo. Overall, our study suggests that Siglec-15 may be a receptor for PEDV infection, which is important for related mechanistic studies and reveals a novel target for anti-PEDV therapeutic development.

Porcine epidemic diarrhea virus (PEDV) infection leads to immunosuppression and clinical symptoms in piglets, including vomiting, watery diarrhea, dehydration, and even death. Mitophagy sustains mitochondrial energy homeostasis and quality through the removal of damaged mitochondria. However, PEDV disrupts mitochondrial homeostasis, which affects cellular energy supply and reproduction. Despite existing research, the mechanisms underlying PEDV pathogenesis and its interaction with the innate immune system remain largely unclear. Therefore, we aimed to clarify the mechanism of PEDV-induced mitophagy and its relationship with apoptosis and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway after PEDV infection. We infected Vero and IPEC-J2 cells with PEDV. Then, we evaluated mitochondrial morphology, structural proteins of PEDV, reactive oxygen species (ROS) levels, and mitochondrial membrane potential using transmission electron microscopy, confocal laser scanning microscopy, and flow cytometry. We identified mitophagy-related proteins through immunoprecipitation and western blotting. We examined the effects of mitophagy on PEDV proliferation and JAK1-STAT1 signaling via western blotting and indirect immunofluorescence. PEDV infection led to mitochondrial damage and the production of mitophagosome-like vesicles. Subsequently, the PEDV structural N protein initiated mitophagy through ubiquitinating mitofusin 2 (MNF2) via the PINK1/Parkin pathway. Moreover, mitophagy promoted PEDV replication. In the early stage of PEDV infection, PEDV infection inhibits apoptosis by promoting mitophagy. PEDV infection significantly decreased the expression of JAK1, STAT1, interferon regulatory factor 9, and phosphorylated STAT1, inhibiting nuclear translocation and promoting replication. Overall, PINK1/Parkin-mediated mitophagy regulated PEDV-induced apoptosis and JAK/STAT1 expression. These findings provide a scientific basis for elucidating the pathogenic and immune escape mechanisms of PEDV.

Porcine epidemic diarrhea virus (PEDV) causes vomiting, dehydration, and diarrhea in piglets, seriously threatening their survival and development and causing huge economic losses to the global pig industry. Current PEDV control relies on vaccines, however, the high mutation rate of PEDV limits vaccine effectiveness, highlighting the need for new antiviral drugs. This study investigated the pharmacological effects of ginsenoside Rb1 (GRb1) on PEDV using network pharmacology, as well as GO and KEGG analyses, to predict its role in modulating the MAPK/ERK pathway. GRb1 downregulated the MAPK/ERK pathway activated by PEDV infection and reduced levels of the apoptotic protein cleaved-caspase-3, thus inhibiting PEDV-induced apoptosis and demonstrating antiviral properties. Further screening showed that the PEDV S1 protein promotes AP-1 nuclear entry and upregulates the MAPK/ERK pathway to induce apoptosis, a process reversed by GRb1. Further in vivo studies revealed that GRb1 treatment significantly reduced viral load in piglet intestinal tissues and anal swabs. GRb1 also alleviated clinical symptoms and intestinal damage in infected piglets, improving their survival rate while also downregulating the levels of inflammatory factors (IL-1β, IL-6, IL-8, and TNF-α). This study is the first to demonstrate that GRb1 effectively inhibits PEDV, uncovering its potential mechanism of action and providing a promising new approach for antiviral treatment in veterinary medicine.

The cross-species infection of coronaviruses has resulted in several major epidemics since 2003. Therefore, it is of great importance to explore the host ranges of coronaviruses and their features among different hosts. In this study, the porcine epidemic diarrhea virus (PEDV), with swine as the only natural reservoir, was detected in rat fecal samples collected from pig farms. Further animal tests showed PEDV can cause systemic infections in neonate mice and rats. The brain, lung intestine and spleen were all targets for PEDV in rodents in contrast to the intestine being targeted in pigs. Morbidity and mortality vary via different infection routes. PEDV was also detectable in feces after infection, suggesting that the infected rodents were potential infectious sources. Moreover, the cerebric tropism of PEDV was verified in piglets, which had not been identified before. In conclusion, our findings demonstrate that PEDV can cross the species barrier to infect mice and rats through different routes. Although it is highly devastating to piglets, PEDV changes the target organs and turns to be milder when meeting with new hosts. Based on these findings, more attention should be paid to the cross-species infection of PEDV to avoid the emergence of another zoonosis.

