In vitro suppression of porcine epidemic diarrhea virus by Panax notoginseng saponins: assessing antiviral potential.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Panax notoginseng saponins (PNS) are bioactive extracts derived from the P. notoginseng plant. In this study, we investigated the anti-PEDV effect of PNS by employing various methodologies to assess their impact on PEDV in Vero cells. Using a CCK-8 (Cell Counting Kit-8) assay, we found that PNS had no significant cytotoxicity below the concentration of 128 µg/mL in Vero cells. Using immunofluorescence assays (IFAs), an enzyme-linked immunosorbent assay (ELISA), and plaque formation assays, we observed a dose-dependent inhibition of PEDV infection by PNS within 24-48 hours postinfection. PNS exerts its anti-PEDV activity specifically at the genome replication stage, and mRNA-seq analysis demonstrated that treatment with PNS resulted in increased expression of various genes, including IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), CFH (complement factor H), IGSF10 (immunoglobulin superfamily member 10), ID2 (inhibitor of DNA binding 2), SPP1 (secreted phosphoprotein 1), PLCB4 (phospholipase C beta 4), and FABP4 (fatty acid binding protein 4), but it resulted in decreased expression of IL1A (interleukin 1 alpha), TNFRSF19 (TNF receptor superfamily member 19), CDH8 (cadherin 8), DDIT3 (DNA damage inducible transcript 3), GADD45A (growth arrest and DNA damage inducible alpha), PTPRG (protein tyrosine phosphatase receptor type G), PCK2 (phosphoenolpyruvate carboxykinase 2), and ADGRA2 (adhesion G protein-coupled receptor A2). This study provides insights into the potential mechanisms underlying the antiviral effects of PNS. Taken together, the results suggest that the PNS might effectively regulate the defense response to the virus and have potential to be used in antiviral therapies.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus in Xinjiang, China, from 2020 to 2022.

In recent years, the pig industry in Xinjiang, China, has been severely impacted by outbreaks of porcine epidemic diarrhea (PED), despite vaccination efforts. In this study, we investigated the genetic characteristics of currently prevalent porcine epidemic diarrhea virus (PEDV) strains in the region. We collected 548 samples from animals with suspected PED on large-scale pig farms in Xinjiang. Of these, 258 tested positive for PEDV by RT-PCR, yielding an overall positivity rate of 47.08%. S1 gene sequencing and phylogenetic analysis were conducted on 23 randomly selected RT-PCR-positive samples. Three endemic strains of PEDV (PEDV/CH/XU/2020, PEDV/CH/XK/2020, and PEDV/CH/XA/2020) were isolated, and their complete genome sequences were analyzed for evidence of genetic recombination. Sequence comparison of the S gene indicated significant variations in the S1 gene of the Xinjiang strains compared to the vaccine strains CV777, AJ1102, and LWL, with 90.2%-98.5% nucleotide sequence identity. Notably, both the N-terminal and C-terminal domains of the S protein showed significant variation. Genetic evolutionary analysis identified the GIIa subtype as the dominant genotype among the epidemic strains in Xinjiang. Recombination analysis revealed inter-subtype recombination events in the PEDV/CH/XK/2020 and XJ1904-34 strains. These findings highlight the extensive genetic variation in the predominant GIIa genotype of PEDV in Xinjiang, which does not match the genotype of the currently used vaccine strains. These data may guide further efforts toward the development of effective vaccines for the control of PED.

