High-affinity monoclonal antibodies against the porcine epidemic diarrhea virus S1 protein.

The porcine epidemic diarrhea virus (PEDV) infection inflicted substantial economic losses upon the global pig-breeding industry. This pathogen can infect all pigs and poses a particularly high fatality risk for suckling piglets. The S1 subunit of spike protein is a crucial target protein for inducing the particularly neutralizing antibodies that can intercept the virus-host interaction and neutralize virus infectivity. In the present study, the HEK293F eukaryotic expression system was successfully utilized to express and produce recombinant S1 protein. Through quantitative analysis, five monoclonal antibodies (mAbs) specifically targeting the recombinant S1 protein of PEDV were developed and subsequently evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry assay (FCA). The results indicate that all five mAbs belong to the IgG1 isotype, and their half-maximal effective concentration (EC50) values measured at 84.77, 7.42, 0.89, 14.64, and 7.86 pM. All these five mAbs can be utilized in ELISA, FCA, and IFA for the detection of PEDV infection. MAb 5-F9 exhibits the highest sensitivity to detect as low as 0.3125 ng/mL of recombinant PEDV-S1 protein in ELISA, while only 0.096 ng/mL of mAb 5-F9 is required to detect PEDV in FCA. The results from antigen epitope analysis indicated that mAb 8-G2 is the sole antibody capable of recognizing linear epitopes. In conclusion, this study has yielded a highly immunogenic S1 protein and five high-affinity mAbs specifically targeting the S1 protein. These findings have significant implications for early detection of PEDV infection and provide a solid foundation for further investigation into studying virus-host interactions.

Lactobacillus rhamnosus GG powder supplementation alleviates intestinal injury in piglets challenged by porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) has become a challenging problem in pig industry worldwide, causing significant profit losses. Lactobacillus rhamnosus GG (LGG) has been regarded as a safe probiotic strain and has been shown to exert protective effects on the intestinal dysfunction caused by PEDV. This study evaluated the effect of LGG on the gut health of lactating piglets challenged with PEDV. Fifteen piglets at 7 days of age were equally assigned into 3 groups (5 piglets per group): 1) control group (basal diet); 2) PEDV group: (basal diet + PEDV challenged); 3) LGG + PEDV group (basal diet + 3×109 CFU/pig/day LGG + PEDV). The trial lasted 11 days including 3 days of adaptation. The treatment with LGG was from D4 to D10. PEDV challenge was carried out on D8. PEDV infection disrupted the cell structure, undermined the integrity of the intestinal tract, and induced oxidative stress, and intestinal damage of piglets. Supplementation of LGG improved intestinal morphology, enhanced intestinal antioxidant capacity, and alleviated jejunal mucosal inflammation and lipid metabolism disorders in PEDV-infected piglets, which may be regulated by LGG by altering the expression of TNF signaling pathway, PPAR signaling pathway, and fat digestion and absorption pathway.

Evolution and virulence of porcine epidemic diarrhea virus following in vitro and in vivo propagation.

Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

Maternal immunization and vitamin A sufficiency impact sow primary adaptive immunity and passive protection to nursing piglets against porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV) causes a highly contagious enteric disease with major economic losses to swine production worldwide. Due to the immaturity of the neonatal piglet immune system and given the high virulence of PEDV, improving passive lactogenic immunity is the best approach to protect suckling piglets against the lethal infection. We tested whether oral vitamin A (VA) supplementation and PEDV exposure of gestating and lactating VA-deficient (VAD) sows would enhance their primary immune responses and boost passive lactogenic protection against the PEDV challenge of their piglets. We demonstrated that PEDV inoculation of pregnant VAD sows in the third trimester provided higher levels of lactogenic protection of piglets as demonstrated by >87% survival rates of their litters compared with <10% in mock litters and that VA supplementation to VAD sows further improved the piglets' survival rates to >98%. We observed significantly elevated PEDV IgA and IgG antibody (Ab) titers and Ab-secreting cells (ASCs) in VA-sufficient (VAS)+PEDV and VAD+VA+PEDV sows, with the latter maintaining higher Ab titers in blood prior to parturition and in blood and milk throughout lactation. The litters of VAD+VA+PEDV sows also had the highest serum PEDV-neutralizing Ab titers at piglet post-challenge days (PCD) 0 and 7, coinciding with higher PEDV IgA ASCs and Ab titers in the blood and milk of their sows, suggesting an immunomodulatory role of VA in sows. Thus, sows that delivered sufficient lactogenic immunity to their piglets provided the highest passive protection against the PEDV challenge. Maternal immunization during pregnancy (± VA) and VA sufficiency enhanced the sow primary immune responses, expression of gut-mammary gland trafficking molecules, and passive protection of their offspring. Our findings are relevant to understanding the role of VA in the Ab responses to oral attenuated vaccines that are critical for successful maternal vaccination programs against enteric infections in infants and young animals.

