scholarly journals Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011–2016

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6843 ◽  
Author(s):  
Supansa Tuanthap ◽  
Sompong Vongpunsawad ◽  
Cherdpong Phupolphan ◽  
Ausanee Duang-in ◽  
Suphot Wattanaphansak ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Phylogenetic Analyses ◽  
Sequence Divergence ◽  
Economic Loss ◽  
Nucleotide Sequencing ◽  
Swine Industry ◽  
N Protein ◽  
Diarrhea Virus ◽  
Swine Farms ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) outbreaks on pig farms have caused significant economic loss in the swine industry since it was first reported in Thailand a decade ago. Anecdotal evidence suggests that PEDV is now endemic in this region, therefore genome information of circulating PEDV is important for molecular surveillance and evaluation of potential benefits of field vaccination. Here, we characterized PEDV infection on commercial Thai swine farms by screening 769 samples of feces and small intestinal contents from pigs with diarrhea between 2011 and 2016. Using reverse-transcription polymerase chain reaction targeting the spike (S) gene, 153 PEDV-positive samples were further subjected to analysis of the open reading frame 3 and nucleocapsid (N) genes. Comparison of 95 samples in which nucleotide sequencing was successfully obtained for all three genes revealed evolutionary diversity among the Thai PEDV strains. Phylogenetic analyses suggest that although some Thai strains changed little from years past, others resembled more closely to the recent strains reported in China. Interestingly, eight Thai PEDV strains possessed amino acid deletions in the N protein. The PEDV sequence divergence may be responsible for driving periodic outbreaks and continued persistence of PEDV on commercial swine farms. Our findings provide important insight into regional PEDV strains in circulation, which may assist future inclusions of suitable strains for future PEDV vaccines.

scholarly journals The interactome of porcine epidemic diarrhea virus nucleocapsid protein

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.

scholarly journals MicroRNA-221-5p Inhibits Porcine Epidemic Diarrhea Virus Replication by Targeting Genomic Viral RNA and Activating the NF-κB Pathway

2018 ◽  
Vol 19 (11) ◽  
pp. 3381 ◽  
Author(s):  
Hongqing Zheng ◽  
Lei Xu ◽  
Yuzhong Liu ◽  
Cheng Li ◽  
Liang Zhang ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Nuclear Translocation ◽  
Regulation Of Gene Expression ◽  
Economic Loss ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Swine Industry ◽  
Regulatory Relationship ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

MicroRNAs (miRNAs) are a class of noncoding RNAs involved in posttranscriptional regulation of gene expression and many critical roles in numerous biological processes. Porcine epidemic diarrhea virus (PEDV), the etiological agent of porcine epidemic diarrhea, causes substantial economic loss in the swine industry worldwide. Previous studies reported miRNA involvement in viral infection; however, their role in regulating PEDV infection remains unknown. In this study, we investigated the regulatory relationship between miRNA-221-5p and PEDV infection, finding that miR-221-5p overexpression inhibited PEDV replication in a dose-dependent manner, and that silencing endogenous miR-221-5p enhanced viral replication. Our results showed that miR-221-5p directly targets the 3′ untranslated region (UTR) of PEDV genomic RNA to inhibit PEDV replication, and that miR-221-5p overexpression activates nuclear factor (NF)-κB signaling via p65 nuclear translocation, thereby upregulating interferon (IFN)-β, IFN-stimulated gene 15, and MX1 expression during CH/HBTS/2017 infection. We subsequently identified NF-κB-inhibitor α and suppressor of cytokine signaling 1, negative regulators of the NF-κB pathway, as miR-221-5p targets. These results demonstrated the ability of miR-221-5p to inhibit PEDV replication by targeting the 3’ UTR of the viral genome and activating the NF-κB-signaling pathway. Our findings will aid the development of preventive and therapeutic strategies for PEDV infection.

scholarly journals The trypsin-enhanced infection of porcine epidemic diarrhea virus is determined by the S2 subunit of the spike glycoprotein