Porcine epidemic diarrhea virus (PEDV) infection can lead to serious acute intestinal infectious disease, bringing huge economic losses to the pig industry. In addition to triggering an extremely high mortality rate for lactating piglets, there is currently a lack of effective treatments and vaccines. Therefore, rapid, accurate, sensitive, and specific detection of PEDV is critical for timely control. In this study, a nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established for the detection of PEDV and performed after optimizing the system (mainly for the design and screening of the LAMP primers and PfAgo gDNA). The optimized system had a detection limit as low as 2.4 copies/μL. To reach more timely on-site detection of PEDV and overcome the reliance on bulky and complex equipment, a lateral flow strip was introduced into the system, which could detect the target as low as 24 copies/μL. This RT-LAMP-PfAgo system took about 35 min to react, and the results could be observed and clarified with the naked eyes. Moreover, the method was highly specific and had no cross-reactivity with other swine pathogens. The detection results for the clinical samples were consistent with those obtained by the gold standard method, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), proving its applicability. In conclusion, the established RT-LAMP-PfAgo system can provide a new solution for the development of a portable, visual PEDV testing platform.

Porcine epidemic diarrhea (PED) is a severe gastrointestinal disease caused by the porcine epidemic diarrhea virus (PEDV), a virus that spreads through the intestinal tract, leading to significant economic losses in the global swine industry. Therefore, compared to traditional injection method, developing vaccines that effectively stimulate the mucosal immune system to induce a protective immune response is crucial for PED prevention. This study evaluated the immunogenicity of recombinant Lactococcus lactis (L. lactis) strains expressing the PEDV S1 and M proteins (MG1363/pMG36e-S1 and MG1363/pMG36e-M) via oral administration in BALB/c mice and neonatal piglets, assessing cellular, humoral, and mucosal immune responses in the host. The results demonstrated that the recombinant strains significantly stimulated lymphocyte proliferation in mice and increased the proportion of CD3+, CD4+, and CD3+, CD8+ double-positive cells in the spleens of mice and the peripheral blood of piglets (p < 0.05). Furthermore, the recombinant strains significantly increased serum IgG, IgA, and mucosal SIgA levels in piglets (p < 0.05). Meanwhile, serum cytokine levels, including IL-4 and IFN-γ, were significantly elevated in piglets when compared to the control group (p < 0.05). In conclusion, the recombinant L. lactis demonstrated promising potential as a novel live vector vaccine against PEDV.

Porcine epidemic diarrhea virus (PEDV) is a pathogenic coronavirus that targets the swine intestinal tract, leading to acute diarrhea and high mortality in neonatal piglets. PEDV is categorized into different genotypes based on genetic variations, especially in the spike (S) gene. The S protein is crucial for viral entry and a major immune target. Significant differences in virulence have been observed among PEDV genotypes, particularly between classical strains and newly emerging strains. In this study, we explored the impact of spike gene variability on PEDV pathogenicity. Using targeted RNA recombination, we generated recombinant PEDV (rPEDV) variants carrying spike genes from contemporary strains (moderately virulent strain UU and highly virulent strain GDU), all within the genetic background of the avirulent DR13 vaccine strain. Pathogenicity was assessed in 3-day-old piglets. The rPEDV carrying the DR13 spike gene was nonpathogenic, with no detectable viral RNA in feces. The rPEDV with the UU spike gene induced mild to severe diarrhea, with moderate viral shedding but no mortality. Conversely, the rPEDV with the GDU spike gene caused severe diarrhea, high viral titers, and high mortality. These findings highlight the critical role of the spike protein in PEDV virulence, informing future development of effective control strategies, including the design of live-attenuated vaccines.IMPORTANCEThis study significantly advances our understanding of how genetic variations in the spike (S) protein of porcine epidemic diarrhea virus (PEDV) influence its ability to cause disease. By engineering viruses with spike genes from different PEDV strains, variations in this protein could be directly linked to differences in disease severity. We found that the spike protein from highly virulent strains caused severe diarrhea and high mortality in piglets, while that from less virulent strains led to milder symptoms. These findings emphasize the central role of the spike protein in determining PEDV virulence, which may enable the design of more effective vaccines to combat PEDV and reduce its impact on the swine industry.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV). The economic impact of PEDV on the global pig industry has been significant, resulting in considerable losses. This paper presents a review of the latest research progress on PEDV genome, molecular epidemiology, vaccine development, and molecular detection methods. It was determined that the genetic diversity of the PEDV spike (S) gene was closely associated with the epidemiological trend of PEDV. The prevalence of S gene variants of different genotypes exhibited variability across regions and pig populations. Epidemiological analyses have demonstrated that PEDV can be transmitted via multiple routes, including direct contact, airborne aerosol, and water source contamination. With regard to vaccine research, the available vaccines can be classified into several categories, including live-attenuated vaccines, inactivated vaccines, subunit vaccines, bacterial vector vaccines, viral vector vaccines, mRNA vaccines, etc. Each of these has distinctive characteristics in terms of immunogenicity, protection efficiency, and safety. Molecular detection methods, including PCR-based methods, isothermal amplification techniques, immunological assays, and biosensors, play an important role in the diagnosis and monitoring of PEDV. Furthermore, this paper examines the current developments in PEDV research and identifies the key areas of future investigation. The objective of this paper is to establish a theoretical foundation for the prevention and control strategies of PED, and to provide a point of reference for further research on the genomics, epidemiology, vaccine development and detection methods of PEDV.