A portable transistor immunosensor for fast identification of porcine epidemic diarrhea virus.

Widespread distribution of porcine epidemic diarrhea virus (PEDV) has led to catastrophic losses to the global pig farming industry. As a result, there is an urgent need for rapid, sensitive and accurate tests for PEDV to enable timely and effective interventions. In the present study, we develop and validate a floating gate carbon nanotubes field-effect transistor (FG CNT-FET)-based portable immunosensor for rapid identification of PEDV in a sensitive and accurate manner. To improve the affinity, a unique PEDV spike protein-specific monoclonal antibody is prepared by purification, and subsequently modified on FG CNT-FET sensor to recognize PEDV. The developed FET biosensor enables highly sensitive detection (LoD: 8.1 fg/mL and 100.14 TCID50/mL for recombinant spike proteins and PEDV, respectively), as well as satisfactory specificity. Notably, an integrated portable platform consisting of a pluggable FG CNT-FET chip and a portable device can discriminate PEDV positive from negative samples and even identify PEDV and porcine deltacoronavirus within 1 min with 100% accuracy. The portable sensing platform offers the capability to quickly, sensitively and accurately identify PEDV, which further points to a possibility of point of care (POC) applications of large-scale surveillance in pig breeding facilities.

Combination of S1-N-Terminal and S1-C-Terminal Domain Antigens Targeting Double Receptor-Binding Domains Bolsters Protective Immunity of a Nanoparticle Vaccine against Porcine Epidemic Diarrhea Virus.

Variants of coronavirus porcine epidemic diarrhea virus (PEDV) frequently emerge, causing an incomplete match between the vaccine and variant strains, which affects vaccine efficacy. Designing vaccines with rapidly replaceable antigens and high efficacy is a promising strategy for the prevention of infection with PEDV variant strains. In our study, three different types of self-assembled nanoparticles (nps) targeting receptor-binding N-terminal domain (NTD) and C-terminal domain (CTD) of S1 protein, named NTDnps, CTDnps, and NTD/CTDnps, were constructed and evaluated as vaccine candidates against PEDV. NTDnps and CTDnps vaccines mediated significantly higher neutralizing antibody (NAb) titers than NTD and CTD recombinant proteins in mice. The NTD/CTDnps in varying ratios elicited significantly higher NAb titers when compared with NTDnps and CTDnps alone. The NTD/CTDnps (3:1) elicited NAb with titers up to 92.92% of those induced by the commercial vaccine. Piglets immunized with NTD/CTDnps (3:1) achieved a passive immune protection rate of 83.33% of that induced by the commercial vaccine. NTD/CTDnps (3:1) enhanced the capacity of mononuclear macrophages and dendritic cells to take up and present antigens by activating major histocompatibility complex I and II molecules to stimulate humoral and cellular immunity. These data reveal that a combination of S1-NTD and S1-CTD antigens targeting double receptor-binding domains strengthens the protective immunity of nanoparticle vaccines against PEDV. Our findings will provide a promising vaccine candidate against PEDV.

Porcine kobuvirus enhances porcine epidemic diarrhea virus pathogenicity and alters the number of intestinal lymphocytes in piglets.

Recent epidemiological studies have discovered that a lot of cases of porcine epidemic diarrhea virus (PEDV) infection are frequently accompanied by porcine kobuvirus (PKV) infection, suggesting a potential relationship between the two viruses in the development of diarrhea. To investigate the impact of PKV on PEDV pathogenicity and the number of intestinal lymphocytes, piglets were infected with PKV or PEDV or co-infected with both viruses. Our findings demonstrate that co-infected piglets exhibit more severe symptoms, acute gastroenteritis, and higher PEDV replication compared to those infected with PEDV alone. Notably, PKV alone does not cause significant intestinal damage but enhances PEDV's pathogenicity and alters the number of intestinal lymphocytes. These results underscore the complexity of viral interactions in swine diseases and highlight the need for comprehensive diagnostic and treatment strategies addressing co-infections.

Virological evaluation of natural and modified attapulgite against porcine epidemic diarrhoea virus.