2021 ◽  
Author(s):  
Yubei Tan ◽  
Limeng Sun ◽  
Gang Wang ◽  
Yuejun Shi ◽  
Wanyu Dong ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Cleavage Site ◽  
Vaccine Development ◽  
Syncytium Formation ◽  
Reverse Genetic System ◽  
Site Mutation ◽  
Swine Industry ◽  
S Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen in the swine industry, causing high mortality in neonatal piglets. Efficient PEDV infection usually relies on the presence of trypsin, yet the mechanism of trypsin dependency is ambiguous. Here, we identified two PEDV strains, trypsin-enhanced YN200 and trypsin-independent DR13, in which the spike (S) protein of YN200 exhibits a stronger ability to induce syncytium formation and cleaved by trypsin than that of DR13. Using a full-length infectious YN200 cDNA clone, we confirmed that the S protein is a trypsin dependency determinant by comparison of rYN200 and rYN200-SDR13. To explore the trypsin-associated sites of the YN200 S protein, we then constructed a series of mutations adjacent to the fusion peptide. The results show that the putative S2’ cleavage site (R892G) is not the determinant for virus trypsin dependency. Hence, we generated viruses carrying chimeric S proteins: the S1 subunit, S2 subunit, and S2720∼892 aa domain (NS2’) were individually replaced by the corresponding DR13 sequences. Intriguingly, only the S2 substitution, not the S1 or NS2’ substitutions, provides trypsin-independent growth of YN200. Additionally, the NS2’ recombinant virus significantly abrogated effective infection, indicating a vital role for NS2’ in viral entry. These findings suggest that the trypsin dependency of PEDV is mainly controlled by mutations in the S2 subunit rather than directly trypsin cleavage site. Importance With the emergence of new variants, PEDV remains a major problem in the global swine industry. Efficient PEDV infection usually requires trypsin, while the mechanism of trypsin dependency is complex. Here, we used two PEDV strains, trypsin-enhanced YN200 and trypsin-independent DR13, and results showed that the S protein determined PEDV trypsin dependency by using a reverse genetic system of YN200. The S2 subunit was verified as the main portion of PEDV trypsin dependency, though the putative S2’ site mutation cannot render trypsin-independent growth of YN200. Finally, these results provide some different insight to the PEDV trypsin dependency and might inspire vaccine development.

scholarly journals Single-Particle Tracking Porcine Epidemic Diarrhea Virus Moving along Microtubules in Living Cells

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 124
Author(s):  
Yangyang Li ◽  
Wei Hou ◽  
Jian Wang ◽  
Fei Liu
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Porcine Epidemic Diarrhea Virus ◽  
Living Cells ◽  
Single Particle Tracking ◽  
Microtubule Organizing Center ◽  
Swine Industry ◽  
Diarrhea Virus ◽  
Organizing Center ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has caused severe damage to the swine industry. Although viruses are believed to hijack the microtubule-based transport system, the exact manner of PEDV moving along microtubules has not been fully characterized. In this study, PEDV was labeled with quantum dots which have great brightness and photostability. By using quantum dot-labeled PEDV and single-particle tracking, we were able to systematically dissect the dynamic behaviors of PEDV moving along the microtubules in living cells. We found that PEDVs maintained a restricted motion mode with a relatively stable speed in the cell membrane region while displaying a slow–fast–slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule-organizing center. The return movements of small amounts of PEDVs were also observed in living cells. Collectively, our work is crucial for understanding the movement of PEDV in living cells; the proposed work also provides important references for further analysis and studies of the infection mechanism of PEDV.

scholarly journals Molecular Characterization of Porcine Epidemic Diarrhea Virus and Its New Genetic Classification Based on the Nucleocapsid Gene

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 790
Author(s):  
Sung-Jae Kim ◽  
Van-Giap Nguyen ◽  
Thi-My-Le Huynh ◽  
Yong-Ho Park ◽  
Bong-Kyun Park ◽  
...  
Keyword(s):  
B Cell ◽  
Porcine Epidemic Diarrhea Virus ◽  
Cell Epitope ◽  
N Gene ◽  
Swine Industry ◽  
Genetic Classification ◽  
Nucleocapsid Gene ◽  
Diarrhea Virus ◽  
B Cell Epitope ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) causes continuous, significant damage to the swine industry worldwide. By RT-PCR-based methods, this study demonstrated the ongoing presence of PEDV in pigs of all ages in Korea at the average detection rate of 9.92%. By the application of Bayesian phylogenetic analysis, it was found that the nucleocapsid (N) gene of PEDV could evolve at similar rates to the spike (S) gene at the order of 10−4 substitutions/site/year. Based on branching patterns of PEDV strains, three main N gene-base genogroups (N1, N2, and N3) and two sub-genogroups (N3a, N3b) were proposed in this study. By analyzing the antigenic index, possible antigenic differences also emerged in both the spike and nucleocapsid proteins between the three genogroups. The antigenic indexes of genogroup N3 strains were significantly lower compared with those of genogroups N1 and N2 strains in the B-cell epitope of the nucleocapsid protein. Similarly, significantly lower antigenic indexes in some parts of the B-cell epitope sequences of the spike protein (COE, S1D, and 2C10) were also identified. PEDV mutants derived from genetic mutations of the S and N genes may cause severe damage to swine farms by evading established host immunities.

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

2020 ◽  
Vol 32 (4) ◽  
pp. 572-576 ◽  
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.

Epidemiological survey of porcine epidemic diarrhea virus in swine farms in Shanghai, China

2013 ◽  
Vol 158 (11) ◽  
pp. 2227-2231 ◽  
Author(s):  
Fei-Fei Ge ◽  
De-Quan Yang ◽  
Hou-Bin Ju ◽  
Jian Wang ◽  
Jian Liu ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Epidemiological Survey ◽  
Diarrhea Virus ◽  
Swine Farms ◽  
Porcine Epidemic Diarrhea

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

2016 ◽  
Vol 91 (2) ◽  
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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