Detection of porcine epidemic diarrhea virus–neutralizing antibody using high-throughput imaging cytometry

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry–based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status ( n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

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Reactivity of Porcine Epidemic Diarrhea Virus Structural Proteins to Antibodies against Porcine Enteric Coronaviruses: Diagnostic Implications

Journal of Clinical Microbiology ◽

10.1128/jcm.02507-16 ◽

2017 ◽

Vol 55 (5) ◽

pp. 1426-1436 ◽

Cited By ~ 26

Author(s):

Luis Gabriel Gimenez-Lirola ◽

Jianqiang Zhang ◽

Jose Antonio Carrillo-Avila ◽

Qi Chen ◽

Ronaldo Magtoto ◽

...

Keyword(s):

Antibody Response ◽

Porcine Epidemic Diarrhea Virus ◽

Herd Immunity ◽

Cross Reactivity ◽

Structural Proteins ◽

Transmissible Gastroenteritis Virus ◽

Serum Samples ◽

Igg Antibody ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

ABSTRACTThe development of porcine epidemic diarrhea virus (PEDV) antibody-based assays is important for detecting infected animals, confirming previous virus exposure, and monitoring sow herd immunity. However, the potential cross-reactivity among porcine coronaviruses is a major concern for the development of pathogen-specific assays. In this study, we used serum samples (n= 792) from pigs of precisely known infection status and a multiplex fluorescent microbead-based immunoassay and/or enzyme-linked immunoassay platform to characterize the antibody response to PEDV whole-virus (WV) particles and recombinant polypeptides derived from the four PEDV structural proteins, i.e., spike (S), nucleocapsid (N), membrane (M), and envelope (E). Antibody assay cutoff values were selected to provide 100% diagnostic specificity for each target. The earliest IgG antibody response, mainly directed against S1 polypeptides, was observed at days 7 to 10 postinfection. With the exception of nonreactive protein E, we observed similar antibody ontogenies and patterns of seroconversion for S1, N, M, and WV antigens. Recombinant S1 provided the best diagnostic sensitivity, regardless of the PEDV strain, with no cross-reactivity detected against transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), or porcine deltacoronavirus (PDCoV) pig antisera. The WV particles showed some cross-reactivity to TGEV Miller and TGEV Purdue antisera, while N protein presented some cross-reactivity to TGEV Miller. The M protein was highly cross-reactive to TGEV and PRCV antisera. Differences in the antibody responses to specific PEDV structural proteins have important implications in the development and performance of antibody assays for the diagnosis of PEDV enteric disease.

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A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus

Journal of Nanobiotechnology ◽

10.1186/s12951-019-0531-x ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 4

Author(s):

Zhiqian Ma ◽

Tianyu Wang ◽

Zhiwei Li ◽

Xuyang Guo ◽

Yangsheng Tian ◽

...

Keyword(s):

Phage Display ◽

Porcine Epidemic Diarrhea Virus ◽

Diagnostic Methods ◽

Watery Diarrhea ◽

Economic Losses ◽

Serum Samples ◽

Phage Display Technology ◽

Diarrhea Virus ◽

Blocking Elisa ◽

Porcine Epidemic Diarrhea

Abstract Background Porcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide. Thus, a rapid and reliable method is required for epidemiological investigations and to evaluate the effect of immunization. However, the current diagnostic methods for PEDV are time-consuming and very expensive and rarely meet the requirements for clinical application. Nanobodies have been used in the clinic to overcome these problems because of the advantages of their easy expression and high level of stability. In the present work, a novel biotinylated nanobody-based blocking ELISA (bELISA) was developed to detect anti-PEDV antibodies in clinical pig serum. Results Using phage display technology and periplasmic extraction ELISA (PE-ELISA), anti-PEDV N protein nanobodies from three strains of PEDV were successfully isolated after three consecutive rounds of bio-panning from a high quality phage display VHH library. Then, purified Nb2-Avi-tag fusion protein was biotinylated in vitro. A novel bELISA was subsequently developed for the first time with biotinylated Nb2. The cutoff value for bELISA was 29.27%. One hundred and fifty clinical serum samples were tested by both newly developed bELISA and commercial kits. The sensitivity and specificity of bELISA were 100% and 93.18%, respectively, and the coincidence rate between the two methods was 94%. Conclusions In brief, bELISA is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PEDV vaccines efficacy and indirect diagnosis of PEDV infection.

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

Journal of Virology ◽

10.1128/jvi.02453-20 ◽

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.

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

Proceedings ◽

10.3390/proceedings2020050124 ◽

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.

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

Viruses ◽

10.3390/v12080790 ◽

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.

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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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Genetic Characterization of Porcine Epidemic Diarrhea Virus in China Between 2014 and 2018: Emergence of the G1c Subtype

Pakistan Veterinary Journal ◽

10.29261/pakvetj/2020.035 ◽

2020 ◽

Vol 40 (04) ◽

pp. 474-478

Author(s):

Wenqiang Jiao

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Phylogenetic Trees ◽

Genetic Characterization ◽

Diagnostic Methods ◽

Clinical Samples ◽

Swine Industry ◽

Diarrhea Virus ◽

Positive Rate ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) has caused substantial economical loss to the Chinese swine industry. To illustrate the genetic characterization of PEDV circulating in China, 205 clinical samples between 2014 and 2018 were collected from 7 provinces in China. 93.17% (191 of 205) of the intestinal and fecal samples were positive for PEDV. 25 S1 amino acid (aa) together with 27 ORF3 genes from 8 provinces were sequenced and analyzed. The phylogenetic trees based on the S1 and ORF3 genes were constructed by the neighbor-joining method using MEGA 7 software. PEDV prevalence was 86.96% (40 of 46) of the swine farms in the 8 provinces and the PEDV positive rate was 93.17% (191 of 205) in the tested samples. Genetic analysis showed CH-JIANGXI-1-2016 CH-JIAGNXI-2-2016, CH-JIANGXI-3-2016 and CH-JIANGXI-2017 had three notable insertions or deletions occurred at aa 59-62, 160, and 139 (140) when compared to all of the strains in this study; moreover, phylogenetic analysis indicated that the four isolates formed a new branch significantly different from G1a, G1b and Indel subtype based on S1 gene: that is the G1c subtype. More research is needed to determine whether the insertions and deletions had biological influence on the virus. The results acquired in the present study showed the genetic diversity of PEDV circulating in 8 provinces, providing information for the development of new diagnostic methods and new vaccines

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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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Construction of a Recombinant Porcine Epidemic Diarrhea Virus Encoding Nanoluciferase for High-Throughput Screening of Natural Antiviral Products

Viruses ◽

10.3390/v13091866 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1866

Author(s):

Wan Li ◽

Mengjia Zhang ◽

Huijun Zheng ◽

Peng Zhou ◽

Zheng Liu ◽

...

Keyword(s):

High Throughput ◽

Porcine Epidemic Diarrhea Virus ◽

High Throughput Screening ◽

Lithocholic Acid ◽

Rapid Screening ◽

Plaque Morphology ◽

Dependent Manner ◽

Strong Positive Correlation ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.

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