BACKGROUND: The Porcine Epidemic Diarrhea Virus (PEDV) has caused significant economic losses in the global swine industry. As a potential drug for treating diarrhea, the antiviral properties of attapulgite deserve further study. METHODS: In this study, various methods such as RT-qPCR, Western blot, viral titer assay, Cytopathic Effect, immunofluorescence analysis and transmission electron microscopy were used to detect the antiviral activity of attapulgite and to assess its inhibitory effect on PEDV. RESULTS: When exposed to the same amount of virus, there was a significant decrease in the expression of the S protein, resulting in a viral titer reduction from 10-5.613 TCID50/mL to 10-2.90 TCID50/mL, which represents a decrease of approximately 102.6 folds. Results of cytopathic effect and indirect immunofluorescence also indicate a notable decrease in viral infectivity after attapulgite treatment. Additionally, it was observed that modified materials after acidification had weaker antiviral efficacy compared to powdered samples that underwent ultrasonic disintegration, which showed the strongest antiviral effects. CONCLUSION: As a result, Attapulgite powders can trap and adsorb viruses to inhibit PEDV in vitro, leading to loss of viral infectivity. This study provides new materials for the development of novel disinfectants and antiviral additives.

Display of porcine epidemic diarrhea virus spike protein B-cell linear epitope on Lactobacillus mucosae G01 S-layer surface induce a robust immunogenicity in mice.

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea.

Matrine exhibits antiviral activities against PEDV by directly targeting Spike protein of the virus and inducing apoptosis via the MAPK signaling pathway.

Porcine Epidemic Diarrhea Virus (PEDV) is a highly contagious virus that causes Porcine Epidemic Diarrhea (PED). This enteric disease results in high mortality rates in piglets, leading to significant financial losses in the pig industry. However, vaccines cannot provide sufficient protection against epidemic strains. Spike (S) protein exposed on the surface of virion mediates PEDV entry into cells. Our findings imply that matrine (MT), a naturally occurring alkaloid, inhibits PEDV infection targeting S protein of virions and biological process of cells. The GLY434 residue in the autodocking site of the S protein and MT conserved based on sequence comparison. This study provides a comprehensive analysis of viral attachment, entry, and virucidal effects to investigate how that MT inhibits virus replication. MT inhibits PEDV attachment and entry by targeting S protein. MT was added to cells before, during, or after infection, it exhibits anti-PEDV activities and viricidal effects. Network pharmacology focuses on addressing causal mechanisms rather than just treating symptoms. We identified the key genes and screened the cell apoptosis involved in the inhibition of MT on PEDV infection in network pharmacology. MT significantly promotes cell apoptosis in PEDV-infected cells to inhibit PEDV infection by activating the MAPK signaling pathway. Collectively, we provide the biological foundations for the development of single components of traditional Chinese medicine to inhibit PEDV infection and spread.

SP2509, a specific antagonist of LSD1, exhibits antiviral properties against Porcine epidemic diarrhea virus.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), a type of coronavirus, is one of the main pathogens that can infect pigs of all ages. It causes diarrhea and acute death of newborn piglets, resulting in massive economic losses to the worldwide swine industry. While vaccination remains the primary approach in combating PEDV, it often fails to address all the challenges posed by the infection, particularly in light of the emergence of evolving mutant strains. Therefore, there is a critical need to identify potent antiviral drugs that can effectively safeguard pigs against PEDV infection. RESULTS: In this study, the antiviral efficacy of SP2509, a specific antagonist of Lysine-specific demethylase 1(LSD1), was evaluated in vitro. The RT-qPCR, Western blot, TCID50, and IFA showed that at a concentration of 1µmol/L, SP2509 significantly inhibited PEDV infection. Additionally, viral life cycle assays showed that SP2509 operates by impeding PEDV internalization and replication rather than attachment and release. Regarding mechanism, in Huh-7 cells, knockdowns LSD1 can suppress PEDV replication. This indicated that the inhibition effect of SP2509 on PEDV largely depends on the activity of its target protein, LSD1. CONCLUSION: Our results in vitro show that SP2509 can inhibit PEDV infection during the internalization and replication stage and revealed a role of LSD1 as a restriction factor for PEDV. These imply that LSD1 might be a target for interfering with the viral infection, and SP2509 could be developed as an effective anti-PEDV agent.

