Investigation of variants in genetics and virulence of Porcine Epidemic Diarrhea Virus after serial passage on Vero cells.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal virus. However, the current PEDV vaccine, which is produced from classical strain G1, offers low protection against recently emerged strain G2. This study aims to develop a better vaccine strain by propagating the PS6 strain, a G2b subgroup originating from Vietnam, on Vero cells until the 100th passage. As the virus was propagated, its titer increased, and its harvest time decreased. Analysis of the nucleotide and amino acid variation of the PS6 strain showed that the P100PS6 had 11, 4, and 2 amino acid variations in the 0 domain, B domain, and ORF3 protein, respectively, compared to the P7PS6 strain. Notably, the ORF3 gene was truncated due to a 16-nucleotide deletion mutation, resulting in a stop codon. The PS6 strain's virulence was evaluated in 5-day-old piglets, with P7PS6 and P100PS6 chosen for comparison. The results showed that P100PS6-inoculated piglets exhibited mild clinical symptoms and histopathological lesions, with a 100% survival rate. In contrast, P7PS6-inoculated piglets showed rapid and typical clinical symptoms of PEDV infection, and the survival rate was 0%. Additionally, the antibodies (IgG and IgA) produced from inoculated piglets with P100PS6 bound to both the P7PS6 and P100PS6 antigens. This finding suggested that the P100PS6 strain was attenuated and could be used to develop a live-attenuated vaccine against highly pathogenic and prevalent G2b-PEDV strains.

Recombinant human adenovirus type 5 based vaccine candidates against GIIa- and GIIb-genotype porcine epidemic diarrhea virus induce robust humoral and cellular response in mice.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus causing severe watery diarrhea, vomiting, dehydration, and death in piglets. However, most commercial vaccines are developed based on the GI genotype strains, and have poor immune protection against the currently dominant GII genotype strains. Therefore, four novel replication-deficient human adenovirus 5-vectored vaccines expressing codon-optimized forms of the GIIa and GIIb strain spike and S1 glycoproteins were constructed, and their immunogenicity was evaluated in mice by intramuscular (IM) injection. All the recombinant adenoviruses generated robust immune responses, and the immunogenicity of recombinant adenoviruses against the GIIa strain was stronger than that of recombinant adenoviruses against the GIIb strain. Moreover, Ad-XT-tPA-Sopt-vaccinated mice elicited optimal immune effects. In contrast, mice immunized with Ad-XT-tPA-Sopt by oral gavage did not induce strong immune responses. Overall, IM administration of Ad-XT-tPA-Sopt is a promising strategy against PEDV, and this study provides useful information for developing viral vector-based vaccines.

Construction and immunogenicity of a trypsin-independent porcine epidemic diarrhea virus variant.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteropathogenic coronavirus that causes high mortality in neonatal piglets. The addition of trypsin plays a crucial role in the propagation of PEDV, but also increases the complexity of vaccine production and increases its cost. Previous studies have suggested that the S2' site and Y976/977 of the PEDV spike (S) protein might be the determinants of PEDV trypsin independence. In this study, to achieve a recombinant trypsin-independent PEDV strain, we used trypsin-dependent genotype 2 (G2) PEDV variant AJ1102 to generate three recombinant PEDVs with mutations in S (S2' site R894G and/or Y976H). The three recombinant PEDVs were still trypsin dependent, suggesting that the S2' site R894 and Y976 of AJ1102 S are not key sites for PEDV trypsin dependence. Therefore, we used AJ1102 and the classical trypsin-independent genotype 1 (G1) PEDV strain JS2008 to generate a recombinant PEDV carrying a chimeric S protein, and successfully obtained trypsin-independent PEDV strain rAJ1102-S2'JS2008, in which the S2 (amino acids 894-1386) domain was replaced with the corresponding JS2008 sequence. Importantly, immunization with rAJ1102-S2'JS2008 induced neutralizing antibodies against both AJ1102 and JS2008. Collectively, these results suggest that rAJ1102-S2'JS2008 is a novel vaccine candidate with significant advantages, including no trypsin requirement for viral propagation to high titers and the potential provision of protection for pigs against G1 and G2 PEDV infections.

Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.

Efficacy of Needle-Less Intradermal Vaccination against Porcine Epidemic Diarrhea Virus.

To prevent diarrhea in suckling piglets infected by porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PED) vaccines are administered mainly through intramuscular (IM) or oral routes. We found that growing pigs vaccinated with an inactivated PEDV vaccine via the intradermal (ID) route had higher neutralizing antibody titers and cytokine (IFN-γ, IL-4, and IL-10) levels than non-vaccinated pigs. In addition, suckling piglets acquired lactogenic immunity from pregnant sows inoculated with an ID PED vaccine. We evaluated the efficacy of vaccination via this route, along with subsequent protection against virulent PEDV. At six days post-challenge, the survival rate of suckling piglets exposed to virulent PEDV was 70% for the ID group and 0% for the mock group (no vaccine). At necropsy, villi length in the duodenum and ileum of piglets with lactogenic immunity provided by ID-vaccinated sows proved to be significant (p < 0.05) when compared with those in piglets from mock group sows. Thus, vaccination using an inactivated PED vaccine via the ID route provides partial protection against infection by virulent PEDV.

