Inactivated Pseudomonas PE(ΔIII) exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice

AbstractPorcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV), is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide. An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010. S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV. In this study, the construction, expression and purification of Pseudomonas aeruginosa exotoxin A (PE) without domain III (PEΔIII) as a vector was performed for the delivery of PEDV S-A or S-B. PE(ΔIII) PEDV S-A and PE(ΔIII) PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice. The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses, but also stimulate PEDV-specific mucosal immune responses in mice. PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.

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Immunogenicity of Recombinant-Deficient Lactobacillus casei with Complementary Plasmid Expressing Alanine Racemase Gene and Core Neutralizing Epitope Antigen against Porcine Epidemic Diarrhea Virus

Vaccines ◽

10.3390/vaccines9101084 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1084

Author(s):

Fengsai Li ◽

Xiaona Wang ◽

Xiaolong Fan ◽

Ling Sui ◽

Hailin Zhang ◽

...

Keyword(s):

Immune Responses ◽

Porcine Epidemic Diarrhea Virus ◽

Lactobacillus Casei ◽

Oral Vaccine ◽

Mucosal Vaccine ◽

Alanine Racemase ◽

Main Function ◽

Neutralizing Epitope ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), has occurred worldwide and poses a serious threat to the pig industry. Intestine is the main function site of PEDV; therefore, it is important to develop an oral mucosal immunity vaccine against this virus infection. Most traditional plasmid delivery vectors use antibiotic genes as a selective marker, easily leading to antibiotic accumulation and gene contamination. In this study, to explore whether the alanine racemase gene (Alr) could be used as a screening marker and develop an efficient oral vaccine against PEDV infection, a recombinant strain was constructed using Lactobacillus casei with Alr deletion (L. casei ΔAlr W56) to deliver the Alr gene and a core-neutralizing epitope (COE) antigen. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in mice. Compared to the other strains, the recombinant bacteria were able to grow without the addition of D-alanine, revealing that Alr in the plasmid could function normally in defective bacteria. This oral mucosal vaccine would provide a useful strategy to substitute the application of antibiotics in the future and induce efficient immune responses against PEDV infection.

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Development of a droplet digital PCR for detection and quantification of porcine epidemic diarrhea virus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638720924753 ◽

2020 ◽

Vol 32 (4) ◽

pp. 572-576 ◽

Cited By ~ 1

Author(s):

Wei W. Cao ◽

Dong S. He ◽

Zhen J. Chen ◽

Yu Z. Zuo ◽

Xun Chen ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Fold Increase ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Economic Losses ◽

Swine Industry ◽

Diarrhea Virus ◽

Viral Loads ◽

Promising Tool ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea, a disease caused by porcine epidemic diarrhea virus (PEDV), results in large economic losses to the global swine industry. To manage this disease effectively, it is essential to detect PEDV early and accurately. We developed a sensitive and accurate droplet digital PCR (ddPCR) assay to detect PEDV. The optimal primer-to-probe concentration and melting temperature were identified as 300:200 nM and 59.2°C, respectively. The specificity of the ddPCR assay was confirmed by negative test results for common swine pathogens. The detection limit for the ddPCR was 0.26 copies/μL, which is a 5.7-fold increase in sensitivity compared to that of real-time PCR (rtPCR). Both ddPCR and rtPCR assays exhibited good linearity, although ddPCR provided higher sensitivity for clinical detection compared to that of rtPCR. Our ddPCR methodology provides a promising tool for evaluating the PEDV viral load when used for clinical testing, particularly for detecting samples with low-copy viral loads.

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Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability

Journal of Virology ◽

10.1128/jvi.01660-16 ◽

2016 ◽

Vol 91 (2) ◽

Cited By ~ 10

Author(s):

Peera Jaru-Ampornpan ◽

Juggragarn Jengarn ◽

Asawin Wanitchang ◽

Anan Jongkaewwattana

Keyword(s):

Cell Culture ◽

Growth Retardation ◽

Porcine Epidemic Diarrhea Virus ◽

Vaccine Development ◽

Wild Type Virus ◽

Economic Losses ◽

Swine Industry ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. IMPORTANCE Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.