Porcine epidemic diarrhoea virus (PEDV) is a porcine enteric coronavirus, outbreaks and epidemics of which have caused huge economic losses to the livestock industry. The disadvantage of existing PEDV vaccines is that the unstable efficacy and high cost limit their widespread use. Therefore, there is an urgent need to develop a recombinant transgenic vaccine candidate for PEDV. In this study, three linear epitopes on the PEDV spike (S) were screened using peptide scanning. The screened epitopes were linked to targeting peptides for lung and intestinal epithelial cells, respectively, and displayed on the M13KE phage to form recombinant phage nanoparticles. Active immunisation experiments showed that a single B-cell epitope delivered by M13KE phage nanoparticles induced the production of specific neutralising antibodies against PEDV in mice. After PEDV stimulation, the immunised mice had significantly higher levels of interferon-γ (IFN-γ) than the control group. Simultaneously, PEDV stimulation caused lymphocyte activation and proliferation in the immunised mice, which is a typical immune response to viral infections. These results suggest that a single linear antigenic epitope delivered by M13KE phage nanoparticles induces significant humoral and cellular immune responses. The constructed recombinant phage nanoparticles are expected to be potential vaccine candidates for PEDV.

Porcine epidemic diarrhea virus (PEDV), particularly the highly virulent G2b strains, has inflicted substantial economic losses on the global swine industry. This study evaluated the prophylactic effects of three Bacillus strains-B. amyloliquefaciens LN, B. licheniformis CK, and B. velezensis AC-against PEDV infection using in vitro and in vivo models. In vitro experiments with Vero cells demonstrated that B. amyloliquefaciens LN increased cell viability, reduced PEDV-N expression, and modulated proinflammatory cytokine responses. In vivo, piglets supplemented with B. amyloliquefaciens LN exhibited alleviated diarrhea symptoms, suppression of fecal viral RNA shedding to below the detection limit, and restoration of gut microbiota balance by increasing Bacteroidetes and reducing Proteobacteria abundance. Mechanistic studies indicated that the measured interferon (IFN)-related genes were not significantly influenced in this study, suggesting that the protective effects of B. amyloliquefaciens LN may involve the modulation of inflammatory responses and the inhibition of viral replication through reduced PEDV-N expression. This study illustrates the potential of using B. amyloliquefaciens LN as a feed additive to prevent PEDV infection.

Porcine epidemic diarrhea (PED) is a significant pig disease causing high mortality in suckling pigs and high morbidity across all age groups. It is highly prevalent in Southeast Asia, posing a threat of transboundary transmission to India. Although antibodies were detected as early as 2003 in Assam, there was no evidence of viral detection or molecular characterization until this study. This study reports the first clinical outbreak of PED in India, followed by the detection and genetic characterization of PED virus (PEDV) during 2022-23.

The outbreak was characterized, and fecal samples (n = 21) were collected from affected pigs. These samples were screened for PEDV using RT-PCR, targeting the N, S, and M genes. Serosurveillance was conducted in eight districts, and serum samples (n = 339) were tested for PEDV antibodies using ELISA. Partial N, S, and M gene sequencing, followed by phylogenetic analysis using MEGA v11.0.13, was performed to identify the prevailing genotype and variations in the coding region.

This study identified the first clinical outbreak of PEDV in India, with morbidity rates of 55-57.1% and symptoms including yellow watery diarrhea, vomiting, and abdominal pain. PEDV was confirmed in 17 of 21 fecal samples by amplifying the N, S, and M genes. Serosurveys showed seropositivity in Mandya (2.8%), Bengaluru Rural (6.6%), and Kolar (21.6%), districts indicating PEDV circulation in the state of Karnataka, India. The phylogenetic analysis of the S and M genes placed our study sequences within the Genotype 2a (G2a) clade, aligning with other known G2a strains. In contrast, the phylogenetic tree of the N gene clustered our sequences within the Genotype 1a (G1a) clade suggesting potential recombination. The Indian PEDV strains clustered with strains of China, with unique amino acid substitutions in the S gene, particularly in the receptor binding region.