In silico and in vitro evaluation of antiviral activity of wogonin against main protease of porcine epidemic diarrhea virus.

The high mortality rate of weaned piglets infected with porcine epidemic diarrhea virus (PEDV) poses a serious threat to the pig industry worldwide, demanding urgent research efforts related to developing effective antiviral drugs to prevent and treat PEDV infection. Small molecules can possibly prevent the spread of infection by targeting specific vital components of the pathogen's genome. Main protease (Mpro, also named 3CL protease) plays essential roles in PEDV replication and has emerged as a promising target for the inhibition of PEDV. In this study, wogonin exhibited antiviral activity against a PEDV variant isolate, interacting with the PEDV particles and inhibiting the internalization, replication and release of PEDV. The molecular docking model indicated that wogonin was firmly embedded in the groove of the active pocket of Mpro. Furthermore, the interaction between wogonin and Mpro was validated in silico via microscale thermophoresis and surface plasmon resonance analyses. In addition, the results of a fluorescence resonance energy transfer (FRET) assay indicated that wogonin exerted an inhibitory effect on Mpro. These findings provide useful insights into the antiviral activities of wogonin, which could support future research into anti-PEDV drugs.`.

Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization.

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there remain no effective drugs against PEDV infection. In this study, we utilized a recombinant PEDV expressing renilla luciferase (PEDV-Rluc) to screen potential anti-PEDV agents from an FDA-approved drug library in Vero cells. Four compounds were identified that significantly decreased luciferase activity of PEDV-Rluc. Among them, niclosamide was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index. It can efficiently inhibit viral RNA synthesis, protein expression and viral progeny production of classical and variant PEDV strains in a dose-dependent manner. Time of addition assay showed that niclosamide exhibited potent anti-PEDV activity when added simultaneously with or after virus infection. Furthermore, niclosamide significantly inhibited the entry stage of PEDV infection by affecting viral internalization rather than viral attachment to cells. In addition, a combination with other small molecule inhibitors of endosomal acidification enhanced the anti-PEDV effect of niclosamide in vitro. Taken together, these findings suggested that niclosamide is a novel antiviral agent that might provide a basis for the development of novel drug therapies against PEDV and other related pathogenic coronavirus infections.

Experimental Infection of Pigs with Recent European Porcine Epidemic Diarrhea Viruses.

Porcine epidemic diarrhea virus (PEDV), belonging to the genus Alphacoronavirus, can cause serious disease in pigs of all ages, especially in suckling pigs. Differences in virulence have been observed between various strains of this virus. In this study, four pigs were inoculated with PEDV from Germany (intestine/intestinal content collected from pigs in 2016) and four pigs with PEDV from Italy (intestine/intestinal material collected from pigs in 2016). The pigs were re-inoculated with the same virus on multiple occasions to create a more robust infection and enhance the antibody responses. The clinical signs and pathological changes observed were generally mild. Two distinct peaks of virus excretion were seen in the group of pigs inoculated with the PEDV from Germany, while only one strong peak was seen for the group of pigs that received the virus from Italy. Seroconversion was seen by days 18 and 10 post-inoculation with PEDV in all surviving pigs from the groups that received the inoculums from Germany and Italy, respectively. Attempts to infect pigs with a swine enteric coronavirus (SeCoV) from Slovakia were unsuccessful, and no signs of infection were observed in the inoculated animals.

Visual and Ultrasensitive Detection of a Coronavirus Using a Gold Nanorod Probe under Dark Field.

Porcine epidemic diarrhea virus (PEDV), a coronavirus that causes highly infectious intestinal diarrhea in piglets, has led to severe economic losses worldwide. Rapid diagnosis and timely supervision are significant in the prophylaxis of PEDV. Herein, we proposed a gold-nanorod (GNR) probe-assisted counting method using dark field microscopy (DFM). The antibody-functionalized silicon chips were prepared to capture PEDV to form sandwich structures with GNR probes for imaging under DFM. Results show that our DFM-based assay for PEDV has a sensitivity of 23.80 copies/µL for simulated real samples, which is very close to that of qPCR in this study. This method of GNR probes combined with DFM for quantitative detection of PEDV not only has strong specificity, good repeatability, and a low detection limit, but it also can be implemented for rapid on-site detection of the pathogens.

A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

PEDV: Insights and Advances into Types, Function, Structure, and Receptor Recognition.