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Construction of Porcine Epidemic Diarrhea Virus-Like Particles and Its Immunogenicity in Mice

Vaccines ◽

10.3390/vaccines9040370 ◽

2021 ◽

Vol 9 (4) ◽

pp. 370

Author(s):

Jihee Kim ◽

Jaewon Yoon ◽

Jung-Eun Park

Keyword(s):

Immune Responses ◽

Genetic Material ◽

Vero Cells ◽

Swine Industry ◽

E Proteins ◽

Virus Like Particles ◽

Diarrhea Virus ◽

Humoral Immune ◽

Neonatal Piglets ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea (PED), a highly contagious and lethal enteric disease in piglets, is characterized by diarrhea, vomiting, and dehydration, with high mortality in neonatal piglets. Despite the nationwide use of attenuated and inactivated vaccines, the outbreak of PED is still a major problem in the swine industry. Virus-like particles (VLPs) are artificial nanoparticles similar to viruses that are devoid of genetic material and are unable to replicate. VLPs have good safety profiles and elicit robust cellular and humoral immune responses. Here, we generated PED VLPs in eukaryotic cells and examined their immune responses in mice. We found that the M protein is essential for the formation of PED VLPs. Interestingly, PED VLP formation was decreased in the presence of E proteins and increased in the presence of N proteins. Both IgG and IgA antibodies were induced in mice immunized with PED VLPs. Moreover, these antibodies protected against PED virus infection in Vero cells. PED VLPs immunization induced Th2-dominant immune responses in mice. Our results indicate that PED VLPs induce strong immune responses in mice, suggesting that the VLP-based vaccine is a promising vaccine candidate.

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The interactome of porcine epidemic diarrhea virus nucleocapsid protein

10.21203/rs.2.12355/v1 ◽

2019 ◽

Author(s):

Min Tan ◽

Guofei Ding ◽

Xinna Cai ◽

Shengliang Cao ◽

Fangyuan Cong ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Cellular Proteins ◽

Economic Losses ◽

N Gene ◽

Effective Vaccine ◽

Label Free ◽

Swine Industry ◽

N Protein ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

Abstract Background Many viral proteins specifically interact with cellular proteins to facilitate virus replication. Understanding these interactions can decipher the viral infection mechanism and provide potential targets for antiviral therapy. Porcine epidemic diarrhea virus (PEDV), the agent of PED, causes numerous economic losses for the swine industry each year. Till now, no effective vaccine or drugs are available to contain this disease. As a result, it is critical urgent to elucidate the PEDV interactome. The nucleocapsid (N) of PEDV plays an important role in viral replication. Results In this study, the N gene was cloned into pEGFP-C1 and transfected into 293T cells. The interactome of N was elucidated by label-free mass spectrometry. A total of 125 cellular proteins interacting with PEDV N protein were discovered, of which 4 cellular proteins, DHX9, NCL, KAP1, TCEA1, were confirmed by pull down, immunoprecipitation, and co-localization. Conclusions The interactome of N protein supplied a powerful tool to explore the role of N in PEDV infection and therapeutic targets.

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Immune Responses in Pregnant Sows Induced by Recombinant Lactobacillus johnsonii Expressing the COE Protein of Porcine Epidemic Diarrhea Virus Provide Protection for Piglets against PEDV Infection

Viruses ◽

10.3390/v14010007 ◽

2021 ◽

Vol 14 (1) ◽

pp. 7

Author(s):

Dianzhong Zheng ◽

Xiaona Wang ◽

Ning Ju ◽

Zhaorui Wang ◽

Ling Sui ◽

...

Keyword(s):