The death domain-associated protein suppresses porcine epidemic diarrhea virus replication by interacting with signal transducer and activator of transcription 1 and inducing downstream ISG15 expression.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute enteric disease in piglets and severely threatens the pig industry all over the world. Death domain-associated protein (DAXX) is a classical chaperone protein involved in multiple biological processes, such as cell apoptosis, transcriptional regulation, DNA damage repair, and host innate immunity. However, whether DAXX functions in the anti-PEDV innate immune responses remains unclear. In this study, we found that PEDV infection upregulated DAXX expression and induced its nucleocytoplasmic translocation in IPEC-J2 cells. Furthermore, we found that DAXX overexpression was inhibitory to PEDV replication, while downregulation of DAXX by RNA interference facilitated PEDV replication. The antiviral activity of DAXX was due to its positive effect on IFN-λ3-STAT1 signaling, as DAXX positively regulated STAT1 activation through their interaction in cytoplasm and enhancing the downstream ISG15 expression. Mutation of tryptophan at 621 to alanine in DAXX increased its abundance in the cytoplasm, leading to the upregulation of STAT1 phosphorylation and ISG15 expression. It indicated that cytoplasmic fraction of DAXX was advantageous for the STAT1-ISG15 signaling axis and PEDV inhibition. In summary, these results show that DAXX inhibits PEDV infection by increasing IFN-λ3-induced STAT1 phosphorylation and the downstream ISG15 expression.

Pyrococcus furiosus Argonaute Based Detection Assays for Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a major cause of diarrhea and diarrhea-related deaths among piglets and results in massive losses to the overall porcine industry. The clinical manifestations of porcine diarrhea brought on by the porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and PDCoV are oddly similar to each other. Hence, the identification of different pathogens through molecular diagnosis and serological techniques is crucial. Three novel detection methods for identifying PDCoV have been developed utilizing recombinase-aided amplification (RAA) or reverse transcription recombinase-aided amplification (RT-RAA) in conjunction with Pyrococcus furiosus Argonaute (PfAgo): RAA-PfAgo, one-pot RT-RAA-PfAgo, and one-pot RT-RAA-PfAgo-LFD. The indicated approaches have a detection limit of around 60 copies/µL of PDCoV and do not cross-react with other viruses including PEDV, TGEV, RVA, PRV, PCV2, or PCV3. The applicability of one-pot RT-RAA-PfAgo and one-pot RT-RAA-PfAgo-LFD were examined using clinical samples and showed a positive rate comparable to the qPCR method. These techniques offer cutting-edge technical assistance for identifying, stopping, and managing PDCoV.

An isotonic protein solution favorably modulated the porcine intestinal immune response and cellular adhesion markers and reduced PEDV shedding in vivo.

Porcine epidemic diarrhea virus (PEDV) causes immensely large economic losses worldwide in the swine industry. PEDV attacks the intestine, disrupts intestinal epithelium morphology and barrier integrity, and results in profound diarrhea and high mortality. A commercially available isotonic protein solution (IPS) (Tonisity Px) has anecdotally been reported to be effective in supportive treatment of piglets with active PEDV infections. This study evaluated the effects of supplementing (or not) the drinking water of 14 day old PEDV-infected piglets with the IPS on the content of E-cadherin, fibronectin, interferon-alpha (IFN-α), and matrix metalloproteinase 9 (MMP-9) in duodenal tissue. The content of PEDV DNA in feces was also measured. Though both groups had similar PEDV shedding at day 1, IPS piglets had significantly lower PEDV shedding at day 5, 14 and 21. The IPS group also had a shorter duration of PEDV virus shedding. Levels of E-cadherin and fibronectin, both of which are structural proteins in the intestine, remained unchanged from baseline in the IPS group, whereas the same molecules decreased significantly in the control group. IFN-α, an antiviral cytokine, and MMP-9, an enzyme that aids in tissue remodeling, were increased at days 5 and 14 post infection, and then decreased at day 21 post-infection in the IPS group compared to control. Overall, the IPS used in this study enhanced epithelial intercellular adhesion (E-cadherin) and extracellular matrix structure (fibronectin), resulted in significantand favorable changes in MMP-9 activity, and favorably modulated IFN-α production. This is the first report of this panel of biomarkers, especially MMP-9 and IFN-α, in the face of in vivo PEDV infection. This is also the first report to investigate a commercially available swine product that does not need to be administered in solid feed, and that is already registered for use throughout Asia, Europe, South America, and North America. Overall, the results of this study serve to clarify the behavior of 4 key biomarkers in the presence of in vivo PEDV infection. The results also indicate that IPS (Tonisity Px) supplementation is a viable intervention to modulate the porcine intestinal immune response with favorable effects on the intestine.

Oral administration of PEDV-dissolved Alg-CS gel induces high and sustained mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and milk is required to decrease losses. Porcine epidemic diarrhea virus (PEDV) was dissolved in calcium alginate (Alg) and combined with chitosan (CS) via electrostatic interactions between cationic chitosan and anionic alginate to create a porous gel (Alg-CS+PEDV). The gel was used to immunize mice orally or in combination with subcutaneous injections of inactivated PEDV vaccine. At 12 and 24 days after immunization, levels of IgA and IgG in Alg-CS+PEDV were higher than with normal PEDV oral administration. At 24 days after immunization, the concentration of IFN-γ in Alg-CS+PEDV was higher than with normal PEDV oral administration. Furthermore, oral administration combining subcutaneous immunization induced higher levels of IgG and IgA than oral administration alone. Our study provides a new method for the preparation and administration of oral vaccines to achieve enhanced mucosal immunity against PEDV.

