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.

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.

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

Genes ◽  
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.

scholarly journals Engineering a Live Attenuated Porcine Epidemic Diarrhea Virus Vaccine Candidate via Inactivation of the Viral 2'-O-Methyltransferase and the Endocytosis Signal of the Spike Protein

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Yixuan Hou ◽  
Hanzhong Ke ◽  
Jineui Kim ◽  
Dongwan Yoo ◽  
Yunfang Su ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Spike Protein ◽  
Economic Losses ◽  
High Dose ◽  
Type I ◽  
Viral Pathogen ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2′-O-methyltransferase (2′-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2′-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A. Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2′-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2′-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

scholarly journals Investigation of the Role of the Spike Protein in Reversing the Virulence of the Highly Virulent Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strains and Its Attenuated Counterpart

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 41 ◽  
Author(s):  
Chi-Fei Kao ◽  
Hui-Wen Chang
Keyword(s):  
Mortality Rate ◽  
Porcine Epidemic Diarrhea Virus ◽  
Rational Design ◽  
Economic Losses ◽  
Diarrhea Virus ◽  
Viral Shedding ◽  
S Gene ◽  
Porcine Epidemic Diarrhea ◽  
Highly Virulent

Porcine epidemic diarrhea virus (PEDV) has continuously caused severe economic losses to the global swine industries; however, no successful vaccine against PEDV has been developed. In this study, we generated four autologous recombinant viruses, including the highly virulent iPEDVPT-P5, attenuated iPEDVPT-P96, and two chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S) with the reciprocally exchanged spike (S) gene, to study the role of the S gene in PEDV pathogenesis. A deeper understanding of PEDV attenuation will aid in the rational design of a live attenuated vaccine (LAV) using reverse genetics system. Our results showed that replacing the S gene from the highly virulent iPEDVPT-P5 led to complete restoration of virulence of the attenuated iPEDVPT-P96, with nearly identical viral shedding, diarrhea pattern, and mortality rate as the parental iPEDVPT-P5. In contrast, substitution of the S gene with that from the attenuated iPEDVPT-P96 resulted in partial attenuation of iPEDVPT-P5, exhibiting similar viral shedding and diarrhea patterns as the parental iPEDVPT-P96 with slightly severe histological lesions and higher mortality rate. Collectively, our data confirmed that the attenuation of the PEDVPT-P96 virus is primarily attributed to mutations in the S gene. However, mutation in S gene alone could not fully attenuate the virulence of iPEDVPT-P5. Gene (s) other than S gene might also play a role in determining virulence.

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

2019 ◽  
Vol 17 (1) ◽  
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.

scholarly journals Development of indirect enzyme-linked immunosorbent assay for detection of porcine epidemic diarrhea virus specific antibodies (IgG) in serum of naturally infected pigs

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Ohnmar Myint ◽  
Ayako Yoshida ◽  
Satoshi Sekiguchi ◽  
Nguyen Van Diep ◽  
Naoyuki Fuke ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Mortality ◽  
Porcine Epidemic Diarrhea Virus ◽  
Indirect Elisa ◽  
Neutralization Test ◽  
Elisa Test ◽  
Watery Diarrhea ◽  
Economic Losses ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Abstract Background Porcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED. Results One hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80–100%, respectively. Conclusions The developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

scholarly journals Characterization and pathogenicity of Vero cell-attenuated porcine epidemic diarrhea virus CT strain

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Yu Wu ◽  
Wei Li ◽  
Qingfeng Zhou ◽  
Qunhui Li ◽  
Zhichao Xu ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Clinical Symptoms ◽  
Vero Cells ◽  
Economic Losses ◽  
Protective Effects ◽  
Diarrhea Virus ◽  
Cytopathic Effects ◽  
Porcine Epidemic Diarrhea ◽  
Piglet Survival

Abstract Background Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains. Methods In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. Results Pathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120. Conclusions Thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.

scholarly journals A Comprehensive View on the Host Factors and Viral Proteins Associated With Porcine Epidemic Diarrhea Virus Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Yi Hu ◽  
Xiaohong Xie ◽  
Lingchen Yang ◽  
Aibing Wang
Keyword(s):  
Virus Infection ◽  
Porcine Epidemic Diarrhea Virus ◽  
Molecular Mechanisms ◽  
Viral Proteins ◽  
Host Factors ◽  
Watery Diarrhea ◽  
Economic Losses ◽  
Target Cells ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV), a coronavirus pathogen of the pig intestinal tract, can cause fatal watery diarrhea in piglets, thereby causing huge economic losses to swine industries around the world. The pathogenesis of PEDV has intensively been studied; however, the viral proteins of PEDV and the host factors in target cells, as well as their interactions, which are the foundation of the molecular mechanisms of viral infection, remain to be summarized and updated. PEDV has multiple important structural and functional proteins, which play various roles in the process of virus infection. Among them, the S and N proteins play vital roles in biological processes related to PEDV survival via interacting with the host cell proteins. Meanwhile, a number of host factors including receptors are required for the infection of PEDV via interacting with the viral proteins, thereby affecting the reproduction of PEDV and contributing to its life cycle. In this review, we provide an updated understanding of viral proteins and host factors, as well as their interactions in terms of PEDV infection. Additionally, the effects of cellular factors, events, and signaling pathways on PEDV infection are also discussed. Thus, these comprehensive and profound insights should facilitate for the further investigations, control, and prevention of 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.

Sign in / Sign up

Export Citation Format

Share Document