Identification of Three Novel Linear B-Cell Epitopes in Non-Structural Protein 3 of Porcine Epidemic Diarrhea Virus Using Monoclonal Antibodies.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic swine coronavirus that causes diarrhea and high mortality in piglets, resulting in significant economic losses within the global swine industry. Nonstructural protein 3 (Nsp3) is the largest in coronavirus, playing critical roles in viral replication, such as the processing of polyproteins and the formation of replication-transcription complexes (RTCs). In this study, three monoclonal antibodies (mAbs), 7G4, 5A3, and 2D7, targeting PEDV Nsp3 were successfully generated, and three distinct linear B-cell epitopes were identified within these mAbs by using Western blotting analysis with 24 truncations of Nsp3. The epitope against 7G4 was located on amino acids 31-TISQDLLDVE-40, the epitope against 5A3 was found on amino acids 141-LGIVDDPAMG-150, and the epitope against 2D7 was situated on amino acids 282-FYDAAMAIDG-291. Intriguingly, the epitope 31-TISQDLLDVE-40 recognized by the mAb 7G4 appears to be a critical B-cell linear epitope due to its high antigenic index and exposed location on the surface of Nsp3 protein. In addition, bioinformatics analysis unveiled that these three epitopes were highly conserved in most genotypes of PEDV. These findings present the first characterization of three novel linear B-cell epitopes in the Nsp3 protein of PEDV and provide potential tools of mAbs for identifying host proteins that may facilitate viral infection.

Development of Glycyrrhizinic Acid-Based Lipid Nanoparticle (LNP-GA) as An Adjuvant That Improves the Immune Response to Porcine Epidemic Diarrhea Virus Spike Recombinant Protein.

Porcine epidemic diarrhea virus (PEDV) has affected the pork industry worldwide and during outbreaks the mortality of piglets has reached 100%. Lipid nanocarriers are commonly used in the development of immunostimulatory particles due to their biocompatibility and slow-release delivery properties. In this study, we developed a lipid nanoparticle (LNP) complex based on glycyrrhizinic acid (GA) and tested its efficacy as an adjuvant in mice immunized with the recombinant N-terminal domain (NTD) of porcine epidemic diarrhea virus (PEDV) spike (S) protein (rNTD-S). The dispersion stability analysis (Z-potential -27.6 mV) confirmed the size and charge stability of the LNP-GA, demonstrating that the particles were homogeneously dispersed and strongly anionic, which favors nanoparticles binding with the rNTD-S protein, which showed a slightly positive charge (2.11 mV) by in silico analysis. TEM image of LNP-GA revealed nanostructures with a spherical-bilayer lipid vesicle (~100 nm). The immunogenicity of the LNP-GA-rNTD-S complex induced an efficient humoral response 14 days after the first immunization (p < 0.05) as well as an influence on the cellular immune response by decreasing serum TNF-α and IL-1ß concentrations, which was associated with an anti-inflammatory effect.

Coinfection and nonrandom recombination drive the evolution of swine enteric coronaviruses.

Coinfection with multiple viruses is a common phenomenon in clinical settings and is a crucial driver of viral evolution. Although numerous studies have demonstrated viral recombination arising from coinfections of different strains of a specific species, the role of coinfections of different species or genera during viral evolution is rarely investigated. Here, we analyzed coinfections of and recombination events between four different swine enteric coronaviruses that infect the jejunum and ileum in pigs, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), and a deltacoronavirus, porcine deltacoronavirus (PDCoV). Various coinfection patterns were observed in 4,468 fecal and intestinal tissue samples collected from pigs in a 4-year survey. PEDV/PDCoV was the most frequent coinfection. However, recombination analyses have only detected events involving PEDV/TGEV and SADS-CoV/TGEV, indicating that inter-species recombination among coronaviruses is most likely to occur within the same genus. We also analyzed recombination events within the newly identified genus Deltacoronavirus and found that sparrows have played a unique host role in the recombination history of the deltacoronaviruses. The emerging virus PDCoV, which can infect humans, has a different recombination history. In summary, our study demonstrates that swine enteric coronaviruses are a valuable model for investigating the relationship between viral coinfection and recombination, which provide new insights into both inter- and intraspecies recombination events among swine enteric coronaviruses, and extend our understanding of the relationship between coronavirus coinfection and recombination.

Unveiling the Role of Protein Kinase C θ in Porcine Epidemic Diarrhea Virus Replication: Insights from Genome-Wide CRISPR/Cas9 Library Screening.

Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.

A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.

An isothermal nucleic acid amplification-based enzymatic recombinase amplification method for dual detection of porcine epidemic diarrhea virus and porcine rotavirus A.

