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 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 Porcine Epidemic Diarrhea Virus and the Host Innate Immune Response

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 367 ◽  
Author(s):  
Shasha Li ◽  
Jinping Yang ◽  
Zixiang Zhu ◽  
Haixue Zheng
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Porcine Epidemic Diarrhea Virus ◽  
Innate Immune ◽  
Viral Rna ◽  
Watery Diarrhea ◽  
Economic Losses ◽  
Diarrhea Virus ◽  
Host Innate Immune Response ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV), a swine enteropathogenic coronavirus (CoV), is the causative agent of porcine epidemic diarrhea (PED). PED causes lethal watery diarrhea in piglets, which has led to substantial economic losses in many countries and is a great threat to the global swine industry. Interferons (IFNs) are major cytokines involved in host innate immune defense, which induce the expression of a broad range of antiviral effectors that help host to control and antagonize viral infections. PEDV infection does not elicit a robust IFN response, and some of the mechanisms used by the virus to counteract the host innate immune response have been unraveled. PEDV evades the host innate immune response by two main strategies including: 1) encoding IFN antagonists to disrupt innate immune pathway, and 2) hiding its viral RNA to avoid the exposure of viral RNA to immune sensors. This review highlights the immune evasion mechanisms employed by PEDV, which provides insights for the better understanding of PEDV-host interactions and developing effective vaccines and antivirals against CoVs.

Development and application of an indirect enzyme-linked immunosorbent assay using recombinant S1 for serological testing of porcine epidemic diarrhea virus

2019 ◽  
Vol 65 (5) ◽  
pp. 343-352
Author(s):  
Ying Shan ◽  
Yajie Liu ◽  
Ziqi Liu ◽  
Guowei Li ◽  
Cong Chen ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Porcine Epidemic Diarrhea Virus ◽  
Fluorescent Antibody ◽  
Area Under The Curve ◽  
Antibody Detection ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) causes severe infectious diseases in all ages of swine and leads to serious economic losses. Serologic tests are widely accepted and used to detect anti-PEDV antibodies that could indicate PEDV infection or vaccination. In this study, PEDV recombinant S1 protein (rS1) was expressed with the Bac-to-Bac system and purified by nickel-affinity chromatography. An indirect enzyme-linked immunosorbent assay based on rS1 (rS1-ELISA) was then developed and optimized by checkerboard assays with serial dilutions of antigen and serum. Serum samples from 453 domestic pigs and 42 vaccinated pigs were analyzed by the indirect fluorescent antibody (IFA) test and rS1-ELISA. Taking IFA as a gold standard, rS1-ELISA produced a high sensitivity (90.7%) and specificity (94.6%) by a receiver operating characteristic (ROC) curve. In addition, ROC analysis also revealed that rS1-ELISA was consistent with IFA (area under the curve 0.9583 ± 0.0082). This rS1-ELISA was then applied to antibody detection in inactivated PEDV vaccinated pigs. The antibody could be detected 2–4 weeks after the first inoculation. These results indicated that the rS1-ELISA established in this study provides a promising and reliable tool for serologic detection of anti-PEDV IgG antibodies in infected or vaccinated pigs.

scholarly journals Origin, Evolution, and Virulence of Porcine Deltacoronaviruses in the United States

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Yuanmei Ma ◽  
Yu Zhang ◽  
Xueya Liang ◽  
Fangfei Lou ◽  
Michael Oglesbee ◽  
...  
Keyword(s):  
United States ◽  
Porcine Epidemic Diarrhea Virus ◽  
Clinical Signs ◽  
The United States ◽  
Immune Serum ◽  
Watery Diarrhea ◽  
Economic Losses ◽  
Genetic Characteristics ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACTA novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.IMPORTANCEPorcine coronaviruses (CoVs) are major viral infectious diseases of swine. Examples of porcine CoVs include porcine transmissible gastroenteritis coronavirus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine respiratory coronavirus (PRCV). In February 2014, another porcine CoV, porcine deltacoronavirus (PdCV), emerged in Ohio and Indiana and subsequently spread rapidly across the United States and Canada,causing significant economic losses. Here, we report the detailed genetic characterization, phylogeny, and virulence of emergent PdCV strains in the United States. We found that PdCV caused severe diarrhea, vomiting, and dehydration in gnotobiotic and conventional piglets, signs that were clinically indistinguishable from those caused by PEDV and TGEV. In addition to extensive intestinal lesions, PdCV caused significant lesions in the stomach and mild pulmonary lesions that have not been reported for TGEV and PEDV. The finding that PdCV is a significant enteric disease of swine highlights the need to develop effective measures to control this disease.

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 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 Full-Length Genome Sequence of Porcine Epidemic Diarrhea Virus Strain CH/GX/2015/750A

2017 ◽  
Vol 5 (27) ◽  
Author(s):  
Yibin Qin ◽  
Bingxia Lu ◽  
Ying He ◽  
Bin Li ◽  
Qunpeng Duan ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Virus Strain ◽  
Complete Genome Sequence ◽  
Porcine Epidemic Diarrhea Virus ◽  
Complete Genome ◽  
Full Length ◽  
Watery Diarrhea ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea ◽  
Full Length Genome

ABSTRACT We report here the complete genome sequence of porcine epidemic diarrhea virus (PEDV) strain CH/GX/2015/750A (750A), which was isolated from a suckling piglet with watery diarrhea in Guangxi, China. The isolate is genetically close to other recent Chinese variant PEDVs and distinct from the classical PEDVs.

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