This study reports the first clinical outbreak of PED in India and identifies the circulating genotype of PEDV. The study emphasizes the need for large-scale surveillance studies to understand the disease's status. Understanding PEDV's genetic diversity and evolution is essential to develop area-specific vaccines to mitigate the disease impact on India's pig population.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea diseases in piglets, which has brought huge economic losses to the pig industry. As the dominant Lactobacillus species in the piglet intestine, the antiviral effect of Limosilactobacillus reuteri (L. reuteri) has been reported. Nine L. reuteri strains were isolated and identified from swine feces in this study. The CCK-8 assay examined the anti-PEDV potential of their cell-free supernatant (CFS). Among the nine L. reuteri isolates examined, LRC8 had a higher inhibition rate to PEDV than the other strains. Thus, the biological properties of the LRC8 strain, such as growth ability, acid production ability, acid and bile salt tolerance, and adhesion to IPEC-J2 cells, were evaluated. Besides, the anti-PEDV activity of LRC8-CFS (LRC8 metabolites, LRM) was assessed using plaque reduction assays, indirect immunofluorescence assays, RT-qPCR, and western blotting. The mRNA relative expression levels of inflammatory factors including IL-1β, IL-6, IL-8, MCP1, and TNF-α were determined by RT-qPCR. The results showed that the LRC8 strain grew well, was resistant to acid, tolerated bile salts, and adhered strongly to IPEC-J2 cells. In addition, treatment with its CFS (LRM) dramatically downregulated the mRNA expression levels of inflammatory cytokines, and in the Vero cell culture, prophylactic, therapeutic, competitive, and direct-inhibitory actions were seen against PEDV. Finally, we explored the anti-PEDV effects of the LRC8 strain in piglets and found that the LRC8 strain effectively relieved the clinical symptoms and intestinal damage of piglets infected by PEDV. To sum up, we found a L. reuteri strain with an anti-PEDV effect.

Porcine epidemic diarrhea virus (PEDV) is a major pathogen that causes serious economic losses to the swine industry. To aid PEDV clinical diagnosis and vaccine development, sensitive and precise serological methods are demanded for rapid detection of (neutralizing) antibodies. Aiming for the development of a novel virus-free hemagglutination inhibition (HI) assay, the N-terminal region of the PEDV S1 subunit, encompassing the sialic acid-binding motif, was first expressed as an Fc-fusion protein with a C-terminal Spy Tag (S10A-Spy). The S10A-Spy protein was then presented on SpyCatcher-mi3 nanoparticles, forming virus-like particles designated S10A-NPs. Electron microscopy and dynamic light scattering analysis confirmed its topology, and the hemagglutination assay showed that S10A-NPs can efficiently agglutinate red blood cells. The HI assay based on S10A-NPs was then validated with PEDV-positive and -negative samples. The results showed that the HI assay had high specificity for the detection of PEDV antibodies. Next, a total of 253 clinical serum samples were subjected to the HI testing along with virus neutralization (VN) assay. The area under the receiver operating characteristic curve with VN was 0.959, and the kappa value was 0.759. Statistical analysis of the results indicated that the HI titers of the samples tested exhibited high consistency with the VN titers. Taken together, a novel virus-free HI assay based on the multivalent display of a chimeric PEDV spike protein upon self-assembling nanoparticles was established, providing a new approach for PEDV serological diagnosis.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus that causes severe diarrhea and high mortality in neonatal piglets. Current control measures, such as inactivated and live-attenuated vaccines, have limitations in providing complete protection. In this study, we evaluate the immunogenicity and protective efficacy of a PEDV S protein subunit vaccine compared to a traditional inactivated vaccine. Piglets and Sows were immunized with either the subunit vaccine or an inactivated vaccine, and serum samples were collected to assess IgG and neutralizing antibody levels. Results demonstrated that the S protein subunit vaccine induced significantly higher IgG and neutralizing antibody levels in both piglets and sows compared to the inactivated vaccine. Piglets born to immunized sows were challenged with a virulent PEDV strain. Piglets from the subunit vaccine group exhibited lower viral shedding, reduced clinical symptoms, and minimal intestinal lesions. These findings suggest that the PEDV S protein subunit vaccine provides enhanced immunity and protection against PEDV, making it a promising candidate for preventing PEDV infections in swine.

Porcine epidemic diarrhea virus (PEDV) is a pathogen that causes a highly contagious intestinal disease in pigs, which causes significant economic losses to the pig industry worldwide. PCR is the most commonly used technique for PEDV diagnosis in practical clinics, however, reported works still suffer from shortcomings, for example, most of them cannot differentiate GI and GII subtypes, they suffer from low sensitivity, and some primer sequences are no longer able to match the mutant strains.

To address these issues, we conducted a comprehensive analysis by comparing the sequences of the PEDV S protein in the existing NCBI database with a recently isolated epidemic strain of PEDV, named SX0818-2022, of subtype GIIa from Shanxi, China. The conserved sequences of GI and GII subtypes were retrieved to design the primers and probe. Leveraging this information, we developed a TaqMan probe-based quantitative real-time PCR (qPCR) assay that is uniquely tailored to detect both PEDV GI and GII subtypes.

Additionally, this qPCR can identify PEDV GI and GII subtypes with high sensitivities of 90 copies/μL and 40 copies/μL, respectively (refers to the number of copies of the DNA target per microliter of template in the reaction system), much higher than the previously reported works and especially suitable for early diagnosis and prevention. Besides, excellent specificity and repeatability of the duplex qPCR were verified, thus supporting its potential applications in practical clinics.

Therefore, this work presents a promising tool for PEDV diagnosis, prevention, and control.