Viral diarrhea is the predominant digestive tract sickness in piglings, resulting in substantial profit losses in the porcine industry. Porcine rotavirus A (PoRVA) and porcine epidemic diarrhea virus (PEDV) are the main causes of grave gastroenteritis and massive dysentery, especially in piglets. PoRVA and PEDV have high transmissibility, exhibit similar clinical symptoms, and frequently co-occur. Therefore, to avoid financial losses, a quick, highly efficient, objective diagnostic test for the prevention and detection of these diseases is required. Enzymatic recombinase amplification (ERA) is a novel technology based on isothermal nucleic acid amplification. It demonstrates high sensitivity and excellent specificity, with a short processing time and easy operability, compared with other in vitro nucleic acid amplification technologies. In this study, a dual ERA method to detect and distinguish between PEDV and PoRVA nucleic acids was established. The method shows high sensitivity, as the detection limits were 101 copies/µL for both viruses. To test the usefulness of this method in clinical settings, we tested 64 swine clinical samples. Our results were 100% matched with those acquired using a commercially available kit. Therefore, we have successfully developed a dual diagnostic ERA nucleic acids method for detecting and distinguishing between PEDV and PoRVA.

Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.

Phylogenetic and Molecular Analysis of the Porcine Epidemic Diarrhea Virus in Mexico during the First Reported Outbreaks (2013-2017).

The characteristics of the whole PEDV genome that has circulated in Mexico from the first outbreak to the present are unknown. We chose samples obtained from 2013 to 2017 and sequenced them, which enabled us to identify the genetic variation and phylogeny in the virus during the first four years that it circulated in Mexico. A 99% identity was found among the analyzed pandemic strains; however, the 1% difference affected the structure of the S glycoprotein, which is essential for the binding of the virus to the cellular receptor. The S protein induces the most efficacious antibodies; hence, these changes in structure could be implicated in the clinical antecedents of the outbreaks. Antigenic changes could also help PEDV avoid neutralization, even in the presence of previous immunity. The characterization of the complete genome enabled the identification of three circulating strains that have a deletion in ORF1a, which is present in attenuated Asian vaccine strains. The phylogenetic analysis of the complete genome indicates that the first PEDV outbreaks in Mexico were caused by INDEL strains and pandemic strains related to USA strains; however, the possibility of the entry of European strains exists, which may have caused the 2015 and 2016 outbreaks.

PEDV-spike-protein-expressing mRNA vaccine protects piglets against PEDV challenge.

Porcine epidemic diarrhea virus (PEDV), a swine enteropathogenic coronavirus, causes severe diarrhea in neonatal piglets, which is associated with a high mortality rate. Thus, developing effective and safe vaccines remains a top priority for controlling PEDV infection. Here, we designed two lipid nanoparticle (LNP)-encapsulated mRNA (mRNA-LNP) vaccines encoding either the full-length PEDV spike (S) protein or a multiepitope chimeric spike (Sm) protein. We found that the S mRNA-LNP vaccine was superior to the Sm mRNA-LNP vaccine at inducing antibody and cellular immune responses in mice. Evaluation of the immunogenicity and efficacy of the S mRNA vaccine in piglets confirmed that it induced robust PEDV-specific humoral and cellular immune responses in vivo. Importantly, the S mRNA-LNP vaccine not only protected actively immunized piglets against PEDV but also equipped neonatal piglets with effective passive anti-PEDV immunity in the form of colostrum-derived antibodies after the immunization of sows. Our findings suggest that the PEDV-S mRNA-LNP vaccine is a promising candidate for combating PEDV infection.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) continues to harm the global swine industry. It is important to develop a highly effective vaccine to control PEDV infection. Here, we report a PEDV spike (S) mRNA vaccine that primes a potent antibody response and antigen-specific T-cell responses in immunized piglets. Active and passive immunization can protect piglets against PED following the virus challenge. This study highlights the efficiency of the PEDV-S mRNA vaccine and represents a viable approach for developing an efficient PEDV vaccine.

PEDV inhibits HNRNPA3 expression by miR-218-5p to enhance cellular lipid accumulation and promote viral replication.