Removal of RNA viruses from swine wastewater using anaerobic membrane bioreactor: Performance and mechanisms.

The effective removal of viruses from swine wastewater using anaerobic membrane bioreactor (AnMBR) is vital to ecological safety. However, most studies have focused only on disinfectants, whereas the capabilities of the treatment process have not been investigated. In this study, the performance and mechanism of an AnMBR in the removal of porcine hepatitis E virus (HEV), porcine kobuvirus (PKoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV) are systematically investigated. The results show that the AnMBR effectively removes the four viruses, with average removal efficiencies of 1.62, 3.05, 2.41, and 1.34 log for HEV, PKoV, PEDV and TGEV, respectively. Biomass adsorption contributes primarily to the total virus removal in the initial stage of reactor operation, with contributions to HEV and PKoV removal exceeding 71.7 % and 68.2 %, respectively. When the membrane is fouled, membrane rejection dominated virus removal. The membrane rejection contribution test shows the significant contribution of membrane pore foulants (23-76 %). Correlation analysis shows that the surface characteristics and size differences of the four viruses contribute primarily to their different effects on biomass adsorption and membrane rejection. This study provides technical guidance for viral removal during the treatment of high-concentration swine wastewater using an AnMBR.

TLR9 mediates IgA production in the porcine small intestine during PEDV infection.

IgA plays a vital role in defending against the infectious pathogens. However, the specific regulatory pathways involved in IgA secretion in the context of PEDV infection have remained elusive. Therefore, in this study, we explore the molecular mechanisms underlying IgA secretion in response to infection, with a particular focus on PEDV, a devastating enteric virus affecting global swine production. Our investigation begins by examining changes in IgA concentrations in both serum and small intestinal contents following PEDV infection in 2- and 4-week-old pigs. Remarkably, a significant increase in IgA levels in these older pigs post-infection were observed. To delve deeper into the regulatory mechanisms governing IgA secretion in response to PEDV infection, isolated porcine intestinal B cells were co-cultured with monocytes derived DCs (Mo-DCs) in vitro. In the intestinal DC-B cell co-cultures, IgA secretion was found to increase significantly after PEDV infection, as well as upregulating the expression of AID, GLTα and PSTα reflecting isotype switching to IgA. In addition, the expression of TLR9 was upregulated in these cultures, as determined by RT-qPCR and western blotting. Moreover, our findings extend to in vivo observations, where we detected higher levels of TLR9 expression in the ileum of pig post PEDV infection. Collectively, our results highlight the ability of PEDV to stimulate the generation of IgA, particularly in elder pigs, and identify TLR9 as a critical mediator of IgA production within the porcine intestinal microenvironment during PEDV infection.

Porcine epidemic diarrhea virus E protein induces formation of stress granules and attenuates protein translation through activation of the PERK/eIF2α signaling pathway.

Porcine epidemic diarrhea virus (PEDV) envelope protein (E) has been characterized as an important structural protein that plays critical roles in the interplay with its host to affect the virus life cycle. Stress granules (SGs) are host translationally silent ribonucleoproteins, which are mainly induced by the phosphorylation of eIF2α in the PERK/eIF2α signaling pathway. Our previous study found that PEDV E protein caused endoplasmic reticulum stress response (ERS)-mediated suppression of antiviral proteins' translation. However, the link and the underlying mechanism by which PEDV induces SGs formation and suppresses host translation remain elusive. In this study, our results showed that PEDV E protein significantly elevated the expression of GRP78, CANX, and phosphorylation of PERK and eIF2α, indicating that the PERK/eIF2α branch of ERS was activated. PEDV E protein localized to the ER and aggregated into puncta to reconstruct ER structure, and further induced SGs formation, which has been caused through upregulating the G3BP1 expression level. In addition, a significant global translational stall and endogenous protein translation attenuation were detected in the presence of E protein overexpression, but the global mRNA transcriptional level remained unchanged, suggesting that the shutoff of protein translation was associated with the translation, not with the transcription process. Collectively, this study demonstrates that PERK/eIF2α activation is required for SGs formation and protein translation stall. This study is beneficial for us to better understand the mechanism by which PEDV E suppresses host protein synthesis, and provides us a new insight into the host translation regulation during virus infection.

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.

Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.