Porcine epidemic diarrhea virus (PEDV) has been endemic in most parts of the world since its emergence in the 1970s. It infects the small intestine and intestinal villous cells, spreads rapidly, and causes infectious intestinal disease characterized by vomiting, diarrhea, and dehydration, leading to high mortality in newborn piglets and causing massive economic losses to the pig industry. The entry of PEDV into cells is mediated by the binding of its spike protein (S protein) to a host cell receptor. Here, we review the structure of PEDV, its strains, and the structure and function of the S protein shared by coronaviruses, and summarize the progress of research on possible host cell receptors since the discovery of PEDV.

Differential Transcriptomics Analysis of IPEC-J2 Cells Single or Coinfected With Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus.

Porcine epidemic diarrhea (PED) and transmissible gastroenteritis (TGE) caused by porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are two highly contagious intestinal diseases in the swine industry worldwide. Notably, coinfection of TGEV and PEDV is common in piglets with diarrhea-related diseases. In this study, intestinal porcine epithelial cells (IPEC-J2) were single or coinfected with PEDV and/or TGEV, followed by the comparison of differentially expressed genes (DEGs), especially interferon-stimulated genes (ISGs), between different groups via transcriptomics analysis and real-time qPCR. The antiviral activity of swine interferon-induced transmembrane protein 3 (sIFITM3) on PEDV and TGEV infection was also evaluated. The results showed that DEGs can be detected in the cells infected with PEDV, TGEV, and PEDV+TGEV at 12, 24, and 48 hpi, and the number of DEGs was the highest at 24 hpi. The DEGs are mainly annotated to the GO terms of protein binding, immune system process, organelle part, and intracellular organelle part. Furthermore, 90 ISGs were upregulated during PEDV or TGEV infection, 27 of which were associated with antiviral activity, including ISG15, OASL, IFITM1, and IFITM3. Furthermore, sIFITM3 can significantly inhibit PEDV and TGEV infection in porcine IPEC-J2 cells and/or monkey Vero cells. Besides, sIFITM3 can also inhibit vesicular stomatitis virus (VSV) replication in Vero cells. These results indicate that sIFITM3 has broad-spectrum antiviral activity.

Killed whole-genome reduced-bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

Genetic characterization of the spike gene of porcine epidemic diarrhea viruses (PEDVs) circulating in Vietnam from 2015 to 2016.

BACKGROUND: Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by the PED virus (PEDV), which is a member of the family Coronaviridae. Since the first outbreaks in Belgium and the United Kingdom were reported in 1971, PED has spread throughout many countries around the world and causing significant economic loss. This study was conducted to investigate the recent distribution of PEDV strains in Vietnam during the 2015-2016 seasons. METHODS: A total of 30 PED-specific PCR-positive intestinal and faecal samples were collected from unvaccinated piglets in Vietnam during the 2015-2016 seasons. The full length of the spike (S) gene of these PEDV strains were analysed to determine their phylogeny and genetic relationship with other available PEDV strains globally. RESULTS: Phylogenetic analysis of the complete S gene sequences revealed that the 28 Vietnamese PEDV strains collected in the northern and central regions clustered in the G2 group (both G2a and G2b sub-groups), while the other 2 PEDV strains (HUA-PED176 and HUA-PED254) collected in the southern region were clustered in the G1/G1b group/sub-group. The nucleotide (nt) and deduced amino acid (aa) analyses based on the complete S gene sequences showed that the Vietnamese PEDV strains were closely related to each other, sharing nt and aa homology of 93.2%-99.9% and 92.6%-99.9%, respectively. The N-glycosylation patterns and mutations in the antigenic region were observed in Vietnamese PEDV strains. CONCLUSIONS: This study provides, for the first time, up-to-date information on viral circulation and genetic distribution, as well as evidence to assist in the development of effective PEDV vaccines in Vietnam.

Virus-like particle vaccine with B-cell epitope from porcine epidemic diarrhea virus (PEDV) incorporated into hepatitis B virus core capsid provides clinical alleviation against PEDV in neonatal piglets through lactogenic immunity.

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope 748YSNIGVCK755 from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC).

Role of Porcine Aminopeptidase N and Sialic Acids in Porcine Coronavirus Infections in Primary Porcine Enterocytes.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been reported to use aminopeptidase N (APN) as a cellular receptor. Recently, the role of APN as a receptor for PEDV has been questioned. In our study, the role of APN in PEDV and TGEV infections was studied in primary porcine enterocytes. After seven days of cultivation, 89% of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to PEDV and TGEV. A significant increase of PEDV and TGEV infection was correlated with a higher expression of APN, which was indicative that APN plays an important role in porcine coronavirus infections. However, PEDV and TGEV infected both APN positive and negative enterocytes. PEDV and TGEV Miller showed a higher infectivity in APN positive cells than in APN negative cells. In contrast, TGEV Purdue replicated better in APN negative cells. These results show that an additional receptor exists, different from APN for porcine coronaviruses. Subsequently, treatment of enterocytes with neuraminidase (NA) had no effect on infection efficiency of TGEV, implying that terminal cellular sialic acids (SAs) are no receptor determinants for TGEV. Treatment of TGEV with NA significantly enhanced the infection which shows that TGEV is masked by SAs.