Porcine epidemic diarrhea virus (PEDV) requires complete dependence on the metabolic system of the host cell to complete its life cycle. There is a strong link between efficient viral replication and cellular lipid synthesis. However, the mechanism by which PEDV interacts with host cells to hijack cellular lipid metabolism to promote its replication remains unclear. In this study, PEDV infection significantly enhanced the expression of lipid synthesis-related genes and increased cellular lipid accumulation. Furthermore, using liquid chromatography-tandem mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) as the interacting molecule of PEDV NSP9. We demonstrated that the expression of HNRNPA3 was downregulated by PEDV-induced miR-218-5p through targeting its 3' untranslated region. Interestingly, knocking down HNRNPA3 facilitated the PEDV replication by promoting cellular lipid synthesis. We next found that the knockdown of HNRNPA3 potentiated the transcriptional activity of sterol regulatory element-binding transcription factor 1 (SREBF1) through zinc finger protein 135 (ZNF135) as well as PI3K/AKT and JNK signaling pathways. In summary, we propose a model in which PEDV downregulates HNRNPA3 expression to promote the expression and activation of SREBF1 and increase cellular lipid accumulation, providing a novel mechanism by which PEDV interacts with the host to utilize cellular lipid metabolism to promote its replication.IMPORTANCEAs the major components and structural basis of the viral replication complexes of positive-stranded RNA viruses, lipids play an essential role in viral replication. However, how PEDV manipulates host cell lipid metabolism to promote viral replication by interacting with cell proteins remains poorly understood. Here, we found that SREBF1 promotes cellular lipid synthesis, which is essential for PEDV replication. Moreover, HNRNPA3 negatively regulates SREBF1 activation and specifically reduces lipid accumulation, ultimately inhibiting PEDV dsRNA synthesis. Our study provides new insight into the mechanisms by which PEDV hijacks cell lipid metabolism to benefit viral replication, which can offer a potential target for therapeutics against PEDV infection.

Pyrococcus furiosus Argonaute-mediated porcine epidemic diarrhea virus detection.

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 virus strain CH/HLJ/18 isolated in China: characterization and phylogenetic analysis.

BACKGROUND: Porcine epidemic diarrhea (PED) is an infectious disease of the digestive tract caused by the porcine epidemic diarrhea virus (PEDV), characterized by vomiting, severe diarrhea, and high mortality rates in piglets. In recent years, the distribution of this disease in China has remarkably increased, and its pathogenicity has also increased. PEDV has been identified as the main cause of viral diarrhea in piglets. This study aimed to understand the genetic evolution and diversity of PEDV to provide a theoretical basis for the development of new vaccines and the prevention and treatment of PED. METHODS: A PEDV strain was isolated from the small intestine of a diarrheal piglet using Vero cells. The virus was identified using reverse transcription-polymerase chain reaction (RT-PCR), indirect immunofluorescence assay (IFA), and transmission electron microscopy. The whole genome sequence was sequenced, phylogenetic analysis was conducted using MEGA (version 7.0), and recombination analysis was performed using RDP4 and SimPlot. The S protein amino acid sequence was aligned using Cluster X (version 2.0), and the S protein was modeled using SWISS-MODEL to compare differences in structure and antigenicity. Finally, the piglets were inoculated with PEDV to evaluate its pathogenicity in newborn piglets. RESULT: PEDV strain CH/HLJ/18 was isolated. CH/HLJ/18 shared 89.4-99.2% homology with 52 reference strains of PEDV belonging to the GII-a subgroup. It was a recombinant strain of PEDV BJ-2011-1 and PEDV CH_hubei_2016 with a breakpoint located in ORF1b. Unique amino acid deletions and mutations were observed in the CH/HLJ/18 S protein. The piglets then developed severe watery diarrhea and died within 7 d of inoculation with CH/HLJ/18, suggesting that CH/HLJ/18 was highly pathogenic to newborn piglets. CONCLUSION: A highly pathogenic recombinant PEDV GII-a strain, CH/HLJ/18, was identified in China, with unique deletion and mutation of amino acids in the S protein that may lead to changes in protein structure and antigenicity. These results will be crucial for understanding the prevalence and variation of PEDV and for preventing and controlling PED.