Dendritic Cell ◽

Immune Responses ◽

Porcine Epidemic Diarrhea Virus ◽

Oral Vaccine ◽

Economic Losses ◽

Antigen Delivery ◽

Lactobacillus Johnsonii ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea (PED) induced by porcine epidemic diarrhea virus (PEDV) is an intestinal infectious disease in pigs that causes serious economic losses to the pig industry. To develop an effective oral vaccine against PEDV infection, we used a swine-origin Lactobacillus johnsonii (L. johnsonii) as an antigen delivery carrier. A recombinant strain pPG-T7g10-COE/L. johnsonii (L. johnsonii-COE) expressing COE protein (a neutralizing epitope of the viral spike protein) was generated. The immunomodulatory effect on dendritic cell in vitro and immunogenicity in pregnant sows was evaluated following oral administration. L. johnsonii-COE could activate monocyte-derived dendritic cell (MoDC) maturation and triggered cell immune responses. After oral vaccination with L. johnsonii-COE, levels of anti-PEDV-specific serum IgG, IgA, and IgM antibodies as well as mucosal secretory immunoglobulin A (SIgA) antibody were induced in pregnant sows. High levels of PEDV-specific SIgA and IgG antibodies were detected in the maternal milk, which provide effective protection for the piglets against PEDV infection. In summary, oral L. johnsonii-COE was able to efficiently activate anti-PEDV humoral and cellular immune responses, demonstrating potential as a vaccine for use in sows to provide protection of their piglets against PEDV.

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Intramuscular Immunization with Chemokine-Adjuvanted Inactive Porcine Epidemic Diarrhea Virus Induces Substantial Protection in Pigs

Vaccines ◽

10.3390/vaccines8010102 ◽

2020 ◽

Vol 8 (1) ◽

pp. 102

Author(s):

Fu-Chun Hsueh ◽

Yen-Chen Chang ◽

Chi-Fei Kao ◽

Chin-Wei Hsu ◽

Hui-Wen Chang

Keyword(s):

Neutralizing Antibodies ◽

Expression System ◽

Inflammatory Cells ◽

Mucosal Immune Response ◽

Swine Industry ◽

Diarrhea Virus ◽

Mammalian Expression ◽

Chemokine Ligand ◽

Cc Chemokines ◽

Porcine Epidemic Diarrhea

Intramuscular (IM) immunization is generally considered incapable of generating a protective mucosal immune response. In the swine industry, attempts to develop a safe and protective vaccine for controlling porcine epidemic diarrhea (PED) via an IM route of administration have been unsuccessful. In the present study, porcine chemokine ligand proteins CCL25, 27, and 28 were constructed and stably expressed in the mammalian expression system. IM co-administration of inactivated PEDV (iPEDV) particles with different CC chemokines and Freund’s adjuvants resulted in recruiting CCR9+ and/or CCR10+ inflammatory cells to the injection site, thereby inducing superior systemic PEDV specific IgG, fecal IgA, and viral neutralizing antibodies in pigs. Moreover, pigs immunized with iPEDV in combination with CCL25 and CCL28 elicited substantial protection against a virulent PEDV challenge. We show that the porcine CC chemokines could be novel adjuvants for developing IM vaccines for modulating mucosal immune responses against mucosal transmissible pathogens in pigs.

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Gene Variations in Cis-Acting Elements between the Taiwan and Prototype Strains of Porcine Epidemic Diarrhea Virus Alter Viral Gene Expression

Genes ◽

10.3390/genes9120591 ◽

2018 ◽

Vol 9 (12) ◽

pp. 591

Author(s):

Tsung-Lin Tsai ◽

Chen-Chang Su ◽

Ching-Chi Hsieh ◽

Chao-Nan Lin ◽

Hui-Wen Chang ◽

...

Keyword(s):

Gene Expression ◽

Porcine Epidemic Diarrhea Virus ◽

The Body ◽

Economic Losses ◽

Viral Gene ◽

Sequence Motif ◽

Cis Acting ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea ◽

In Cis

In 2013, the outbreak of porcine epidemic diarrhea (PED) in Taiwan caused serious economic losses. In this study, we examined whether the variations of the cis-acting elements between the porcine epidemic diarrhea virus (PEDV) Taiwan (TW) strain and the prototype strain CV777 alter gene expression. For this aim, we analyzed the variations of the cis-acting elements in the 5’ and 3’ untranslated regions (UTRs) between the PEDV TW, CV777, and other reference strains. We also determined the previously unidentified transcription regulatory sequence (TRS), a sequence motif required for coronavirus transcription, and found that a nucleotide deletion in the TW strain, in comparison with CV777 strain, immediately downstream of the leader core sequence alters the identity between the leader TRS and the body TRS. Functional analyses using coronavirus defective interfering (DI) RNA revealed that such variations in cis-acting elements for the TW strain compared with the CV777 strain have an influence on the efficiency of gene expression. The current data show for the first time the evolution of PEDV in terms of cis-acting elements and their effects on gene expression, and thus may contribute to our understanding of recent PED outbreaks worldwide.

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