Porcine epidemic diarrhea virus (PEDV) continues to spread globally, causing clinical symptoms in piglets, including watery diarrhea, vomiting, and dehydration. Its exceptionally high morbidity and mortality rate contributes significantly to the economic losses of the swine industry. The continuous genetic mutations of PEDV have compromised the effectiveness of classical strain vaccines. Early detection and accurate diagnosis are therefore crucial for controlling its further spread. Developing a detection method that is user-friendly, highly sensitive, and efficient is crucial for disease control. In this study, a point-of-care rapid detection method for PEDV was successfully established using reverse transcription-recombinase-aided amplification (RT-RAA) technology. This method enables results to be obtained within 20 min of amplification at a constant temperature of 42 °C. It demonstrates high sensitivity, with a detection limit as low as 1 copy/μL, and shows strong specificity, with no cross-reactivity observed with seven other common swine pathogens. When applied to clinical samples, the results were 100% consistent with those obtained by RT-qPCR. This method is distinguished by its portable instrumentation and simple operation, making it particularly suitable for resource-constrained settings.

Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen that causes acute diarrhoea, dehydration and high mortality rates in suckling pigs. Tripartite motif 8 (TRIM8) has been shown to play multiple roles in the host's defence against viral infections. However, the functions of TRIM8 in regulating PEDV infection are still not well understood. In our study, we found a significant upregulation of TRIM8 following PEDV infection. We created TRIM8 knockout and overexpression cell lines and discovered that TRIM8 can inhibit PEDV replication within host cells. Co-immunoprecipitation assays revealed that TRIM8 directly interacts with the nucleocapsid protein (N) of PEDV, specifically within the coiled-coil structural domain of TRIM8. Furthermore, TRIM8 was shown to reduce the expression of the PEDV N protein in a dose-dependent manner. Mechanistically, TRIM8 inhibits the expression of PEDV N through K48-linked ubiquitin proteasome degradation. Transcriptomics analysis revealed that TRIM8 facilitates the expression of genes associated with several pathways, including the IL-17 signalling pathway, chemokine signalling pathway, and cytokine-cytokine receptor interaction. This suggests that TRIM8 plays a crucial role in boosting antiviral immune responses against PEDV infection. Our findings provide new insights into the functions and mechanisms of TRIM8 in regulating PEDV infection and highlight its potential as a molecular target for the prevention and control of this virus.

SADS-CoV, a recently identified Rhinolophus bat coronavirus HKU2-associated swine coronavirus, is a malignant pathogen that causes acute diarrhea, severe diarrhea, and weight loss in infected piglets. The virus was first detected in Guangdong Province, China, in 2017 and has since been observed in Jiangxi, Fujian, and Guangxi Provinces. In 2023, the virus was detected in Henan Province, in inland China. This virus can infect various cell lines, including human cell lines, showing significant potential for cross-species transmission and posing a possible zoonotic threat. However, the molecular biology of SADS-CoV remains largely unknown, and there are no commercially available therapeutics or vaccines to prevent SADS-CoV infection. In this review, an update on progress in SADS-CoV research is provided, with a focus on the history of outbreaks, the characteristics of the virus, its interactions with the host, and developments in therapeutics and vaccines.

The role of coronaviral nucleocapsid (N) protein in regulating viral translation remains poorly understood. Here, we showed that the N protein of porcine epidemic diarrhea virus (PEDV) enhances the translation of both virus-like genomic RNA (gRNA) and messenger RNA. Further characterization of the gRNA translation upregulation showed that the N-terminal domain (NTD) + Linker region plays a major role. The stem-loop 1 in the 5' untranslated region (UTR) and the budged stem loop in the 3'UTR are required for viral translation upregulation by PEDV N protein. The signaling kinase Akt exists in three isoforms. We found that Akt1 enhances viral gRNA translation upregulation by the N protein dependent on its kinase activity. We further showed an interaction between Akt1 and PEDV N, that is abolished by the NTD + Linker region. This suggested that the enhancing effect of Akt1 on translation upregulation by the N protein does not require interaction between these two proteins.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

Swine Enteric Coronaviruses (SECoVs), with high lethality and infectiousness, are the main pathogens causing fatal and watery diarrhea in piglets and spreading globally. Moreover, these SECoVs can cause similar clinical manifestations and are often co-infected, requiring an accurate assay suitable for rapid, in situ, and differential detection. Here, we developed a multiplexed fluorescent-based lateral flow immunoassay (mFB-LFIA) for the detection of three SECoVs, including porcine delta coronaviruses (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), in swine fecal samples. Thanks to the filter pad design and reasonable optimization, the mFB-LFIA was achieved within 15 min for three SECoVs detection simultaneously and improved the tolerance of the strips for feces samples. The limit of detection (LoD) of detecting PDCoV, TGEV, and PEDV were 2.1 × 104 TCID50 mL-1, 3.4 × 102 TCID50 mL-1, and 3.6 × 102 TCID50 mL-1, respectively. Additionally, the proposed assay was successfully applied to the detection of PDCoV, TGEV, and PEDV in swine feces with high accuracy. Compared with the gold standard nucleic acid testing, the total coincidence rate of the proposed assay was more than 90 %. Moreover, the mFB-LFIA performed excellent stability and repeatability. The proposed mFB-LFIA allows for rapid, in situ, more cost-effective and simultaneous detection of PDCoV, TGEV, and PEDV compared with nucleic acid testing. To the best of our knowledge, this is the first report to describe a multiplexed point-of-care assay capable of detecting PDCoV, TGEV, and PEDV in swine fecal samples. We believe our approach has a great potential for application to pig farm.

Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and "collateral" promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT - qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, induces severe vomiting and acute watery diarrhea in unweaned piglets. The pig industry has suffered tremendous financial losses due to the high mortality rate of piglets caused by PEDV. Consequently, a simple and rapid on-site diagnostic technology is crucial for preventing and controlling PEDV. This study established a detection method for PEDV using recombinase-aided amplification (RAA) and Pyrococcus furiosus Argonaute (PfAgo), which can detect 100 copies of PEDV without cross-reactivity with other pathogens. The entire reaction of RAA and PfAgo to detect PEDV does not require sophisticated instruments, and the reaction results can be observed with the naked eye. Overall, this integrated RAA-PfAgo cleavage assay is a practical tool for accurately and quickly detecting PEDV. KEY POINTS: • PfAgo has the potential to serve as a viable molecular diagnostic tool for the detection and diagnosis of viral genomes • The RAA-PfAgo detection technique has a remarkable level of sensitivity and specificity • The RAA-PfAgo detection system can identify PEDV without needing advanced equipment.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), has been frequently occurring in the southwestern region of China over the past few years, continuously affecting the development of the swine industry. However, the genetic diversity and prevalence of PEDV strains circulating in the swine population in southwestern China in recent years have not been well studied. To address this gap, a total of 478 clinical samples were collected from 125 pig farms experiencing piglet diarrhea in 18 cities in southwestern China. The detection results revealed that 227 out of 478 samples tested positive for PEDV nucleic acid, with a positivity rate of 47.49%. Complete S gene sequences of 28 PEDV strains were obtained and classified into four subgroups, G1-a subgroup (classical strain), G1-b subgroup (S-INDEL), and two G2 subgroups (G2-a and G2-b), accounting for 17.86% (5/28), 3.57% (1/28), 35.71% (10/28), 42.86% (12/28) of the total sequenced strains, respectively. The coexistence of multiple genotypes indicates the complex genetic background and prevalence of PEDV in southwest China. Amino acid comparisons of the S proteins showed that the 28 PEDV strains sequenced in the study showed different patterns of variation in the epitope domains compared to vaccine strains belonging to different genotypes and contained many unique amino acid mutations compared to the reference strains, which might lead to immune escape of PEDV. The complex epidemiology of PEDV with multiple subgroups co-circulating in Southwest China underscores the importance of selecting appropriate vaccine strains based on locally prevalent strains and the ongoing need for epidemiological surveillance of PEDV. The emergence of new variant strains also highlights the urgency of developing updated vaccines, and effective management practices remain crucial for controlling PED outbreaks in pig farms.

Piglet diarrhea poses significant economic losses to the pig industry, posing a worldwide challenge that urgently needs to be addressed in pig breeding practices. Porcine rotavirus (PoRV) is an important viral diarrhea pathogen in piglets, with a high incidence rate and a tendency to cause growth retardation. To enhance the sensitivity and specificity of PoRV detection, we sequenced the NSP3 gene of G5 and G9 genotypes of rotavirus A (RVA), enabling simultaneous detection of the two serotypes. Subsequently, we developed a rapid PoRV detection method using a combination of recombinase-aided amplification (RAA) and CRISPR/Cas12a. In this method, Cas12a binds to RAA amplification products, guided by CRISPR-derived RNA (crRNA), which activates its cleavage activity and releases fluorescence by cutting FAM-BHQ-labeled single-stranded DNA (ssDNA). In the optimized reaction system, the recombinant plasmid PoRV can achieve a highly sensitive reaction within 30 min at 37 °C, with a detection limit as low as 2.43 copies/μL, which is ten times higher in sensitivity compared to the qPCR method. Results from specificity testing indicate that no cross-reactivity was observed between the RAA-CRISPR/Cas12a analysis of PoRV and other viral pathogens, including PoRV G3, PoRV G4, porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV). In the clinical sample detection using the RAA-CRISPR/Cas12a method and qPCR, Cohen's Kappa value reached as high as 0.952. Furthermore, this approach eliminates the need for large-scale instrumentation, offering a visual result under an ultraviolet lamp through fluorescence signal output.