Pathological and immunohistochemical assessment of the impact of three different strains of swine enteric coronaviruses in the intestinal barrier.

Swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV) or transmissible gastroenteritis virus (TGEV), have risen concern for the porcine industry and research community due to the increase in their virulence, their potential recombination capacity and the emergence of new variants. This in vivo study aims to compare the impact of three different strains of swine enteric coronaviruses [(two G1b (S-INDEL) PEDV strains and a recombinant TGEV-PEDV or Swine enteric coronavirus (SeCoV)] in the intestine of 3-weeks-old infected piglets, focusing on the pathology and main components of the intestinal barrier, including the number of goblet cells, and the expression of IgA as well as FoxP3, a regulatory T cell marker. Severity of lesions was evidenced in the three infected groups and was highly correlated with the viral load in feces and the frequency of viral antigen-positive cells. Furthermore, higher cellular death together with an increase in the expression of the FoxP3 marker was detected in the duodenum and jejunum of infected animals at 3 days post-infection. Our results highlight a recruitment of FoxP3+ cells in the small intestine of infected animals which may represent a response to the tissue damage caused by viral replication and cell death. Further studies should be addressed to determine the potential role of these cells during swine enteric coronavirus infections.

Immunogenicity and protective efficacy of recombinant adenovirus expressing a novel genotype G2b PEDV spike protein in protecting newborn piglets against PEDV.

IMPORTANCE: Porcine epidemic diarrhea (PED) is a highly infectious and economically significant gastrointestinal disorder that affects pigs of all ages. Preventing and controlling PED is achieved by immunizing sows with vaccines, enabling passive piglet immunization via colostrum. The prevalence of G2b porcine epidemic diarrhea virus (PEDV) continues in China despite the use of commercial vaccines, raising questions regarding current vaccine efficacy and the need for novel vaccine development. Adenovirus serotype 5 (Ad5) has several advantages, including high transduction efficiency, a wide range of host cells, and the ability to infect cells at various stages. In this study, we expressed the immunogenic proteins of spike (S) using an Ad5 vector and generated a PED vaccine candidate by inducing significant humoral immunity. The rAd5-PEDV-S prevented PED-induced weight loss, diarrhea, and intestinal damage in piglets. This novel vaccine candidate strain possesses the potential for use in the pig breeding industry.

Drug repurposing screens identify Tubercidin as a potent antiviral agent against porcine nidovirus infections.

The emergence of new coronaviruses poses a significant threat to animal husbandry and human health. Porcine epidemic diarrhea virus (PEDV) is considered a re-emerging porcine enteric coronavirus, which causes fatal watery diarrhea in piglets. Currently, there are no effective drugs to combat PEDV. Drug repurposing screens have emerged as an attractive strategy to accelerate antiviral drug discovery and development. Here, we screened 206 natural products for antiviral activity using live PEDV infection in Vero cells and identified ten candidate antiviral agents. Among them, Tubercidin, a nucleoside analog derived from Streptomyces tubercidicus, showed promising antiviral activity against PEDV infection. Furthermore, we demonstrated that Tubercidin exhibited significant antiviral activity against both classical and variant PEDV. Time of addition assay showed that Tubercidin displayed a significant inhibitory effect on viral post-entry events but not during other periods. Molecular docking analysis indicated that Tubercidin had better docking efficiency and formed hydrophobic interactions with the active pocket of RNA-dependent RNA polymerase (RdRp) of PEDV and other nidoviruses. Additionally, Tubercidin can effectively suppress other porcine nidoviruses, such as SADS-CoV and PRRSV, demonstrating its broad-spectrum antiviral properties. In summary, our findings provide valuable evidence for the antiviral activity of Tubercidin and offer insights into the development of new strategies for the prevention and treatment of coronavirus infections.

Feeding with 4,4'-diaponeurosporene-producing Bacillus subtilis enhances the lactogenic immunity of sow.