Major histocompatibility complex class I (MHC-I) plays crucial roles against viral infections not only by initiating CD8+ T cell immunity but also by modulating natural killer (NK) cell cytotoxicity. Understanding how viruses precisely regulate MHC-I to optimize their infection is important; however, the manipulation of MHC-I molecules by porcine epidemic diarrhea virus (PEDV) remains unclear. In this study, we demonstrate that PEDV infection promotes the transcription of NLRC5, a key transactivator of MHC-I, in several porcine cell lines and in vivo. Paradoxically, no increase in MHC-I expression is observed after PEDV infection both in vitro and in vivo. Mechanistic studies revealed that PEDV infection inhibits the translation of PEDV-elicited NLRC5 mRNA and the expression of downstream MHC-I proteins, without affecting the expression of physiological NLRC5 and MHC-I proteins. Through viral protein screening, we identified PEDV nonstructural protein 1 (nsp1) as the critical antagonist that inhibits NLRC5-mediated upregulation of MHC-I, and the nsp1's inhibitory effect on MHC-I requires the motif of 15 amino acids at its C-terminus. Notably, our results revealed that the cytotoxic ability of NK cells against PEDV-infected cells is similar to that against healthy cells. Collectively, our findings uncover an immune evasion mechanism by which PEDV-infected cells masquerade as healthy cells to evade NK and T cell immunity. This is achieved by targeting NLRC5, a key MHC-I transcriptional regulator, via nsp1.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that inflicts substantial financial losses on the swine industry. Major histocompatibility complex class I (MHC-I) is a critical factor influencing both CD8+ T cell and natural killer (NK) cell immunity. However, how PEDV manipulates MHC-I expression to optimize its infection process remains largely unknown. In this study, we demonstrate that PEDV's nonstructural protein 1 (nsp1) inhibits virus-mediated induction of MHC-I expression by directly targeting NLRC5, a key MHC-I transactivator. Intriguingly, nsp1 does not reduce physiological NLRC5 and MHC-I expression. This selective inhibition of virus-elicited NLRC5 mRNA translation allows PEDV-infected cells to masquerade as healthy cells, thereby evading CD8+ T cell and NK cell cytotoxicity. Our findings provide unique insights into the mechanisms by which PEDV evades CD8+ T cell and NK cell immunity.

Porcine epidemic diarrhea virus (PEDV) is a member of the genus α-coronavirus and causes severe diarrhea in piglets, leading to enormous economic losses in the pig industry. To understand the epidemic variation of PEDV strains in Yunnan province, three PEDV strains (YN2021, YNLP 2022, and YNBS 2022) and one commercially available attenuated vaccine strain (Attenuated AJ1102-R) that were previously isolated were sequenced and compared with the representative PEDV strains. NJ phylogenetic analysis showed that YN2021 strain and Attenuated CV777 strain were clustered into GI-b subtype, while YNLP 2022 and YNBS 2022 belong to GII-b subtype, accompanying ZJCZ4 and Attenuated AJ1102-R. RDP analysis revealed that YNLP 2022 was a genome recombination from both GII-b strain PEDV-7C and GII-a strain YN1, of which the recombination region is in the range nt4994-7605. YNBS 2022 strain was another recombination originated from GII-b subtype strain 17GXZC-1ORF3c and GII-a subtype strain PEDV-CHZ, of which the counterpart is in the range nt16399-22326. The Yunnan strain of PEDV was analyzed for the first time from the whole-genome perspective, and comprehensive analysis showed that the Yunnan strains have high genetic variation. This study may shed new light on the current PEDV infections in Yunnan and pave the way toward further control of PEDV infections.

Porcine epidemic diarrhea has emerged as a significant threat to the global swine industry. The shedding and exposure status of porcine epidemic diarrhea virus (PEDV) in intensive farms is not completely understood. In this study, a total of 56,598 clinical samples collected from 256 intensive pig farms in 20 provinces in China from 2022 to 2023, were evaluated for PEDV using quantitative real-time PCR. The overall PEDV prevalence was 11.78 % and 28.45 % at the sample and farm levels, respectively, which are relatively high in Northern China and the fourth and first quarter of the year. The PEDV-positive rates and viral loads in suckling piglet herds were higher than those in growing-finishing pigs and multiparous sows. Meanwhile, 15.61 % of pig pens, 9.51 % of corridors, 9.4 % of office areas, 9.23 % of production personnel, and 8.33 % of pig cart driver samples were positive for PEDV, indicating potential biosafety gaps in intensive pig farms. In addition, 93.41 % of inguinal lymph node tissue samples contained viral nucleic acids, revealing a possible persistent infection of PEDV in pig herds. Our study presents the first report of the large-scale detection of PEDV in intensive pig farms, which constitutes indirect evidence of virus circulation in pig herds. This study provides valuable data for preventing and controlling PEDV infection in the future.

Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), primarily affects the jejunum and ileum of pigs. Interferons, glycoproteins with high species specificity and potent antiviral activity, are crucial in defending against viral infections. Unlike other interferons, interferon-lambda (IFN-λ) mainly acts on mucosal epithelial cells and exhibits robust antiviral activity at mucosal surfaces. However, the high cost limits the use of naturally extracted interferons in farming. In this study, we expressed recombinant porcine interferon-lambda 1 (rpIFN-λ1) in eukaryotic cells, demonstrating effective antiviral activity against PEDV in Vero E6 and IPI-FX cells. In vivo, rpIFN-λ1 alleviated clinical symptoms and intestinal damage, enhanced antioxidant capacity, reduced inflammation, and significantly improved the survival rate of piglets following PEDV infection. Both in vitro and in vivo studies confirmed that rpIFN-λ1 upregulated interferon-stimulated genes (ISGs) via the JAK-STAT pathway, thereby exerting antiviral effects. In conclusion, rpIFN-λ1 significantly inhibited PEDV replication and alleviated clinical symptoms. The selectivity of rpIFN-λ1 for intestinal cells and its ability to reduce viral shedding suggest that this agent is a promising antiviral for enteric viruses such as PEDV. Our findings highlight rpIFN-λ1 as a cost-effective, efficient, and novel strategy for antiviral treatment of PEDV.

Porcine epidemic diarrhea (PED) is an acute, highly contagious, and infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV can affect pigs of all ages, with 50~100% mortality in neonatal piglets and substantial economic losses in the swine industry. In the present study, 347 fecal and intestinal samples were collected from seven regions in China during 2020-2022. A comprehensive molecular investigation of the spike (S) gene of PEDV strains was carried out, which included phylogenetic analysis of the obtained PEDV sequences. Epidemiological surveillance data indicate that the GIIc subgroup strains are widely distributed among pigs. A PEDV strain was successfully isolated from positive small intestine samples and identified through RT-PCR detection using specific N gene primers of PEDV, indirect immunofluorescence assay (IFA), TEM analysis, genome sequencing, and full-length S gene analysis, named PEDV/SC/2022. RDP and SimPlot analysis showed that the isolate originated from the recombination of PEDV/AH2012 and PEDV/AJ1102. In conclusion, our findings contribute to the current understanding of PEDV epidemiology and provide valuable information for the control of PED outbreaks in China.

Porcine epidemic diarrhea virus (PEDV) causes the third most important disease in the pig industry, after African swine fever and porcine reproductive and respiratory syndrome, and leads to illness or death of the entire litter, causing significant economic losses. In this study, three PEDV strains (HN-1, HN-2, and SC2023) were isolated from swine farms with suspected PEDV infections in Sichuan and Henan provinces. Phylogenetic analysis based on complete S gene sequences showed that all three strains belonged to the G2c subgroup. HN-1 adapted readily to cell culture, grew to a viral titer as high as 2 × 108 TCID50/mL in Vero cells, and caused the formation of large syncytia. We analyzed the amino acid sequence of the HN-1 isolate and found that its S1 subunit contained a three-amino-acid insertion (355KRL358). A seven-amino-acid-deletion (1377FEKVHVQ1383) in the S2 subunit resulted in the partial deletion of the endocytosis signal YxxΦ and the complete deletion of the endoplasmic reticulum retrieval signal (ERRS) KVHVQ in the cytoplasmic tail of the S protein. Consequently, HN-1 is predicted to be less pathogenic than its parent strain, an attribute that facilitates rapid cell-to-cell spread by enhancing syncytium formation. In addition, strain HN-1 was found to have the mutation 884-885SG→RR, which may favor adaptation to cell culture by providing new trypsin cleavage sites. These results suggest that HN-1 is a G2c subtype variant that adapts well to cell culture and can be used to study the adaptive mechanisms of PEDV and develop attenuated vaccines.

Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus that causes substantial economic loss to the global pig industry. The emergence of PEDV variants has increased the need for new vaccines, as commercial vaccines confer inferior protection against currently circulating strains. It is well established that the induction of mucosal immunity is crucial for PEDV vaccines to provide better protection against PEDV infection. In this study, we constructed a recombinant adenovirus expressing the core neutralization epitope (COE) of G2b PEDV based on human adenovirus serotype 5 (Ad5). We evaluated the effects of different administration routes and doses of vaccine immunogenicity in Balb/c mice. Both intramuscular (IM) and intranasal (IN) administration elicited significant humoral responses, including COE-specific IgG in serum and mucosal secretions, along with serum-neutralizing antibodies. Moreover, IN delivery was more potent than IM in stimulating IgA in serum and mucosal samples and in dampening the immune response to the Ad5 vector. The immune response was stronger after high versus low dose IM injection, whereas no significant difference was observed between high and low IN doses. In summary, our findings provide important insights for developing novel PEDV vaccines.IMPORTANCEPorcine epidemic diarrhea (PED) is a highly contagious disease that has severe economic implications for the pork industry. Developing an effective vaccine against PEDV remains a necessity. Here, we generated a recombinant adenovirus vaccine based on Ad5 to express the COE protein of PEDV (rAd5-PEDV-COE) and systematically evaluated the immunogenicity of the adenovirus-vectored vaccine using different administration routes (intramuscular and intranasal) and doses in a mouse model. Our results show that rAd5-PEDV-COE induced potent systemic humoral response regardless of the dose or immunization route. Notably, intranasal delivery was superior to induce peripheral and mucosal IgA antibodies compared with intramuscular injection. Our data provide valuable insights into designing novel PEDV vaccines.