Specific antibodies produced sow by oral porcine epidemic diarrhea virus (PEDV) vaccines would transfer to newborn piglets via colostrum, and it is an effective strategy to prevent porcine epidemic diarrhea (PED). However, there is a lag in the development of corresponding vaccines due to the rapid mutation of PEDV, which could increase the difficulty of PED prevention and control in pig farms. Hence, congenital lactogenic immunity was assessed by feeding 4,4'-diaponeurosporene-producing Bacillus subtilis (B.S-Dia) to sow on the 80th day of gestation in order to protect newborn piglets from PEDV infection. Firstly, we found that the quantities of T lymphocytes and monocytes in the blood and colostrum after oral administration of B.S-Dia were significantly increased as observed by flow cytometry, whereas the proliferative activity of T lymphocytes in colostrum was also markedly increased. Furthermore, enzyme-linked immunosorbent assay (ELISA) results revealed that levels of TGF (Transforming growth factor) -ß, Interleukin (IL) -6, lysozyme and lactoferrin were significantly increased. Finally, it was found in the piglets' challenge protection test that offspring pigs of the sows feeding B.S-Dia during pregnancy did not develop diarrhea symptoms and intestinal pathological changes at 48 h after infection with PEDV, and PEDV load in the jejunum and ileum was significantly reduced, but offspring pigs of the sows taking orally PBS during pregnancy developed pronounced diarrhea symptoms and extensive PEDV colonization was noted both in the jejunum and ileum. In summary, sow by oral administration of B.S-Dia substantially increased congenital lactogenic immunity, thereby preventing newborn piglets from being infected with PEDV.

[Effects of host proteins interacting with non-structural protein nsp9 of porcine epidemic diarrhea virus on viral replication].

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic virus that can cause acute intestinal infectious diseases in both piglets and fattening pigs. The virus encodes at least 16 non-structural proteins, including nsp9, which has been shown to bind to single-stranded RNA. However, its function and mechanism remain unclear. In this study, we aimed to identify potential host proteins that interact with PEDV nsp9 using immunoprecipitation combined with mass spectrometry. The interactions were then confirmed by co-immunoprecipitation (Co-IP) and confocal laser scanning fluorescence techniques. The results showed that nsp9 interacts with HSPA8, Tollip, HSPA9 and TOMM70. Among them, overexpression of HSPA8 resulted in caused first upregulated and then down-regulated expression of nsp9, and promoted the proliferation of PEDV. Overexpression of Tollip significantly upregulated the expression of nsp9 and inhibited the proliferation of PEDV. Overexpression of TOMM70 significantly reduced the expression of nsp9, but did not show significant effect on the proliferation of PEDV. Overexpression of HSPA9 did not show significant effect on the expression of nsp9 and the proliferation of PEDV. These findings may facilitate further investigating the role of nsp9-interacting proteins in PEDV infection.

The impact and complete genome characterisation of viruses involved in outbreaks of gastroenteritis in a farrow-to-finish holding.

Viral enteric pathogens continuously burden intensive pig farming, causing gastrointestinal diseases of epidemic and endemic nature. The present study investigated two diarrhoea outbreaks on a large farrow-to-finish holding and subsequent circulation of outbreak-related enteric viruses. These viruses were characterised by whole genome sequencing, and statistical evaluation of the impact on specific production metrics was performed. The results provided evidence that the Porcine epidemic diarrhoea virus-swine enteric coronavirus (PEDV-SeCoV) S gene recombinant strain was responsible for the first outbreak, whilst Rotavirus A (RVA) in a mixed infection with Rotavirus B (RVB) and porcine kobuvirus (PKV) probably caused the second diarrhoea outbreak. Whole genome characterisation revealed a porcine origin of all viruses involved and significant heterogeneity of RVB strain, proposing four novel genotypes and changes in RVB VP1 genotype classification. The statistical evaluation confirmed only a minor disturbance in the number of weaned pigs per sow, with statistical forecasting showing positive trends. A follow-up study corroborated the endemicity of RVA and PKV, in contrast to PEDV-SeCoV. Punctual, comprehensive and timely investigation of diarrhoea outbreaks is a prerequisite for applying adequate pig health and biosecurity management. Calculating such outbreaks' impact on production metrics can potentially shape future decisions on management improvements.